The objectives of this project were to determine the bacterial diversity in a heavily contaminated metal region of Portugal. Both traditional and molecular based methods were used to identify tolerant strains and species. The most tolerant species were subsequently identified and utilized for examining the potential for using them to immobilize specific metals from contaminated waste streams by comparing different support materials. Heterotrophic bacterial populations were isolated and characterized from a contaminated industrial area in Northern Portugal. In a first sampling, 278 strains were isolated in different solid media. To assess the diversity of this ecological site and to select representative strains, the isolates were screened by using Random Amplified Polymorphic DNA (RAPD)-PCR profiles. Phenotypic characterization, phylogenetic analysis by sequencing the 16S rRNA genes and metal tolerance tests with zinc (Zn), cadmium (Cd) and arsenic (As) were performed with the selected strains. Recovered gram-positive isolates were related to class Actinobacteria and Bacilli. The majority of the isolates were related to genera Microbacterium and Bacillus. Strains from the genus Arthrobacter were also well represented. 16S rRNA gene sequence similarity of the gram-negative isolates showed that they were related to classes γ-Proteobacteria, ß-Proteobacteria, a-Proteobacteria and Flavobacteria. The most frequently isolated taxa were γ-Proteobacteria, related with the genus Pseudomonas, where a large number of isolates were clustered. These genera are common in metal contaminated environments. Many of the strains (approx. 17) had a high level of tolerance to the heavy metals tested. A total of 13 isolates were not able to grow when metals were present. In a second sampling the soil rhizosphere was screened for bacterial populations, using metal-based selective media for isolation. About 42 strains were recovered when metal supplemented media was used. The gram-positive population were predominantly Bacilli and Actinobacteria members. Bacillus, Microbacterium and Arthrobacter were the most common gram-positive genera. Gram-negative genera were from the same classes as in the first sampling however Sphingobacteria was present. γ-Proteobacteria and ß-Proteobacteria were the most common taxa. The isolates were shown to be very resistant to Zn and As, with about half of the isolates able to grow with Cd present. Interestingly, no strains could grow in the presence of metal mixtures. Despite the number of strains recovered in both samplings the majority of the isolates were clustered within a very small number of genera. During the sampling periods two strains showing low similarity to other bacteria were isolated. These strains were characterized and studied in detail justifying their classification as representing two novel species of the genus Chryseobacterium. The names proposed for these organisms are Chryseobacterium palustre sp. nov. (type strain 3A10[type strain]) and Chryseobacterium humi sp. nov. (type strain ECP37[type strain]). Three isolates 1C2, 1ZP4 and EC30 belonging to genera Cupriavidus, Sphingobacterium and Alcaligenes respectively, showing high tolerance to heavy metals, were selected for further study in immobilised systems for Zn and Cd removal. In most cases, matrices (alginate, pectate and a synthetic cross-linked polymer) with immobilised bacteria showed better metal removal. 1C2, a strain belonging to the Cupriavidus genera, was able to increased the removal of Zn; EC30, a bacteria related to Alcaligenes, was the most promising candidate for Cd removal, especially when combined with the synthetic polymer. Removal of metals as single or in binary mixtures was also assessed. Cd removal was most effective when single metal solutions were tested using immobilised bacteria and examining metal matrixes. Based on the strains used and the matrices tested, best results were obtained for removal of Zn from binary mixtures with Cd. Potential exists for further studies to exploit these bacterial strain to develop effective bioremediation approaches for the removal of heavy metals from waste water streams.

The treatment of wastewater and monitoring of its toxicity is essential before discharging it to the environment. This study focuses on assessing the toxicity of wastewater following treatment using the electrochemical peroxidation process (ECP). Three categories of low-cost toxicity assays were used based on plant cells, microorganisms and invertebrates, all of which do not require ethical approval or a special licence. In addition, a novel cost-effective device was developed for assessing wastewater toxicity at low concentrations. In Chapter 2, the problem of high turbidity of the ECP-treated samples is addressed, by accelerating particulate settling of the existent compounds. This was achieved by storing the ECP treated samples at low temperatures. This is an essential step for enhancing the clarity of the processed samples in order to improve the performance of the toxicity assays.

