Grantee Research Project Results

We examined the toxicity of 10-nm diameter Fe₃O₄ (magnetite) and CoFe₂O₄ (cobalt ferrihydrite) nanoparticles functionalized with carboxymethyl dextran (CMDx) to developing zebrafish and found them to be relatively non-toxic both in the absence and presence of simulated sunlight illumination.

 

We found that 5-day exposure of zebrafish embryos to unfunctionalized TiO₂ NPs produced malformation and death, but only if the fish are also illuminated. The effects were seen at high NP concentrations. Damage to macromolecules by reactive oxygen species (ROS) produced when TiO₂ NPs are illuminated, however, can be continuous and cumulative. We hypothesized that given sufficient time, lower, more environmentally relevant, concentrations of TiO₂ NPs would produce sufficient ROS damage to be manifested as toxic responses in a whole organism. To test this hypothesis, we examined growth and survival of zebrafish exposed to aqueous suspensions of the same commercial preparation of TiO₂ NPs over a 23-day period, with and without simulated sunlight illumination. This exposure period encompasses embryogenesis, larval development, and metamorphosis from the larval into the juvenile/adult form. The TiO₂ NPs produced toxicity that was photo-dependent at concentrations as low as 1 ng∙mL^-1. Toxicity manifested as decreased survival, and retarded growth, delayed metamorphosis, malformed tails and jaws. In general, zebrafish are easier to culture than other fish, perhaps because they are resistant to the stresses of an artificial environment. Our results, using these hardy zebrafish, and an exposure period of less than 1 month, indicate significant mortality and toxicity in the low ng∙mL^-1 range. The environmental implications of this result are worth consideration.

 

We hypothesized that engineered surface functionalizations influence NP uptake by and toxicity to developing zebrafish. We tested these hypotheses using 6-nm diameter anatase TiO₂ NPs functionalized with small organic ligands (citrate, ascorbate, or 3,4-dihydroxybenzaldehyde (DHBA)). Ascorbate is an antioxidant. We expected TiO₂ NPs functionalized with this molecule to exhibit lower ROS production under simulated sunlight illumination than those functionalized with citrate or DHBA. Measured ROS concentrations were consistent with this expectation. For this set of functionalized TiO₂ NPs, we observed only modest toxicity to zebrafish embryos/larvae. Toxicity was enhanced under simulated sunlight illumination only for the NPs functionalized with DHBA consistent with the higher level of uptake of these nanoparticles. Exposure to simulated sunlight altered NP surface ligands and caused aggregation.

 

We hypothesized that coatings of natural organic matter acquired in the environment alter the uptake and (photo-enhanced) toxicity of TiO₂ NPs. We examined the influence of humic acid (HA) on TiO₂ NP suspension stability, and uptake by and toxicity toward developing zebrafish. We found that in the absence of simulated sunlight, HA increased suspension stability and decreased TiO₂ NP exposure. Despite this, a small but statistically significant increase in lethality was observed in fish exposed to TiO₂ NPs in the presence of HA. With exposure to simulated sunlight, photocatalytic oxidation of HA reduced suspension stability resulting in increased TiO₂ NP exposure in the presence of HA. Mortality increased significantly with HA relative to those exposed to TiO₂ NPs in the absence of HA. This study demonstrates the importance of considering environmental factors (i.e., exposure to sunlight, adsorption of HA), which can influence nanoparticle suspension stability when assessing the risks posed by engineered nanomaterials in the environment.