Aluminium (Al) and silver (Ag), through human activities, are present in the environment at concentrations sufficient to cause toxicity. The aim of this study was to administer Al and Ag to the short lived model organism Drosophila melanogaster, so that developmental toxicity and potential ameliorative interventions could be examined over a compressed timescale relative to mammalian models. Aluminium was administered to Drosophila in food as either the chloride salt or citrate complex at concentrations of 1, 10 and 100 mM and various developmental parameters were assessed. The lowest concentration to delay pupation relative to the control was 10 mM but this depended upon the food in which it was administered. Higher whole body tissue levels of Al were seen following Al citrate administration compared to AlCl3, but Al citrate was less toxic as this did not did not impair larval viability at 100 mM; 100 mM AlCl3 resulted in 100% mortality. Eclosion success was significantly impaired with either form of Al at 10 mM, but no difference was seen between the forms of Al. When Drosophila were fed AlCl3 over their entire lifespan, a small but significant reduction in the lifespan of male flies was seen. No behavioural toxicity could be demonstrated. Existing studies have demonstrated significant tissue Al concentrations and toxicity whereas these have been minimal in this study. It is suggested that these differences may have a genetic component, with food composition exerting an influence also. Silver, either as AgNO3 or Ag nanoparticles (AgNPs) was administered in concentrations up to 500 micromolar and 10 mM, respectively. Either form of Ag, at 50 micromolar was sufficient to significantly retard pupation rate, although pupation or eclosion success was not impaired until 100 micromolar. The concentration-response relationship for AgNO3 was steep with pupation success dropping to nearly zero by 300 micromolar; Drosophila in this study were far more sensitive to AgNO3 than those in other reports. Animals exposed to AgNPs were still able to pupate at 500 micromolar, but these pupae were almost all non-viable when exposed to 400 micromolar AgNPs. At 1 mM and above, AgNPs, however, showed reduced toxicity compared to lower concentrations. The reasons for this are unclear. Both forms of Ag caused de-pigmentation in adults after larval exposure that may be explainable by inhibition of polyphenol oxidase enzymes by Ag (I) ions. The de-pigmentation was preventable by pre-loading larvae with Cu. Ascorbate prevented the de-pigmentation caused by AgNPs but not AgNO3 suggesting that AgNP toxicity is due to Ag (I) ion release. Oxidation of AgNPs was found to be greatly accelerated by Fe (III) and Cu (II) ions in the presence of Cl- ions. Although some of the results here conflict with the literature, developmental toxicity has been observed here, for both Al and Ag, and the variability across studies may provide an opportunity for dissecting the mechanisms behind Al and Ag toxicity through identification of the traits that confer sensitivity or resistance.

Many studies addressing the toxicity of silver nanomaterials have found that smaller sized silver nanoparticles are usually more toxic to organisms and in cell culture than particles of larger sizes yet it is not entirely clear why. We investigated the size dependent toxicity of silver nanoparticles by measuring the response of embryonic zebrafish (Danio rerio) following exposure to a library of thirteen distinct silver nanoparticle size distributions with mean diameters between 8.9 nm and 112.6 nm. Data analysis using dose���response modeling revealed that silver nanoparticles (AgNP) induced embryo toxicity that is dependent on the total surface area and not on the mass or particle number in solution. Included in this study is a comparison between embryo toxicity induced by silver nitrate (AgNO���) and AgNPs for cardiovascular endpoints, as well as an investigation into the influence of the chorion on AgNP toxicity. This study demonstrates the importance of using alternative dose metrics in nanotoxicology, and highlights the value of using the embryonic zebrafish to explore nanomaterial structure activity relationships. / Graduation date: 2013