A significant route for Endocrine disrupting chemicals (EDCs) such as natural and synthetic estrogens and nonylphenolics to enter the environment is via sewage treatment. Nanogram per litre levels of these compounds have been demonstrated to cause feminisation of aquatic organisms. The utilisation of treatments such as advanced oxidation and activated carbon could remove these compounds from effluent; however these technologies are expensive and energy intensive. Determining which operating parameters control the biodegradation of EDCs during activated sludge treatment, removal could be more sustainable. This study investigates which process parameters are responsible for controlling biodegradation of estrogens and nonylphenolics during activated sludge treatment. The role ammonia oxidising bacteria upon the breakdown of estrogens was also investigated. Field-based sampling campaigns were undertaken at three activated sludge plants and pilot-scale porous pot bioreactors were utilised to further examine the individual influences of loading, hydraulic retention time (HRT) and sludge retention time (SRT) upon EDC biodegradation.

Pharmaceuticals represent important and indispensable elements in modern society and their usage is considerable. Post consumption and body-elimination, pharmaceuticals are not completely removed in sewage treatment works (STWs) and as such, have been detected at low levels in STW effluents, surface waters, seawaters, ground waters and some drinking waters. Accordingly, pharmaceutical toxicity has been detected in several aquatic organisms. To date, environmental risk assessments (ERA) examine for toxicity using a series of chronic toxicity assays that examine for standard physiological responses in algae, Daphnia and fish and do not address pharmaceutical mode of action. Therefore, using the fathead minnow (Pimephales promelas) and the β-blocking pharmaceutical propranolol as the test-species and test-drug, respectively, the aim of this study was to establish an intelligent targeted 4-phased ERA using molecular, in vivo exposure, metabolomic and quantitative expression analytical techniques. The first phase established that the fathead minnow expressed the β3bi-adrenergic receptor (AR), which is a target receptor for propranolol in humans. The in vivo pair-breeding assay suggested that at 1mgL-1 and 10mgL-1, propranolol levels in fish blood plasma exceeded the human therapeutic concentration and caused 80% and 100% mortality, respectively. The most likely causes of mortality were liver failure and central nervous system toxicity. It was not possible to identify a robust biomarker of propranolol exposure using proton nuclear magnetic resonance (1H NMR) as there was considerable metabolic variation between male liver tissues within the same treatment groups. β3bi¬¬-AR expression was significantly lower at 1mgL-1 in the brain and liver, which was most likely the result of desensitisation in response to elevated levels of epinephrine and cortisol. β3bi¬¬-AR expression was significantly increased in the heart at the environmentally relevant concentration of 0.001mgL 1, however it was not possible to link β3bi¬¬-AR expression to a toxic response. Propranolol is unlikely to pose a threat to the aquatic environment as the concentrations measured in the environment are approximately 1000-fold lower than those that induced a toxic response. The proposed ERA represents a marked improvement over the existing ERA as it addresses pharmaceutical mode of action and both subtle and physiological toxicity responses, however it still requires further validation studies to address both metabolomic and gene expression variation.

No description available.

