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This thesis examines the vibrational structures of six phytoestrogen isoflavones, namely, daidzein, genistein and their methoxy derivatives: formononetin, biochanin A, prunetin and 4’,7-dimethoxygenistein, with a particular focus on surface-enhanced Raman spectroscopy (SERS). The purpose of this work is to develop a fundamental understanding of some of the structural and vibrational aspects of isoflavones, thereby providing a foundation for applications of vibrational spectroscopy in future phytoestrogen research. Following the first 3 introductory chapters on phytoestrogens, vibrational spectroscopy and experimental techniques, the results are presented over Chapters 4-7, of which, Chapters 4-6 are based on published or accepted articles. Chapter 7, based on unpublished work, applies the knowledge acquired in the earlier chapters to the interpretation of SERS on alternative substrates. Theoretical density functional theory (DFT) calculations are also extensively applied in aiding the interpretation of all spectra explored in this thesis, demonstrating the benefits of computational chemistry in studying vibrational spectroscopy. In Chapter 4, infrared and Raman spectra were applied in the characterization of the structural and vibrational properties of the isoflavones. The analysis was performed through a combined approach, incorporating DFT calculations, vibrational mode correlations between the isoflavone derivatives and also between their sub-structural motifs: phenol and anisole. This provides the necessary background for understanding SERS of the isoflavones in the ensuing chapters. In Chapter 5, investigations of the SERS of daidzein, which has two hydroxyl groups (7-OH and 4’-OH), and its 4’-methoxy derivative, formononetin, on citrate-reduced silver colloids (cit-AgC) are reported. In SERS, the nature of the interaction between the analyte and the metal surface is critical for the effective enhancement of the Raman signals. Through pH dependence and comparative SERS analysis of the two isoflavones, it was revealed that the OH groups needed to be deprotonated in order to interact with the silver surface and become SERS active. Accordingly, the deprotonated 7-OH acts as the site of interaction with cit-AgC on both daidzein and formononetin, and also through 4’-OH deprotonation on daidzein. Bands near 1482 and 1493 cm-1 are identified as characteristic of the anionic interactions with silver via the 7-OH and 4’-OH groups, respectively. The study is extended in Chapter 6 to a series of 5-hydroxyisoflavones: genistein (Figure T1), biochanin A, prunetin and 4’,7-dimethoxygenistein, to reveal a similar pH dependent, anionic interaction via the 7-OH and 4’-OH groups. However, the 5-OH group was found not to be strongly SERS active. Limited solubility in the finely controlled SERS conditions and steric hindrance are suggested as possible reasons for their lack of interaction. Finally, in Chapter 7, a selection of alternative SERS substrates is examined for their effectiveness in aiding isoflavone analysis. Three substrates were selected on the basis of commercial availability and partial reusability: sol-gel SERS substrates, oblique angle deposition (OAD) SERS substrates and Klarite® SERS Detection substrates. Different substrates can have distinct surface environments, which can consequently affect the analyte-metal interactions and the resultant SERS profile. On sol-gel and OAD substrates, which contain silver as the SERS active surface, the isoflavones were found to undergo similar interactions as on cit-AgC, as evidenced by similarities in the measured spectra and, in particular, a number of marker bands. In contrast, the isoflavones were found to adsorb in the neutral form on the gold surface of Klarite, being weakly physisorbed via 7-OH and π-cloud of the chromone group. On all three substrates, the analysis is facilitated by the fundamental understanding acquired in the earlier chapters, thereby demonstrating the applicability of the vibrational characterisations in this work on alternative substrates, and in effect, different environments.

