In recent decades, there has been a rapid expansion in the use of manufactured nanoparticles (MNPs). Experimental evidence and material flow models predict that MNPs enter wastewater treatment plants and partition to sewage sludge and majority of that sludge is land applied as biosolids. During wastewater treatment and after land application, MNPs undergo biogeochemical transformations (aging). The primary transformation process for silver MNPs (Ag-MNPs) is sulfidation, while zinc oxide MNPs (ZnO-MNPs) most likely undergo phosphatation and sulfidation. Our overall goal was to assess bioavailability and toxicogenomic impacts of both pristine, defined as-synthesized, and aged Ag- and ZnO-MNPs, as well as their respective ions, to a model organism, the soil nematode Caenorhabditis elegans. We first investigated the toxicity of pristine Ag-MNPs, sulfidized Ag-MNPs (sAg-MNPs), and AgNO3 to identify the most sensitive ecologically relevant endpoint in C. elegans. We identified reproduction as the most sensitive endpoint for all treatments with sAg-MNPs being about 10-fold less toxic than pristine Ag-MNPs. Using synchrotron x-ray microspectroscopy we demonstrated that AgNO3 and pristine Ag-MNPs had similar bioavailability while aged sAg-MNPs caused toxicity without being taken up by C. elegans. Comparisons of the genomic impacts of both MNPs revealed that Ag-MNPs and sAg-MNPs have transcriptomic profiles distinct from each other and from AgNO3. The toxicity mechanisms of sAg-MNPs are possibly associated with damaging effects to cuticle. We also investigated the effects pristine zinc oxide MNPs (ZnO-MNPs) and aged ZnO-MNPs, including phosphatated (pZnO-MNPs) and sulfidized (sZnO-MNPs), as well as ZnSO4 have on C. elegans using a toxicogenomic approach. Aging of ZnO-MNPs reduced toxicity nearly 10-fold. Toxicity of pristine ZnO-MNPs was similar to the toxicity caused by ZnSO4 but less than 30% of responding genes was shared between these two treatments. This suggests that some of the effects of pristine ZnO-MNPs are also particle-specific. The genomic results showed that based on Gene Ontology and induced biological pathways all MNP treatments shared more similarities than any MNP treatment did with ZnSO4. This dissertation demonstrates that the toxicity of Ag- and ZnO-MNPs to C. elegans is reduced and operates through different mechanisms after transformation during the wastewater treatment process.

There has been an increase in the incorporation of silver nanomaterials into consumer products due to their antimicrobial properties. Therefore there is potential for silver nanoparticles (Ag-NPs) to leach out into the environment during different life-cycle stages of these nanomaterial-containing products. Concern about the toxicity of Ag-NPs has led to investigations into their toxic effects on a variety of organisms mainly using acute and sub-chronic, single-generation exposures. The focus of this project was to understand the effects of long-term continuous multigenerational exposure to AgNO3 and Ag-NPs in both pristine and environmentally transformed forms, on the model organism, Caenorhabditis elegans, a soil nematode. A previous multigenerational C. elegans study, showed increased sensitivity in terms of reproductive toxicity, in response to AgNO3 and Ag-NPs, but not sulfidized Ag-NPs (sAg-NPs), with increasing generations of exposure. The reproductive toxicity persisted in subsequently unexposed generations even after rescue from the exposure. We hypothesized that genomic mutations and/or epigenetic changes were possible mechanisms by which the reproductive toxicity was inherited. We investigated the potential for induction of germline mutations in C. elegans after exposures for ten generations to AgNO3, Ag-NPs, and sAg-NPs using whole genome DNA sequencing. Epigenetic changes at histone methylation markers, (H3K4me2 and H3K9me3), and DNA methylation at adenosine (N6-methyl-2’-deoxyadenosine) were investigated after multigenerational exposure as well as after rescue from the exposure using enzyme-linked immunosorbent assays (ELISA) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), respectively. Expression levels of the genes of methyltransferases and demethylases, associated with the histone methylation markers and DNA methylation, were also examined. Our results for germline mutations reveal no significant differences between the nematodes exposed to AgNO3 or pristine Ag-NPs when compared to controls. The significant increase in the number of transversion was observed only for sAg-NPs. However, a trend toward an increase in the total number of mutations was observed in all Ag treatments with some of those mutations having a predicted moderate or high impact. This potentially contributed towards reproductive as well as growth toxicity shown previously after ten generations of exposure in every treatment.. These results did not entirely support the multigenerational reproductive toxicity observed previously. Epigenetic responses at histone methylation markers revealed opposite patterns between pristine and transformed Ag-NPs with Ag-NPs causing a significant increase while exposure to sAg-NPs resulted in significant decrease in methylation at H3K4me2 mark. The increase in H3K4me2 levels was also inherited by subsequent unexposed generations rescued from Ag-NP exposure. Only sAg-NPs caused a significant decrease in methylation at H3K9me3 mark. Changes in mRNA levels for histone methyltransferases and demethylase corresponded with the histone methylation levels affected by Ag-NPs and sAg-NPs. For DNA methylation, a significant increase was observed only for AgNO3, which was not inherited after the rescue. In conclusion, while germline mutations with a high or moderate impact may affect reproduction, our results do not support this as a mechanism for the heritable increase in C. elegans sensitivity to reproductive toxicity from AgNO3 and pristine Ag-NPs. The epigenetic changes, however, do show partial correlation with the observed reproductive toxicity. The reproductive multigenerational effects of AgNO3 can be attributed to changes in DNA methylation whereas that of Ag-NPs can be attributed to changes in histone methylation. Further studies, focused on the investigation of changes in histone and DNA methylation levels at specific loci using chromatin immunoprecipitation sequencing (ChIP-Seq) and methylated DNA immunoprecipitation sequencing (MeDIP-Seq), respectively, are warranted for a better understanding of the impact of such changes.

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