The sustained high price of crude oil is increasing the viability of producing oils and tarry bitumens which have high acid contents and which would have been uneconomical to produce previously. The acidity of these oils is due to the presence of a class of compounds known as Naphthenic Acids (NAs) which are thought to arise from the biodegradation of some of the crude oil hydrocarbons. Some oils contain as much as 3% by weight NAs. The acids cause engineering and production difficulties through corrosion of refinery plant and deposition as salts in pipelines. NAs also cause environmental problems because they are known toxicants. For example, the processing of the vast tar sands deposits of Alberta, Canada results in tailings pond waters containing very high concentrations of NAs. The ponds are estimated to exceed a billion m3 by 2025 and are already visible from space. Bioremediation is an attractive option for reducing the toxicity of such NA wastes. However, an understanding of the biodegradation potential of NAs has previously been hindered by the lack of knowledge of the molecular structure of individual NAs and by the lack of appropriate suitable surrogate acids for detailed mechanistic studies. Although it is known that NAs are complex mixtures of alkyl substituted, mainly alicyclic, carboxylic acids fitting the general formula C H2 _ZO2, where Z denotes the hydrogen deficiency resulting from ring formation, beyond this, comparatively little was known of NA chemistry, prior to the present study. The current study describes the synthesis of a series of novel monocyclic surrogate NAs containing both alkyl and alkanoate side-chains (viz: butylcyclohexylbutanoic acids, BCHBAs). The branching in the butyl chain was varied in a systematic way from nthrough sec-, iso- and tent-butyl. The surrogates were synthesised in high purity (>94%) and rigorously characterised at each synthetic stage by various spectroscopic techniques. A toxicity assay in which developing oyster (Crassostrea gigas) embryos were inoculated with the individual monocyclic surrogate NAs demonstrated EC50 (Effective Concentrations for 50% of population) values ranging from 0.11 mg L1 to 0.49 mg L'. The results exhibited a clear quantitative structure-activity relationship (QSAR) between the degree of branching in the alkyl chain and the toxic effect, with the less branched NAs exhibiting the highest toxicity. A detailed biodegradation study of the monocyclic surrogate NAs was made. Again a clear QSAR was established. In this instance, n-BCHBA was the most readily degraded with 97% degradation by day 9, and tert-BCHBA the most resistant with only 2% degradation by day 30, whereas iso-BCHBA and sec-BCHBA were 77% and 53% degraded after 30 days, respectively. The degradation of the monocyclic surrogate NAs proceeded with the production of major metabolites consistent with ß-oxidation of the alkanoic acid side chain. The metabolites were tentatively identified by mass spectrometry as the ethanoic acid analogues of the butanoic acids. These resisted further biodegradation. That biodegradation proceeded via 8-oxidation was confirmed by the synthesis and subsequent biodegradation experiments with iso-butylcyclohexylmethylbutanoic acid, where the iv additional methyl branch in the butanoic acid side chain effectively halted biodegradation. The QSAR observed in the toxicity and biodegradation experiments is thought to be related to the hydrophobicity of the individual NA surrogates, which determines their bioavailability. These results with surrogate compounds help to explain previous reports of the biodegradation of natural NAs in which reductions in toxicity during initial biodegradation have been observed. The surrogate NAs synthesised herein were also used to devise a unique derivatisation procedure which then facilitated the analysis of synthetic and natural NAs by liquid chromatography-multistage mass spectrometry (LC-MS°). Analysis of the resulting naphthenic amides (NAds) allowed an improvement in detection of over two orders in magnitude by positive ion electrospray ionisation-mass spectrometry (ESI-MS) compared to negative ion ESI-MS. The improved response allowed multistage LC-ESI-MS° experiments to be carried out and detailed mass spectral fragmentation pathways for individual NAds to be deduced. Analysis of derivatised natural NA mixtures then allowed a more detailed molecular characterisation of individual NAs than has been possible hitherto. The use of the surrogate NAs synthesised herein has thus improved significantly our understanding of NA chemistry and the influence of NA structure on the potential for NA bioremediation.

