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The introduction of lux genes, able to express bioluminescence, into terrestrial bacteria enabled the optimisation of a bioluminescence-based bioassay that was environmentally relevant. Individual assay parameters such as growth phase, cell washing, lyophilisation, pH tolerance and temporal response to a range of metal and xenobiotic pollutants were evaluated. The effects of a range of pollutants upon the metabolic response of the lux-marked organisms were assessed using declines in bioluminescence. The lux -based bioassay proved more sensitive to the sub-lethal effects of metal pollutants than tests relying on culturability. Uncontaminated soils were spiked with metal and xenobiotic solutions both as single pollutants and in combination with other contaminants. Relative toxicity of metal and xenobiotic pollutants in soil systems were investigated using ecotoxicity assays based upon lux-marked constructs of Rhizobium leguminosarum biovar trifolii (an important associative nitrogen fixer) and the respiration of the microbial community. The lux-marked bioassay proved to be more sensitive than the community microbial assay to the presence of multiple contaminants at sub-lethal concentrations. The relative toxicities of metal and organic xenobiotic compounds were shown to be time dependent and better represented using chronic assaying of lux-marked microorganisms. Following a field trial involving the application of paper mill sludge to land and subsequent crop failure a rapid diagnosis of soil pollutants was required. A suite of ecotoxicity assays including lux-based bioassays, respirometry and enzyme activity were used to assess the toxicity of paper mill sludge to the soil microbial biomass. The selected lux-marked soil bacteria showed potential for use as rapid, field-based screening techniques to provide early warning of the potential hazards of waste application.

by Wong Pik-kwan. / Thesis submitted in: September 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 116-139). / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENT --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF TABLES --- p.viii / LIST OF FIGURES --- p.ix / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER TWO --- LITERATURE REVIEW / Chapter 2.1 --- Introduction --- p.4 / Chapter 2.2 --- Heavy metals --- p.6 / Chapter 2.3 --- Mechanisms of metal toxicity --- p.9 / Chapter 2.4 --- Toxic effects of metals on marine organisms --- p.10 / Chapter 2.4.1 --- Cadmium --- p.10 / Chapter 2.4.2 --- Chromium --- p.10 / Chapter 2.4.3 --- Copper --- p.11 / Chapter 2.4.4 --- Lead --- p.12 / Chapter 2.4.5 --- Nickel --- p.12 / Chapter 2.4.6 --- Zinc --- p.13 / Chapter 2.5 --- Metal uptake and elimination in marine organisms --- p.14 / Chapter 2.5.1 --- Uptake of metals --- p.14 / Chapter 2.5.2 --- Elimination of metals --- p.15 / Chapter 2.5.3 --- Metal detoxification system in fish --- p.16 / Chapter 2.6 --- Heavy metals in marine fish --- p.17 / Chapter 2.7 --- Bioaccumulation --- p.20 / Chapter 2.7.1 --- Models of metal accumulation --- p.21 / Chapter 2.7.2 --- Compartment model --- p.21 / Chapter 2.7.3 --- Physiologically based pharmacokinetic (PB-PK) model --- p.22 / Chapter 2.8 --- The influence of environmental factors on bioaccumulation of metals --- p.23 / Chapter 2.8.1 --- Temperature --- p.23 / Chapter 2.8.2 --- Salinity --- p.23 / Chapter 2.8.3 --- Organic matter --- p.24 / Chapter 2.8.4 --- pH --- p.25 / Chapter 2.8.5 --- Chelators and surfactants --- p.25 / Chapter 2.8.6 --- Other metals --- p.26 / Chapter 2.9 --- Biological effects of