Refereed/Peer-reviewed Redox-sensitive trace elements, such as vanadium, are frequently used as paleoredox proxies to infer the redox status of ancient marine depositional environments. When applied to vanadium, this approach relies on an accurate understanding of vanadium geochemistry in marine sediments. This, in turn, requires the application of analytical techniques capable of providing reliable chemical information on vanadium host phases of vanadium and thus the ability to distinguish authigenic from detrital phases. The increasing availability of synchrotron-based X-ray spectroscopic techniques capable of providing chemical speciation information at both the bulk and micro-scale presents an opportunity to refine our understanding of vanadium geochemistry in ancient marine sediments. Here, we show the utility of synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy and scanning X-ray fluorescence microscopy (SXFM) for investigating vanadium host phases in ancient marine sediments. The studied samples from the 1400 Ma Xiamaling Formation came from strongly contrasting depositional settings (ranging from oxic to euxinic) with a wide range of vanadium enrichments (enrichment factors from 0.7 to 9.6), yet vanadium speciation determined by XANES was consistently found to be phyllosilicate-hosted vanadium (III). High-resolution elemental imaging by SXFM coupled with global statistical colocalization analyses revealed strong associations between vanadium and potassium, consistent with vanadium hosted by potassium-rich clay minerals. These findings, while demonstrating the power of synchrotron-based X-ray methods, imply that differentiating authigenic and detrital vanadium in ancient marine sediments is unlikely to be possible given that post-depositional processes result in similar chemical speciation for both fractions. This will also limit the reliability of vanadium isotope system studies that depend on differentiating authigenic and detrital fractions using selective extraction procedures.

The objective of this study was to test the original speciation of silver (Ag) in eight different commercially available personal care products and investigate the chemical transformation of Ag during exposure to two types of synthetic greywater. The antimicrobial activity of the products was examined to determine the relationship between Ag content and speciation with the antibacterial functionality of the products. The Ag content of each product was quantified and X-ray absorption near-edge structure (XANES) analysis was used to investigate the initial speciation in the products and the changes occurring upon mixture with greywater. The results showed that the total Ag concentration in the products ranged from 17 to 30 mgkg−1, and was usually below the value reported on the label. Analyses revealed the complexity of Ag speciation in these products and highlighted the importance of characterisation studies to help elucidate the potential risks of nano-Ag in the environment. The antibacterial results confirmed that the antibacterial efficacyof the products depends on the concentration, form and speciation of Ag in the products, but is also significantly affected by product formulation. For instance, many of the products contained additional bactericidal ingredients, making it difficult to determine how much of the bactericidal effect was due directly to the Ag content/species. This paper offers some suggestions for standard methodologies to facilitate cross-comparison of potential risks across different studies and nano-enabled products. Refereed/Peer-reviewed

With the growing availability and use of copper-based nanomaterials (Cu-NMs), there is increasing concern regarding their release and potential impact on the environment. In this study, the short-term (≤ 5 d) aging profile and the long-term (135 d) speciation of dissolved Cu, copper oxide, and copper sulfide nanoparticles (CuO-NPs and CuS-NPs) were investigated in five different soils using X-ray absorption spectroscopy. Soil pH was found to strongly influence the short-term chemistry of the Cu-NMs added at 100 mg kg ⁻¹ above background. Low pH soils promoted rapid dissolution of CuO-NPs that effectively aligned their behavior to that of dissolved Cu within 3 d. In higher pH soils, CuO-NPs persisted longer due to slower dissolution in the soil and resulted in contrasting short-term speciation compared with dissolved Cu, which formed copper hydroxides and carbonates that were reflective of the soil chemistry. Organic matter appeared to slow the dissolution process, but in the long term, the speciation of Cu added as dissolved Cu, CuO-NPs, and CuS-NPs were found to be same for each soil. The results imply that, in the short term, Cu-NMs may exhibit unique behavior in alkaline soils compared with their conventional forms (e.g., in the event of an adverse leaching event), but in the long term (≥ 135 d), their fates are dictated by the soil properties, are independent of the initial Cu form, and are likely to present minimal risk of nanospecific Cu-NM impact in the soil environment for the concentration studied here. Refereed/Peer-reviewed