There is an emergent need to increase protection of nearshore resources from a growing human population, which is deteriorating coral reef ecosystems through coastal development, overfishing and destructive fishing practices. A possible solution involves increasing the number of smaller Marine Protected Areas (MPAs), creating a network of reserves with greater fisheries potential, while locally remaining small enough not to overly impinge on fishers available fishing grounds. Coral reefs are often found in developing countries, where governments financially struggle to establish successfully managed MPAs. A growing number of Hotel Managed Marine Reserves (HMMRs) have partly therefore, recently been established. Hotels arguably often have adequate funding, resources, and incentive to protect adjacent coastal areas - an HMMR could allow hotels to establish a market niche for a growing environmentally aware tourist. The principals of an Ecosystem-based Management (EBM) approach was adopted to test protection potential of an HMMR in Vietnam (Whale Island Resort: WIR) from a biological and socioeconomic point of view. Biannual visual fish census surveys (October 2005-April 2007) were conducted at the two marine reserves adjacent to WIR. The 6-year protected Whale Island Bay Reserve (WIB: 11 ha) showed significantly higher fish densities, richness, average size and number of fish >15 cm compared with two unprotected control sites. Fish stocks at the second, newer reserve, Whale Island Bay Peninsula Reserve (WIBP: 5 ha), quickly increased following protection. Fish assemblages at the 5 Artificial Reefs (ARs), made from clay pots (AR areas: 4.2-14.9 m^) in WIB, were greater than adjacent area-equivalent Natural Reefs (NRs) (11.15 greater biomass). showing larger fish assemblages with increasing AR size, adding to local fish stocks enhancement. Surveys were conducted with local fishermen to gauge socioeconomic impacts and management performance of the HMMRs. Fishermen mainly dependent on beach seining mostly opposed the HMMRs, while fishermen using other fishing techniques were generally in favour of the HMMRs, welcoming more protection and confirming spillover of fish, including large food fishes. In a Willingness to Pay (WTP) survey (n=211). 97.5 % of tourists at WIR supported HMMRs and 86.3 % were willing to pay an extra 10 % of the average room rate to stay at such hotels. In a worldwide survey of existing HMMRs, protecting areas from 1-700 ha (average 110 ha +/-13.22 SE), the average management rating attained was high (Good - HMMR is enforced). The accumulated findings from WIR and HMMRs globally, support the great potential of HMMRs as an added tool to protecting a part of our nearshore natural resources.

There is growing concern that the invertebrate test organisms commonly employed in the field of aquatic ecotoxicology may not be sufficient to accurately screen for the possible deleterious effects of contaminants discharged into the marine environment. The use of echinoderms has been proposed to redress this problem, due to their ecological importance and their evolutionary closeness to the chordates. But to date, there is a paucity of data in the published literature which has utilised the adult stages of echinoderms in laboratory based toxicology studies. The present studies aimed to fill this lack of information. A suite of biomarkers which operated at different levels of biological organisation (sub-cellular, cellular and individual level) were identified for use with different echinoderm species (the common sea star, Asterias rubens, the purple sea urchin, Paracentrotus lividus and the common brittle star, Ophiothrix fragilis). These biomarkers used were micronucleus induction, the Comet assay, the modified Comet assay, phagocytosis, neutral red retention, clearance rate and righting time. Concurrent exposures showed that echinoderms were more sensitive to model contaminants than a commonly used sentinel ecotoxicological test species, namely the blue mussel, Mytilus edulis. These contaminants included: the reference toxicants hydrogen peroxide (Chapter 2) and methyl methanesul phonate (Chapter 3); a pharmaceutical, cyclophosphamide (Chapter 3); a polycyclic aromatic hydrocarbon, benzo(a)pyrene (Chapter 4) and a heavy metal, cadmium (Chapter 5). The results for all the exposures showed that the biomarkers which operated at the lower levels of biological organisation (i. e. at the subcellular level – namely the micronucleus and Comet assays) were the most sensitive at detecting the deleterious effects of the contaminants. But, interestingly, some strong correlations were found between these sub-cellular consequences and those that operated at higher levels of biological organisation (for example, between righting time and both micronucleus induction and Comet assay in Asterias rubens following cyclophosphamide exposure). Theses correlations suggest that biomarkers which operate at the whole organism level (namely righting time and clearance rate) may serve as rapid and accurate indicators of possible damage induced by xenobiotics in echinoderms and bivalve molluscs.

