Date of Award

Spring 2019

Document Type

Open Access Dissertation

Department

Environmental Health Sciences

First Advisor

Jamie R. Lead

Abstract

In marine systems, silver is regarded as being one of the most toxic and readily accumulated metals, and is known to be taken up and accumulate within marine invertebrates, fish, phytoplankton and seaweeds. Reported open ocean concentrations of silver range from 0.1 – 2 ng L-1 while silver concentrations in coastal waters are typically 5 to 10 times higher than open ocean values (0.1 - 50 ng L-1). The determination of silver in marine and coastal waters presents several challenges including low concentrations and matrix complexity of seawater, which creates interferences and other challenges for many instrumental techniques creating a requirement for additional separation steps. On-line matrix removal of the salt matrix prior to inductively coupled plasma mass spectrometry (ICP-MS) analysis has been developed to overcome these problems. A solid phase extraction method for the quantification of dissolved silver in seawater was developed using Dowex 1-x8 anion exchange resin, 0.1 M thiourea/0.05% nitric acid solution, and quantification by ICP-MS. Recoveries of 102.8 ± 4.1% were obtained for 25 µg L-1 silver spiked natural seawater samples. Using a 50 mL sample, a preliminary method detection limit of 66 ng L-1 (n=14, DI water samples) was achieved.

Silver nanoparticles (AgNPs) have become a central component in a wide range of commercial and consumer products including electronic, biomedical, and pharmaceutical drug delivery applications. Due to their unique properties, such as electronic constraint and high specific surface area, AgNPs exhibit novel behaviors that may affect biological processes such as uptake and accumulation, which might be significantly different to that of the bulk material or free silver (Ag) ion. Due to their partial solubility, studies show little agreement as to whether the effects and behavior of AgNPs are controlled by ions from dissolution of particles, or the particles themselves.

Bi-metallic nanoparticles (NPs), specifically core-shell NPs, offer a higher degree of functionality compared to single element NPs; Au and Ag have been used in this study in a core-shell conformation to allow better understanding of the impact of Ag solubility on bioavailability and bioaccumulation. The inner (Au or Ag107) core is protected from interacting with the media due to the Ag or Au/Ag109 outer shell. Therefore, Au or Ag107 can be used as a standard measurement of particle behavior and ratios of Ag and Au or isotopic ratios of Ag can be used to help explain the mechanisms influencing Ag and AgNP uptake. In this study we use Au@Ag NPs for exposure studies with an estuarine dinoflagellate, Prorocentrum minimum, and 107Ag@Au@109Ag NPs for exposure studies with an estuarine bivalve, Crassostrea virginica. This allowed the quantification of the contribution of ion and NP Ag bioavailability, ie., to understand the relative roles of ion and NP in AgNP uptake and accumulation.

Results in all cases showed that 60-85% of added Ag remained in aqueous suspension, while 10-30% of added Ag was detected in the dissolved phase. After exposure to Au@Ag P. minimum accumulated less Ag mass than seen in the AgNO3 exposures after 72 hours with 0.17 µg-Ag 100,000 cells-1 and 0.5 µg-Ag 100,000 cells-1, respectively. Cell densities decreased in a dose-dependent manner after exposure to Au@Ag and we speculate that algae are able to recover from initial exposure to Au@Ag after 24 hours and continue to increase in cell density. Although algae were able to recover, densities remained 10-20% below the control growth densities at the same time point. Conversely, there was continual decrease in cell densities after exposure to AgNO3 with a 94.9% decrease in the 50 µg L-1 exposure. Thus, we speculate that the potential mechanisms of AgNP uptake and accumulation are likely to be associated with NP dissolution and the interaction of dissolved Ag in the form of silver-chloride complexes, ripened AgNPs, or smaller NPs formed from the nucleation of dissolved Ag. In addition, there is a nano-specific result in accumulation of Ag as shown by the Ag:Au ratios associated with the algal pellet and dissolution results (3 kDa, ultrafiltered fraction) which confirms Au did not dissolve and remained as a NP.

After exposure to 107Ag@Au@109Ag, the average Ag uptake by C. virginica was a small fraction (8.6 – 9.0 ± 3.4 %) of the total available Ag. Compared to the isotopic ratios measured for the original core-shell AgNPs, we found increased accumulation of 107Ag in the 1 µg L-1 exposures and the opposite trend (increased accumulation of 109Ag) in the 50 µg L-1 exposures. There was variability among individual oysters both within a size class and across size classes. The hepatopancreas accumulated the most Ag in the 1 µg L-

1. The same trend was not seen in the 50 µg L-1 exposures. The F2 size class (reproductively mature females) accumulated the most Ag in the 1 and 50 µg L-1 exposures, as compared to the other size classes. We interpreted results as preferential uptake of NPs with a reduced Ag109 outer layer at 1 µg L-1 107Ag@Au@109Ag exposures. Furthermore, the increased accumulation in the hepatopancreas suggests that oysters are selecting AgNPs for ingestion. We explain higher 109Ag accumulation in 50 µg L-1 exposures as preferential uptake of nucleated AgNPs (containing only 109Ag) formed from dissolution of the 109Ag outer layer. Individual variability may be due to differences in filtration rate (1-1.5 L h-1 g- tissue weight-1), where larger oysters filter a larger volume of water which may results in greater amounts of Ag accumulated in tissues. Differences could also be caused by differences in energetic demands between oyster size classes or sex. For example, reproductively active individuals (M2 and F2) may require more nutrients to replenish energy spent on creating gametes as compared to not reproductively active individuals (M1 and F1).

The main reason for this study was to identify the mechanisms that facilitate AgNP uptake and accumulation by measuring ionic and NP Ag in estuarine organisms after exposure to AgNPs. We have shown that Ag is taken up and accumulated (or strongly bound) within both organisms after exposure to AgNPs with differences in uptake between the particles and ions observed. In both algae and oyster experiments, at low exposure concentrations (1 µg L-1) there was either less Ag than Au accumulation or less 109Ag than 107Ag suggesting that Ag is take up in the NP form. In exposure concentrations of 50 µg L-1 there was more Ag than Au uptake or more 109Ag than 107Ag for algae and oysters, respectively. This suggests that at higher concentrations, ionic Ag is preferentially taken up, most likely in a complexed or nucleated form. Consistent with previous research, our results confirm that Ag is taken up and accumulated by algae and oysters in a dose and time dependent manner when exposed to AgNO3 and has a greater accumulation and inhibition than seen in AgNP exposures. From these data and at current environmental levels of AgNPs, however, no immediate risk of AgNPs to P. minimum or C. virginica is indicated. Further testing and mechanistic understanding of AgNP interactions with marine algae and bivalves should be continued in order to fully understand the mechanisms of AgNP uptake and accumulation and the subsequent effect on estuarine ecosystems.

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