A wide range of methods are used to estimate the plant-availability of soil phosphorus (P). Published research has shown that the diffusive gradients in thin films (DGT) technique has a superior correlation to plant-available P in soils compared to standard chemical extraction tests. In order to identify the plant-available soil P species, we combined DGT with infrared and P K- and L2,3-edge X-ray adsorption near-edge structure (XANES) spectroscopy. This was achieved by spectroscopically investigating the dried binding layer of DGT devices after soil deployment. All three spectroscopic methods were able to distinguish between different kinds of phosphates (poly-, trimeta-, pyro- and orthophosphate) on the DGT binding layer. However, infrared spectroscopy was most sensitive to distinguish between different types of adsorbed inorganic and organic phosphates. Furthermore, intermediates of the time-resolved hydrolysis of trimetaphosphate in soil could be analyzed Refereed/Peer-reviewed

Silver nanoparticles (Ag-NPs) are used in a wide variety of products, prompting concerns regarding their potential environmental impacts. To accurately determine the toxicity of Ag-NPs it is necessary to differentiate between the toxicity of the nanoparticles themselves and the toxicity of ionic silver (Ag) released from them. This is not a trivial task given the reactive nature of Ag in solution, and its propensity for both adsorption and photo reduction. In the experiments reported here, we quantified the loss of silver from test solutions during standard ecotoxicity testing conducted using a variety of different test container materials and geometries. This sensitive 110mAg isotope tracing method revealed a substantial underestimation of the toxicity of dissolved Ag to the green algae Pseudokirchneriella subcapitata when calculated only on the basis of the initial test concentrations. Furthermore, experiments with surface-functionalized Ag-NPs under standard algal growth inhibition test conditions also demonstrated extensive losses of Ag-NPs from the solution due to adsorption to the container walls, and the extent of loss was dependent on Ag-NP surface-functionality. These results hold important messages for researchers engaged in both environmental and human nanotoxicology testing, not only for Ag-NPs but also for other NPs with various tailored surface chemistries, where these phenomena are recognized but are also frequently disregarded in the experimental design and reporting. Refereed/Peer-reviewed

DNA double strand breaks (DSBs) are deadly lesions that can lead to genetic defects and cell apoptosis. Techniques that directly detect DNA DSBs include scanning electron microscopy, atomic force microscopy (AFM), and fluorescence based approaches. While these techniques can be used to identify DSBs they provide no information on the molecular events occurring at the break. Tip-enhanced Raman scattering (TERS) can provide molecular information from DNA at the nanoscale and in combination with AFM provides a new way to visualize and characterize the molecular structure of DSBs. DSBs result from cleavage at the 3'- and 5'-bonds of deoxyribose upon exposure to UVC radiation based on the observation of P-O-H and methyl/methylene deformation modes enhanced in the TERS spectra. It is hypothesized that strand fragments are hydrogen-terminated at the lesion, indicating the action of free radicals during photon exposure. Refereed/Peer-reviewed

The reaction mechanisms of phosphate-bearing mineral phases from sewage sludge ash-based fertilizers in soil were determined by Raman and synchrotron infrared microspectroscopy. Different reaction mechanisms in wet soil were found for calcium and magnesium (pyro-) phosphates. Calcium orthophosphates were converted over time to hydroxyapatite. Conversely, different magnesium phosphates were transformed to trimagnesium phosphate. Since the magnesium phosphates are unable to form an apatite structure, the plant-available phosphorus remains in the soil, leading to better growth results observed in agricultural pot experiments. The pyrophosphates also reacted very differently. Calcium pyrophosphate is unreactive in soil. In contrast, magnesium pyrophosphate quickly formed plant-available dimagnesium phosphate. Refereed/Peer-reviewed