heavy metals on man --- p.26 / Chapter 2.10 --- The use of biological indicator organisms for metal pollution --- p.28 / Chapter CHAPTER THREE --- HEAVY METAL CONCENTRATIONS IN CULTURED MARINE FISH IN HONG KONG / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.36 / Chapter 3.2.1 --- Sampling --- p.36 / Chapter 3.2.2 --- Water analysis --- p.36 / Chapter 3.2.3 --- Sediment analysis --- p.39 / Chapter 3.2.4 --- Mussel analysis --- p.40 / Chapter 3.2.5 --- Fish analysis --- p.40 / Chapter 3.2.6 --- Quality control and statistical analysis --- p.41 / Chapter 3.3 --- Results --- p.42 / Chapter 3.3.1 --- Seawater --- p.42 / Chapter 3.3.2 --- Sediment --- p.46 / Chapter 3.3.3 --- Mussel --- p.46 / Chapter 3.3.4 --- Fish --- p.50 / Chapter 3.4 --- Conclusion --- p.67 / Chapter 3.4.1 --- "Metal concentration in seawater, sediment, green mussel and fish" --- p.67 / Chapter 3.4.2 --- Accumulation of heavy metals in different tissues of cultured fish --- p.69 / Chapter 3.4.3 --- Relationship between the body weight and metal accumulation --- p.71 / Chapter 3.4.4 --- Heavy metal pollution in fish culture sites --- p.72 / Chapter 3.4.5 --- Selection of fish culture site --- p.72 / Chapter CHAPTER FOUR --- ACUTE AND SHORT-TERM EFFECTS OF COPPER(II) IONS ON SPARUS SARBA / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Experimental animals --- p.79 / Chapter 4.2.2 --- Determination of the 96 hour median lethal concentrations --- p.19 / Chapter 4.2.3 --- Determination of growth rate --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- Determination of the 96 hour median lethal concentrations --- p.82 / Chapter 4.3.2 --- Determination of growth rate --- p.82 / Chapter 4.3.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.82 / Chapter 4.4 --- Conclusion --- p.91 / Chapter 4.4.1 --- Determination of the 96 hour median lethal concentrations --- p.91 / Chapter 4.4.2 --- Determination of growth rate --- p.93 / Chapter 4.4.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.94 / Chapter CHAPTER FIVE --- ACCUMULATION AND ELIMINATION OF COPPER(II) IONS TO SPARUS SARBA / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Materials and methods --- p.98 / Chapter 5.2.1 --- Experimental animals --- p.98 / Chapter 5.2.2 --- Uptake and elimination of Cu ion in S. sarba during continuous exposure to waterborne Cu --- p.98 / Chapter 5.3 --- Results --- p.100 / Chapter 5.4 --- Conclusion --- p.108 / Chapter CHAPTER SIX --- GENERAL CONCLUSION --- p.112 / CHAPTER SEVEN REFERENCES --- p.116

Heavy metals and hydrocarbons are persistent pollutants in the environment. Problems associated with the cleanup of sites contaminated by metals and hydrocarbons have demonstrated the need to develop remediation technologies that are feasible, quick, and effective in a wide range of physical settings. Experiments were conducted to investigate the efficiency of silica encapsulation and the factors that influence its performance. Analysis was done by ICP-OES and GC-FID for metals and hydrocarbons respectively. This technology was tested using sodium silicate and ChemcapTM. Soils and water contaminated with hydrocarbons and heavy metals were successfully remediated by silica encapsulation. The silica coating was stable under both acidic and alkaline conditions. A new product that is based on sodium silicate formulation was developed and was more effective at encapsulating hydrocarbons and heavy metals. Laboratory tests indicated that it is more effective in an acidic medium and it continues to strengthen with time. Metal encapsulation was affected by the sizes of metal ions and the presence of hydrocarbons.