Silver nanoparticles (AgNPs) are known to cause ecotoxic effects, but there are no existing derived ambient water quality criteria (AWQC) for these nanomaterials to protect freshwater aquatic life due to insufficient toxicological data. We exposed Hyalella azteca to silver nitrate, citrate-coated AgNPs (citrate-AgNPs), and polyvinylpyrrolidone-coated AgNPs (PVP-AgNPs) in a 10-day and 28-day water-only static renewal system with clean sand as a substrate for the amphipods and compared their point estimates with the United States Environmental Protection Agency (USEPA) AWQC for silver. We observed that all treatments decreased the survival, growth, and biomass of H. azteca, and the order of toxicity was AgNO > citrate-AgNPs > PVP-AgNPs. The LC50s of AgNO, citrate-AgNPs, and PVP-AgNPs were 3.0, 9.6, and 296.0 µg total Ag L, respectively, for the acute exposure and 2.4, 3.2, and 61.4 µg total Ag L, respectively, for the chronic exposure. Acute and chronic EC20s of citrate-AgNPs ranged from 0.5 to 3.5 µg total Ag L while that of PVP-AgNPs ranged from 31.2 to 175 µg total Ag L for growth and biomass. Both Ag released from AgNPs and the nanoparticles contributed to the observed toxicity. The dissolution and toxicity of AgNPs were influenced by surface coating agents, particle size, and surface charge. Most point estimates for AgNPs were above AWQC for silver (4.1 µg L) and the lowest concentration (0.12 µg/L) at which Ag is expected to cause chronic adverse effects to freshwater aquatic life. Our study demonstrates that the current AWQC for silver, in general, is protective of freshwater aquatic life against AgNPs tested in the present study.

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In freshwater, there is more opportunity for silver (Ag) to interact with organic ligands than in seawater. Triclosan is an antibiotic agent which resembles thyroid hormone T3 and is finding its way into aquatic systems. Preliminary toxicology studies for the frogSCOPE program suggest that triclosan and nanosilver (nanoAg), also used as an antibiotic agent, may be chemically interacting, as they seem to synergistically increase the endocrine-disrupting abilities already observed independently in each chemical. Ag speciation methods can be used to determine if triclosan or thyroid hormone T3 are interacting with Ag ion (Ag+), which gets released over time by nanoAg. To fully utilize Ag speciation methods, however, total Ag in the sample must also be independently analyzed. Here we investigated a new total Ag analysis using cadmium sulfide quantum dots (CdS QDs) as fluorescence probes in solution. This method promises results in a fraction of the time of the established competitive ligand equilibration-solvent extraction (CLE-SE) technique utilizing PDC- and DDC- to bind Ag and bring it out of solution. Following this investigation were a series of experiments using CLE-SE for total Ag and Ag speciation in well water used to house bullfrog tadpoles in frogSCOPE Ag exposure studies. CLE-SE for Ag speciation was also applied to well water samples containing the two levels of nanoAg or Ag+ used in frogSCOPE Ag exposures, and used in ligand competition experiments to examine the potential of triclosan or T3 to act as strong Ag-binding ligands, as compared to glutathione and EDTA, two known Ag-binding ligands. The results of the latter experiments could be used to determine if either of these could be forming complexes with Ag which increase or decrease their delivery to amphibian cells. The fluorometric method using CdS QDs showed no ideal analytical response to nanomolar Ag+, even when commercial QDs were modified and used, so it could not be applied to our samples. Using CLE-SE for total Ag, the well water used as a base for toxicity studies in frogSCOPE contained Ag below the method detection limit of 5 pM. Using the speciation variation of the CLE-SE method, no evidence of naturally-occurring ligands which could produce extractable (hydrophobic) or non-extractable (hydrophilic) Ag complexes was found in this well water. EDTA and glutathione responded as model Ag-binding ligands to form non-extractable hydrophilic Ag complexes in fresh water. T3 behaved like these model ligands, while triclosan enhanced the extractability of Ag in the presence of certain concentrations of the added ligand, DDC-. In another set of experiments, coordination of Ag by triclosan or T3 was not detectable within that analytical window. These results suggest that ionic Ag released over time by nanoAg may be binding T3 and preventing it from reaching its receptor, but confirming the interaction of triclosan and Ag+ will require additional experiments using different analytical windows.