This thesis describes a study of the remobilisation of heavy metals from sediments by three aminopolycarboxylic acids (APCAs). They are nitrilotriacetic acid, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. The investigation is introduced by examining the sources, uses and chemistry of these acids. The introduction also includes a discussion of what is known about the inclusion of heavy metals into sediments and their remobilisation from sediments. Typical concentrations of APCAs in natural waters and sediments have been catalogued from the literature. The advantages and disadvantages of various laboratory techniques employed for the remobilisation of heavy metals by APCAs from sediments are assessed, as is the use of such experiments in quantifying the role of APCAs in the remobilisation of heavy metals from sediments. Sediments from three areas were sampled for this study; they were the Alexandra Canal, Captains Flat and Jenolan Caves in New South Wales, Australia. In each area several sites were sampled. For each site there is a brief description of the catchment geology and hydrology. Selected sediment-associated waters in the areas were analysed for their metal concentrations as well as for ultratrace levels of APCAs employing a method developed in the present study. The waters were analysed for the major ions Ca2+, Mg2+, K+, Na+, Cl-, NO3- and SO42-. The sediments from selected sites in each of the areas were dried and fractionated. The dry total and fine sediments were analysed for their metal content and the latter was found to adequately represent the former in this respect. Water samples from the three areas showed different chemistries and exhibited more subtle differences between sites. In general, the Alexandra Canal waters are saline and alkaline and are a mixture of urban runoff and seawater; the Captains Flat waters are acidic and contain high sulfate from acid mine and tailings drainage; the Jenolan Caves waters are neutral and have the features characteristic of waters draining through limestone. The APCA contamination in all water samples when ranked against other global sites is very low. Although the current APCA levels in the waters appear low, it was concluded that they should be closely monitored so that efforts can be made to minimise the risk of APCAs being hazardous environmental contaminants and also that any remobilisation of heavy metals from sediments by APCAs can be controlled. Agitation and column laboratory-scale experiments were carried out in order to obtain an understanding of the remobilisation of metals by contamination levels of APCAs in water, both as the individual APCAs and as a mixture of APCAs. Complimentary experiments were carried out using a molar excess of APCAs calculated from the metal concentrations obtained by acid digestion (assuming 1:1 metal complex formation). Both types of remobilisation experiments were designed to investigate the role that redox potential (Eh) and concentration of APCAs in natural waters have on the remobilisation of heavy metals from the sediments. The agitation experiments were employed to assess metal remobilisation for the situation where the sediments are disturbed while the column experiments explored metal remobilisation for the case where the sediments are left undisturbed in situ. The major conclusions from the agitation experiments that used fine sediment from the Alexandra Canal were that 100 ppm APCA solutions will remobilise metals from the sediments under oxic conditions but only remobilise infinitesimally small amounts of metal under anoxic conditions. The use of fine sediments for the duplicate agitation experiments was found to give adequate duplication of results. A mixture of APCAs in solution acts similarly to the average of the three individual APCA solutions, showing that there are no antagonistic or synergistic effects likely to occur when they are found together in the environment. It was found that the mmoles of the metals remobilised exceeded the mmoles of the APCAs added when 500.0 mL of 100 ppm APCA solution was used on 50.00 g of sediment. This might be due to APCAs remobilising metals from the sediments in ways other than by complexation. Even though an excess of APCAs was available, metal remobilisation was not complete when the experiments were forced to terminate. During the 14 days of the experiment, only one quarter of the metals liberated from the sediment by HNO3 and 30 % H2O2 digestion were remobilised by the APCAs. Therefore an excess of free APCAs remains in solution. Fine sediments from Alexandra Canal, Captains Flat and Jenolan Caves were employed in the oxic agitation experiments using excess APCAs in solution. These experiments resulted in the following major conclusion: when producing an APCA remobilisation signature for trace and ultratrace metals, the geochemistry of the site is of secondary importance to the source of the contaminating metals. This is a feature of the trace and ultratrace metal speciation in the source rather than their concentration in it. From the different levels of calcium present in the three areas it was found that calcium is unlikely to form stable 1:1 APCA complexes at the pH values employed and is unlikely to compete with the heavy metal remobilisation by APCAs. Total sediments from Alexandra Canal and 100 ppm APCA solutions were employed for the column leaching experiments. From mass, pore water volumes and flow measurements it was shown that the ten mini cores taken from the same site were not true replicates. Despite this, when the sediments have settled and the pore waters removed from the cores, the levels of metal being leached stabilise and may represent a clearer picture of the in situ metal leaching from sediment with time. The levels of metal leached from the cores in 14 days suggest that during this period the cores are essentially anoxic, with the oxygen supplied from the oxic leaching solutions used for inorganic and microbial processes in the sediments. Agitation experiments appeared to yield an adequate picture of what would happen if free APCA solution came in contact with fine sediments suspended in the water column. Column leaching experiments employing total sediment were found to be only of limited value in assessing heavy metal remobilisation from undisturbed sediment. These experiments do not give a reliable assessment of the bioavailability of heavy metals and further testing of the acute and chronic toxicity of the sediments is recommended. APCA solutions that have been used in sediment and soil washing under conditions related to those used in the present study may contain an excess of the free APCAs as well as APCA heavy metal complexes and hence may be toxic to biota.