In this research, the determination of trace concentrations of heavy metal ions was investigated using peptide modified electrochemical biosensors. The biosensor has several advantages over atomic absorption spectroscopy and inductively coupled plasma mass spectrometry by offering greater simplicity in use and the possibility of determining the bioavailability of heavy metals. Oligopeptides were modified on the electrode surface through the spontaneous self-assembly of thiols on gold. Firstly, 3-mercaptopropionic acid (MPA) was self-assembled onto the gold surface followed by activation of the carboxyl groups using a combination of carbodiimide and succinimide chemistry for coupling of the N-terminus of the peptide to occur. Using this generic strategy, Gly-Gly-His was used for the determination of copper ions. Cu2+ was accumulated at the MPA-Gly-Gly-His modified electrode at open circuit potential followed by electrochemical measurements. The reduction of Cu2+ to form underpotential deposited copper in the Osteryoung square wave voltammogram was used for quantification. The influence of various factors on the performance was investigated and after the optimal conditions had been identified, the biosensor was used for Cu2+ calibration and was applied to the analysis of a real sample. For Cd2+ detection, two different peptides covalently attached to MPA were investigated, g-Glu-Cys-Gly (GSH) and His-Ser-Gln-Lys-Val-Phe, with the latter sensor exhibiting a lower Cd2+ detection limit, higher sensitivity and greater selectivity. Although the success of MPA as a peptide linker to the gold surface had been shown for the detection of Cu2+ and Cd2+, a more viable approach was necessary for the stable detection of a wider range of metal ions. A more stable self-assembled monolayer of thioctic acid (TA) was identified in which human angiotensin I was attached. This alternate modification procedure was superior to MPA-angiotensin I for Pb2+ detection in terms of stability and reusability with the drawback being sensitivity. The newly identified strategy was also applied to the determination of Ag+ using TA-methionine enkephalin modified electrodes. A sensor array for Cu2+ was also investigated as well as an extension to the simultaneous determination of multianalytes using four different modified electrodes. Combining a soft-modelling approach, the responses of Cu2+, Cd2+ and Pb2+ could be deconvoluted.

Urbanisation in coastal catchments has significantly increased not only the input of terrestrial sediment to the marine environment but also the input of contaminants. In Tamaki Estuary, Auckland, heavy metals have accumulated in the upper estuarine muddy sediments and metal contamination has been detected on downstream intertidal sandflats. Sub-lethal levels of heavy metal contamination may affect the growth and behaviour of benthic organisms, which in turn may influence key ecosystem processes and productivity. The aim of this study was to examine whether the burial rate of an ecologically important bivalve species (Austrovenus stutchburyi) differed between a contaminated and a lesser-contaminated site and whether burial rates were affected by density. A secondary aim was to determine whether the burial of Austrovenus affected sediment transport and consequently if this was affected by density. This study demonstrated no consistent difference in burial time between source populations (sites). This was explained by a lack of measured difference in the condition index and heavy metal tissue loading of Austrovenus used throughout this study. The present range of contamination measured in Tamaki Estuary, Auckland, did not have negative biological consequences on the key ecosystem engineer, Austrovenus stutchburyi. Contamination levels in Tamaki Estuary may not be high enough to cause major physiological or behaviour changes to infaunal organisms, such as Austrovenus. Sediment erodability was not significantly correlated with any measured environmental and biotic factors. Austrovenus density was the only predictor variable that could be used to explain any variation in sediment erodability. There was no significant density effects observed between the amounts of sediment eroded for densities gt; 150 ind. m-2. There was a significant difference between sediment void of Austrovenus (0 ind. m-2; smooth, flat undisturbed sediment surface) and sediment containing Austrovenus (gt;150ind. m-2; physical structure on/in the sediment surface, increase in bed roughness). These results indicate that there is little or no effect of Austrovenus on the critical erosion threshold, suggesting that in the absence or presence of Austrovenus the current required to erode 10 g m-2 of sediment would remain somewhere between 28.5 and 30.5 cm s-1. This study found that there was considerable variation in the burial rate of individuals and the greatest variation was recorded in the lowest density treatments (150 ind. m-2), which corresponded to the same density that had the greatest variation in sediment erodability. Further investigations are needed to gain a better understanding into the important roles (the importance of the various feedbacks and limitations and interrelationships) that Austrovenus play in the soft-sediment ecosystem, as losses of this species are likely to have large-scale impacts on the wider soft-sediment communities and ecosystem functioning.