The widespread use of silver nanoparticles (Ag-NPs) results in their movement into wastewater treatment facilities and subsequently to agricultural soils via application of contaminated sludge. On-route, the chemical properties of Ag may change, and further alterations are possible upon entry to soil. In the present study, we examined the long-term stability and (bio)availability of Ag along the "wastewater sludge soil" pathway. Synchrotron based X-ray absorption spectroscopy (XAS) revealed that ca. 99% of Ag added to the sludge reactors as either Ag-NPs or AgNO3 was retained in sludge, with _>:79% of this being transformed to Ag2S, with the majority (>= 87%) remaining in this form even after introduction to soils at various pH values and Cl concentrations for up to 400 days. Diffusive gradients in thin films (DGT), chemical extraction, and plant uptake experiments indicated that the potential (bio)availability of Ag in soil was low but increased markedly in soils with elevated Cl, likely due to the formation of soluble AgClx complexes in the soil solution. Although high Cl concentrations increased the bioavailability of Ag markedly, plant growth was not reduced in any treatment. Our results indicate that Ag-NPs entering soils through the wastewater-sludge soil pathway pose low risk to plants due to their conversion to Ag2S in the wastewater treatment process, although bioavailability may increase in saline soils or when irrigated with high-Cl water. Refereed/Peer-reviewed

A series of 5-hydroxy-isoflavones—genistein, biochanin A, prunetin, and 4',7-dimethoxygenistein—have been studied by surface-enhanced Raman spectroscopy (SERS). Citrate reduced silver colloids were employed as a standard technique to measure SER spectra over a range of pH and concentrations. Density functional theory calculations were used to assist in determining the mode of interaction of isoflavones with the silver nanoparticles. It is revealed that biochanin A and prunetin interact with the silver nanoparticles upon deprotonation of the 7- and the 4'-OH groups, respectively, to show SERS activity. Correlations of their spectra with SERS of genistein strongly support the presence of multiple interaction modes involving both of the OH groups in genistein, in a similar manner to daidzein. Surprisingly, however, under these conditions, the 5-OH group was found to be noninteractive as revealed by attempts to measure SERS of 4',7-dimethoxygenistein. This was attributed partly to the low solubility and, more importantly, to the influence of steric hindrance, caused by the position of the pendant phenyl ring, which prevented interaction with the Ag colloid surface. These results complement recent work on daidzein and formononetin and provide further insight into understanding the SER spectra of isoflavones. Refereed/Peer-reviewed

The chemical state of thermochemically treated chromium(III) oxide (Cr2O3) with various carbonates was analyzed by far-infrared (far-IR) spectroscopy (spectral region 700–25 cm−1). Non-toxic Cr2O3 was oxidized with potassium, sodium, and calcium carbonate, respectively, to toxic Cr(VI) and Cr(V) compounds during thermal treatment at 1000 °C. In reverse, thermochemical treatment of Cr2O3 with magnesium carbonate lead to the formation of the Cr(III) compound MgCr2O4. Higher temperatures (>1200 °C) or reducing atmospheric conditions prevent the formation of Cr(VI)/Cr(V) compounds, too. Additionally, it was found that polyethylene powder with a low particle size (

Zero-valent iron nanoparticles (nZVI) have been widely tested as they are showing significant promise for environmental remediation. However, many recent studies have demonstrated that their mobility and reactivity in subsurface environments are significantly affected by their tendency to aggregate. Both the mobility and reactivity of nZVI mainly depends on properties such as particle size, surface chemistry and bulk composition. In order to ensure efficient remediation, it is crucial to accurately assess and understand the implications of these properties before deploying these materials into contaminated environments. Many analytical techniques are now available to determine these parameters and this paper provides a critical review of their usefulness and limitations for nZVI characterisation. These analytical techniques include microscopy and light scattering techniques for the determination of particle size, size distribution and aggregation state, and X-ray techniques for the characterisation of surface chemistry and bulk composition. Example characterisation data derived from commercial nZVI materials is used to further illustrate method strengths and limitations. Finally, some important challenges with respect to the characterisation of nZVI in groundwater samples are discussed. Refereed/Peer-reviewed