Small islands dependent on reef fisheries, farming and tourism are vulnerable to rising human and natural pressures and may target "sustainable" development. Marine Protected Areas (MPA) have a proven ability to meet ecological goals, such as restoring fisheries and preserving ecosystem function across scales. However, there is a comparative lack of scientific baseline data and social research which may help local MPA to overcome failures in achieving mixed conservation and development goals in populated coastal areas facing intense pressure. This fieldwork-based case study researched in French and Creole languages in the sub-tropical, mid-Oceanic island of Rodrigues (Mauritius, Indian Ocean) addresses two main questions: "Are conservation and development compatible goals for MPA in small islands reliant on fisheries and tourism?", and, "Do social-ecological resilience concepts help clarify related issues of sustainability?". Results from two quantitative surveys with fishers {n=93) and tourists (n=351) on one level support a "win-win" scenario for conservation and development. Local fishers' knowledge suggested marine fish species including large predators of ecological and economic significance had been in decline for decades. Tourists' stated willingness to pay to use Marine and Coastal Protected Areas could help fund consen/ation of biodiversity and fishery enhancement, with fee options ranging from MPA up to island-level. Beyond this, downside risks emerged from qualitative interviews with key informants (n=70) and historical analysis of island-level social-ecological resilience testing the explanatory value of the conceptual Adaptive Cycle model (Holling and Gunderson 2002). A recent crisis catalysed by severe drought (1970s) led to deep social and ecological changes (collapse in farming, migration and external dependence), while subsequent policies failed to address key drivers, instead creating negative feedbacks ensuring degradation extended outward from the coast. Remote and vulnerable small islands with few resources (forests, soil, water, energy) need significant capital inputs from higher scales which are seldom taken into account in determining the balance of winners and losers in conservation and development policy at MPA or island level. A lack of interisland trust (social capital), water scarcity, climate change and migration arise as critical issues for the future. Rodrigues characterises the secondary importance of island regions within larger Island states, and underlines the cross-scale and cross-temporal nature of sustainability in resilience terms. This thesis' main contribution lies in its first demonstration of shifting baselines in an island reef fishery or MPA context. Findings contributed to the establishment of MPA In the fieldwork site of Rodrigues, and are of broad relevance for MPA policy across the tropics and beyond. More studies are needed across other ecosystems and cultures.

Human activities are degrading marine ecosystems and undermining the ecological functions and processes which provide valued goods and services. European and UK marine policy developments aim to implement the Ecosystem Approach to support better management of activities and maintain the health of regional seas. Current public perceptions of the UK marine environment are overwhelmingly negative, creating a barrier to engaging society with marine environmental issues and policy. This thesis conducts a study of the attributes of a suite of 72 UK marine species to identify those which contribute most to marine ecological health. The findings show that structurally complex species are most important and are recommended as species to assess and monitor Good Environmental Status as defined by the EU Marine Strategy Framework Directive. Existing conservation policies are biased towards large vertebrate species, with ecologically important species being underprotected. A survey of public perceptions of the marine environment. revealed conflicting perceptions of charismatic megafauna. Charismatic species were the most interesting species but least important as measures of marine health. Ecologically important species were the least interesting, but ecological health concepts were considered important measures of marine health. Perceptions of the marine environment varied with socio-demographic and social value factors. By integrating these studies, barriers and opportunities to engaging society with the marine environment were identified. Communication strategies which address these are proposed, including a suite of Spokes Species, potential high profile species to champion the marine environment. These include puffin, cod, basking shark and seagrass. A series of themes are proposed which implement other key findings such as the importance of personal experience in building connections with marine species. Communication strategies are supported by ecologically defined assessments of marine environmental health, are relevant to current policy developments and will resonate with social values of the marine environment.