Concentrations of heavy metals (Cu, Cd, Cr, Zn, Mn, Fe and Pb) in the water columns, aquatic plants and sediments of fourteen lakes of varied levels of pollution were measured. Correlation analysis was carried out between heavy metal concentrations in aquatic plants and heavy metal concentrations in water and sediment. The aquatic plants which accumulated heavy metals in their tissues in proportion to that in water and sediments were identified. The aquatic plants studied were: 8/yxa auberti Rich, Cabomba caroliniana Gray, Ceratophyllum demersum L, Ceratopteris thalictroides (L.) Bron, Chara globularis, Eichhornia crassipes Solmn, Hydrilla verticillata Royle, Ipomoea aquatica Forsk, Limnophila aromatica (Lam.) Merr., Ludwigia adscendens (L) Hara, Nelumbo nucifera Gaertn, Nymphaea stallata Linn, Nymphoides indica (L.) Kuntze, Typha angustata Bony & Chaub and Utricularia aurea Lour. Metal uptake by aquatic plants varied between different species and within the same species depending on lake water contamination levels. The level of metal uptake to a great extent was a function of the environment water metal concentration. Results showed that Utricularia accumulated Mn, Zn, Cr, Cd and Pb in direct proportion to the overlying waters (r2 = 0.69, 0.63, 0.69, 0.65 and 0.39 respectively). Hydrilla accumulated Cu, Mn, Zn, Fe, Cr, Cd and Pb in direct proportion to the overlying waters (r2 = 0.65, 0.66, 0.44, 0.72, 0.38, 0.63, and 0.73 respectively). Blyxa leaves accumulated Zn, Fe, Cr, Cd and Pb in direct proportion to the overlying waters (r2 = 0.74, 0.74, 0.72, 0.60 and 0.82 respectively). Echhornia leaf accumulated only Cr in direct proportion to the overlying waters r2 = 0.81. Nymphaea leaf and Chara did not accumulate any metal in direct proportion to the overlying waters. Roots of Blyxa auberti, Ceratopteris thalictroides, and Eichhornia crassipes contained higher concentrations of heavy metals than their leaves. Roots of Blyxa accumulated Cr, Cd and Pb in direct proportion to the overlying waters (r2 = 0.91, 0.65 and 0.69 respectively). Echhornia root accumulated Cd in direct proportion to the overlying waters with r2 = 0.90. Nymphaea stem showed no significant correlations between the metal concentrations in the waters and in the plant. Utricularia accumulated Zn, Fe, Cr, Cd and Pb in direct proportion to the metals in the underlying sediment extracted by cold hydrochloric acid (r2 = 0.84, 0.51, 0.47, 0.68 and 0.80 respectively). Hydrilla accumulated Cu, Zn, Cr, Cd and Pb in direct proportion to the underlying sediment (r2 = 0.34, 0.37, 0.91, 0.49 and 0.96 respectively). Blyxa accumulated Zn, Fe, Cr, Cd and Pb in direct proportion to the underlying sediments (r2 = 0.99, 0.61, 0.82, 0.75 and 0.64 respectively) . Echhornia leaf showed significant correlation between the Cu (r2 = 0.83) and Cr (i2 = 0.88) concentration in underlying sediment and the plant. Nymphaea leaf showed a significant correlation between the Zn (r2 = 0.83) concentration in the plant and the underlying sediments. Roots of Blyxa showed significant correlation between concentrations of Cu, Cr and Pb in sediment extracted by hydrochloric acid and plant (r2 = 0.9, 0.7 and 0.9 respectively). Roots of Echhornia had no significant correlation with the sediment metal concentrations (hydrochloric acid extractable). Two techniques (cold hydrochloric acid extractable and nitric acid extractable) to extract metals from sediment were compared. Based on correlations of metal concentrations in plant tissue and metal extracted from the sediment, it was concluded that the cold hydrochloric acid extractable metal technique is more suitable for determining bioavailable sediment metal concentration in environmental studies. Laboratory studies investigations on the bioaccumulation of Zn and Cu in Hydrilla confirmed that Hydrilla is a good bioindicator of Cu as it accumulated 20360 ug/g dry weight of Cu in 72 hours. Hydrilla showed higher bioaccumulation factor with low concentration of Cu in the solution, in the laboratory studies. Hydrilla was determined to be the best indicator species as it reflected the heavy metal concentration in the environment which was supported by the laboratory studies.