Silver nanoparticles (NPs) are used in more consumer products than any other nanomaterial and their release into the environment is unavoidable. Of primary concern is the wastewater stream in which most silver NPs are transformed to silver sulfide NPs (Ag2S-NPs) before being applied to agricultural soils within biosolids. While Ag2S-NPs are assumed to be biologically inert, nothing is known of their effects on terrestrial plants. The phytotoxicity of Ag and its accumulation was examined in short-term (24 h) and longer-term (2-week) solution culture experiments with cowpea (Vigna unguiculata L. Walp.) and wheat (Triticum aestivum L.) exposed to Ag2S-NPs (0–20 mg Ag L−1), metallic Ag-NPs (0–1.6 mg Ag L−1), or ionic Ag (AgNO3; 0–0.086 mg Ag L−1). Although not inducing any effects during 24-h exposure, Ag2S-NPs reduced growth by up to 52% over a 2-week period. This toxicity did not result from their dissolution and release of toxic Ag+ in the rooting medium, with soluble Ag concentrations remaining below 0.001 mg Ag L−1. Rather, Ag accumulated as Ag2S in the root and shoot tissues when plants were exposed to Ag2S-NPs, consistent with their direct uptake. Importantly, this differed from the form of Ag present in tissues of plants exposed to AgNO3. For the first time, our findings have shown that Ag2S-NPs exert toxic effects through their direct accumulation in terrestrial plant tissues. These findings need to be considered to ensure high yield of food crops, and to avoid increasing Ag in the food chain.

Surface enhanced Raman scattering (SERS) is a powerful tool with great potential to provide improved bio-sensing capabilities. The current 'gold-standard' method for diagnosis of malaria involves visual inspection of blood smears using light microscopy, which is time consuming and can prevent early diagnosis of the disease. We present a novel surface-enhanced Raman spectroscopy substrate based on gold-coated butterfly wings, which enabled detection of malarial hemozoin pigment within lysed blood samples containing 0.005%and 0.0005% infected red blood cells Refereed/Peer-reviewed

The use of nanoscale Ag in textiles is one the most often mentioned uses of nano-Ag. It has previously been shown that significant amounts of the Ag in the textiles are released upon washing. However, the form of Ag present in the textiles remains largely unknown as product labelling is insufficient. The aim of this study was therefore to investigate the solid phase speciation of Ag in original and washed silver textiles using XANES. The original Ag speciation in the textiles was found to vary greatly between different materials with Ag(0), AgCl, Ag2S, Ag-phosphate, ionic Ag and other species identified. Furthermore, within the same textile a number of different species were found to coexist. This is likely due to a combination of factors such as the synthesis processes at industrial scale and the possible reaction of Ag with atmospheric gases. Washing with two different detergents resulted in marked changes in Ag-speciation. For some textiles the two detergents induced similar transformation, in other textiles they resulted in very different Ag species. This study demonstrates that in functional Ag textiles a variety of different Ag species coexist before and after washing. These results have important implications for the risk assessment of Ag textiles because they show that the metallic Ag is only one of the many silver species that need to be considered Refereed/Peer-reviewed

The transformation and environmental fate of engineered nanomaterials (ENMs) is the focus of intense research due to concerns about their potential impacts in the environment as a result of their uniquely engineered properties. Many approaches are being applied to investigate the complex interactions and transformation processes ENMs may undergo in aqueous and terrestrial environments. However, major challenges remain due to the difficulties in detecting, separating, and analyzing ENMs from environmental matrices. In this work, a novel technique capable of in situ study of ENMs is presented. By exploiting the functional interactions between surface modified silver nanoparticles (AgNPs) and plasma-deposited polymer films, AgNPs were immobilized on to solid supports that can be deployed in the field and retrieved for analysis. Either negatively charged citrate or polyethylene glycol, or positively charged polyethyleneimine were used to cap the AgNPs, which were deployed in two field sites (lake and marina), two standard ecotoxicity media, and in primary sewage sludge for a period of up to 48 h. The chemical and physical transformations of AgNPs after exposure to different environments were analyzed by a combination of XAS and SEM/EDX, taken directly from the substrates. Cystine- or glutathione-bound Ag were found to be the dominant forms of Ag in transformed ENMs, but different extents of transformation were observed across different exposure conditions and surface charges. These results successfully demonstrate the feasibility of using immobilized ENMs to examine their likely transformations in situ in real environments and provide further insight into the short-term fate of AgNPs in the environment. Both the advantages and the limitations of this approach are discussed. Refereed/Peer-reviewed