This thesis describes a study of the remobilisation of heavy metals from sediments by three aminopolycarboxylic acids (APCAs). They are nitrilotriacetic acid, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. The investigation is introduced by examining the sources, uses and chemistry of these acids. The introduction also includes a discussion of what is known about the inclusion of heavy metals into sediments and their remobilisation from sediments. Typical concentrations of APCAs in natural waters and sediments have been catalogued from the literature. The advantages and disadvantages of various laboratory techniques employed for the remobilisation of heavy metals by APCAs from sediments are assessed, as is the use of such experiments in quantifying the role of APCAs in the remobilisation of heavy metals from sediments. Sediments from three areas were sampled for this study; they were the Alexandra Canal, Captains Flat and Jenolan Caves in New South Wales, Australia. In each area several sites were sampled. For each site there is a brief description of the catchment geology and hydrology. Selected sediment-associated waters in the areas were analysed for their metal concentrations as well as for ultratrace levels of APCAs employing a method developed in the present study. The waters were analysed for the major ions Ca2+, Mg2+, K+, Na+, Cl-, NO3- and SO42-. The sediments from selected sites in each of the areas were dried and fractionated. The dry total and fine sediments were analysed for their metal content and the latter was found to adequately represent the former in this respect. Water samples from the three areas showed different chemistries and exhibited more subtle differences between sites. In general, the Alexandra Canal waters are saline and alkaline and are a mixture of urban runoff and seawater; the Captains Flat waters are acidic and contain high sulfate from acid mine and tailings drainage; the Jenolan Caves waters are neutral and have the features characteristic of waters draining through limestone. The APCA contamination in all water samples when ranked against other global sites is very low. Although the current APCA levels in the waters appear low, it was concluded that they should be closely monitored so that efforts can be made to minimise the risk of APCAs being hazardous environmental contaminants and also that any remobilisation of heavy metals from sediments by APCAs can be controlled. Agitation and column laboratory-scale experiments were carried out in order to obtain an understanding of the remobilisation of metals by contamination levels of APCAs in water, both as the individual APCAs and as a mixture of APCAs. Complimentary experiments were carried out using a molar excess of APCAs calculated from the metal concentrations obtained by acid digestion (assuming 1:1 metal complex formation). Both types of remobilisation experiments were designed to investigate the role that redox potential (Eh) and concentration of APCAs in natural waters have on the remobilisation of heavy metals from the sediments. The agitation experiments were employed to assess metal remobilisation for the situation where the sediments are disturbed while the column experiments explored metal remobilisation for the case where the sediments are left undisturbed in situ. The major conclusions from the agitation experiments that used fine sediment from the Alexandra Canal were that 100 ppm APCA solutions will remobilise metals from the sediments under oxic conditions but only remobilise infinitesimally small amounts of metal under anoxic conditions. The use of fine sediments for the duplicate agitation experiments was found to give adequate duplication of results. A mixture of APCAs in solution acts similarly to the average of the three individual APCA solutions, showing that there are no antagonistic or synergistic effects likely to occur when they are found together in the environment. It was found that the mmoles of the metals remobilised exceeded the mmoles of the APCAs added when 500.0 mL of 100 ppm APCA solution was used on 50.00 g of sediment. This might be due to APCAs remobilising metals from the sediments in ways other than by complexation. Even though an excess of APCAs was available, metal remobilisation was not complete when the experiments were forced to terminate. During the 14 days of the experiment, only one quarter of the metals liberated from the sediment by HNO3 and 30 % H2O2 digestion were remobilised by the APCAs. Therefore an excess of free APCAs remains in solution. Fine sediments from Alexandra Canal, Captains Flat and Jenolan Caves were employed in the oxic agitation experiments using excess APCAs in solution. These experiments resulted in the following major conclusion: when producing an APCA remobilisation signature for trace and ultratrace metals, the geochemistry of the site is of secondary importance to the source of the contaminating metals. This is a feature of the trace and ultratrace metal speciation in the source rather than their concentration in it. From the different levels of calcium present in the three areas it was found that calcium is unlikely to form stable 1:1 APCA complexes at the pH values employed and is unlikely to compete with the heavy metal remobilisation by APCAs. Total sediments from Alexandra Canal and 100 ppm APCA solutions were employed for the column leaching experiments. From mass, pore water volumes and flow measurements it was shown that the ten mini cores taken from the same site were not true replicates. Despite this, when the sediments have settled and the pore waters removed from the cores, the levels of metal being leached stabilise and may represent a clearer picture of the in situ metal leaching from sediment with time. The levels of metal leached from the cores in 14 days suggest that during this period the cores are essentially anoxic, with the oxygen supplied from the oxic leaching solutions used for inorganic and microbial processes in the sediments. Agitation experiments appeared to yield an adequate picture of what would happen if free APCA solution came in contact with fine sediments suspended in the water column. Column leaching experiments employing total sediment were found to be only of limited value in assessing heavy metal remobilisation from undisturbed sediment. These experiments do not give a reliable assessment of the bioavailability of heavy metals and further testing of the acute and chronic toxicity of the sediments is recommended. APCA solutions that have been used in sediment and soil washing under conditions related to those used in the present study may contain an excess of the free APCAs as well as APCA heavy metal complexes and hence may be toxic to biota.

Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.