Master of Science / Investigations of fluvial and estuarine sediments have indicated stormwater is an important source of heavy metals to Port Jackson estuary and high concentrations of these sedimentary contaminants are a threat to the healthy functioning of the estuarine ecosystem. Stormwater remediation devices have been installed in stormwater channels entering the estuary, however these devices are mainly for removing gross pollutants and are ineffective in removing heavy metals from stormwater. A thorough characterisation of heavy metal inputs and behaviour has been undertaken by sampling, analysing and modelling heavy metals in stormwater entering Port Jackson estuary to provide a rigorous data base for future remediation efforts. A conceptual model of transport and fate of heavy metals in stormwater entering Port Jackson estuary has also been developed to identify heavy metals, subcatchments and flow regimes requiring remediation, and to assist in designing remediation devices for optimum removal of heavy metals from stormwater. Modelling of stormwater using the Model for Urban Stormwater Improvement Conceptualisation (MUSIC) indicated that the average annual discharge of stormwater from the Port Jackson catchment was 215,307 ML. Average annual loadings of arsenic, cadmium, chromium, copper, nickel, lead and zinc in stormwater discharging to Port Jackson estuary were 0.8, 0.5, 1.7, 3.2, 1.1, 3.6 and 17.7 tonnes per year, although comparison to other studies in the catchment suggests these values may be underestimations of actual loadings by 1.3 to 10 times. The proportion of heavy metals discharged under low-flow conditions (<5mm of rainfall in 24 hours), medium-flow conditions (between 5 and 50mm in 24 hours), and high-flow conditions (>50 mm of rainfall in 24 hours) was 6.5%, 62.5% and 31%, respectively. The conceptual model indicates stormwater loadings of copper, lead and zinc pose a risk to the health of riverine and estuarine ecosystems in the catchment and these metals should be targeted for remediation. Stormwater channels which should be prioritised for remediation include the channels entering southern embayments west of Darling Harbour; Duck, Parramatta and Lane Cove Rivers; and the channels and rivers entering Neutral, Long and Sugarloaf Bays. Stormwater loadings of lead are predominantly associated with suspended particulates, whereas loadings of copper and zinc are equally partitioned between dissolved and particulate phases. Stormwater remediation strategies should target both dissolved and particulate phases to ensure effective removal of copper, lead and zinc. Research suggests heavy metals in stormwater discharged to the estuary under high-flow conditions are rapidly exported seaward and bypass the estuary. Preliminary research also suggests that under medium-flow conditions, particulate heavy metals bypass the embayments of Port Jackson and are deposited in the main channel. Once deposited in the main channel, particulate heavy metals are likely to be remobilised and removed from the estuary through multiple phases of resuspension. Although further research is required in this area, this preliminary research suggests remediation should target low-flow conditions. The findings of the current research could be used to identify appropriate remediation strategies for dissolved and particulate phase heavy metals in stormwater discharging to Port Jackson estuary. However, in designing stormwater remediation devices, consideration should also be given to the range of contaminants that may be present in stormwater entering Port Jackson estuary (including suspended solids, nutrients, pesticides and organics).