Performance evaluation of onsite sewage treatment : Evaluation of current research

Efficient management of domestic wastewater is a primary requirement for human well being. Failure to adequately address issues of wastewater collection, treatment and disposal can lead to adverse public health and environmental impacts. The increasing spread of urbanisation has led to the conversion of previously rural land into urban developments and the more intensive development of semi urban areas. However the provision of reticulated sewerage facilities has not kept pace with this expansion in urbanisation. This has resulted in a growing dependency on onsite sewage treatment. Though considered only as a temporary measure in the past, these systems are now considered as the most cost effective option and have become a permanent feature in some urban areas. This report is the first of a series of reports to be produced and is the outcome of a research project initiated by the Brisbane City Council. The primary objective of the research undertaken was to relate the treatment performance of onsite sewage treatment systems with soil conditions at site, with the emphasis being on septic tanks. This report consists of a ‘state of the art’ review of research undertaken in the arena of onsite sewage treatment. The evaluation of research brings together significant work undertaken locally and overseas. It focuses mainly on septic tanks in keeping with the primary objectives of the project. This report has acted as the springboard for the later field investigations and analysis undertaken as part of the project. Septic tanks still continue to be used widely due to their simplicity and low cost. Generally the treatment performance of septic tanks can be highly variable due to numerous factors, but a properly designed, operated and maintained septic tank can produce effluent of satisfactory quality. The reduction of hydraulic surges from washing machines and dishwashers, regular removal of accumulated septage and the elimination of harmful chemicals are some of the practices that can improve system performance considerably. The relative advantages of multi chamber over single chamber septic tanks is an issue that needs to be resolved in view of the conflicting research outcomes. In recent years, aerobic wastewater treatment systems (AWTS) have been gaining in popularity. This can be mainly attributed to the desire to avoid subsurface effluent disposal, which is the main cause of septic tank failure. The use of aerobic processes for treatment of wastewater and the disinfection of effluent prior to disposal is capable of producing effluent of a quality suitable for surface disposal. However the field performance of these has been disappointing. A significant number of these systems do not perform to stipulated standards and quality can be highly variable. This is primarily due to houseowner neglect or ignorance of correct operational and maintenance procedures. The other problems include greater susceptibility to shock loadings and sludge bulking. As identified in literature a number of design features can also contribute to this wide variation in quality. The other treatment processes in common use are the various types of filter systems. These include intermittent and recirculating sand filters. These systems too have their inherent advantages and disadvantages. Furthermore as in the case of aerobic systems, their performance is very much dependent on individual houseowner operation and maintenance practices. In recent years the use of biofilters has attracted research interest and particularly the use of peat. High removal rates of various wastewater pollutants have been reported in research literature. Despite these satisfactory results, leachate from peat has been reported in various studies. This is an issue that needs further investigations and as such biofilters can still be considered to be in the experimental stage. The use of other filter media such as absorbent plastic and bark has also been reported in literature. The safe and hygienic disposal of treated effluent is a matter of concern in the case of onsite sewage treatment. Subsurface disposal is the most common and the only option in the case of septic tank treatment. Soil is an excellent treatment medium if suitable conditions are present. The processes of sorption, filtration and oxidation can remove the various wastewater pollutants. The subsurface characteristics of the disposal area are among the most important parameters governing process performance. Therefore it is important that the soil and topographic conditions are taken into consideration in the design of the soil absorption system. Seepage trenches and beds are the common systems in use. Seepage pits or chambers can be used where subsurface conditions warrant, whilst above grade mounds have been recommended for a variety of difficult site conditions. All these systems have their inherent advantages and disadvantages and the preferable soil absorption system should be selected based on site characteristics. The use of gravel as in-fill for beds and trenches is open to question. It does not contribute to effluent treatment and has been shown to reduce the effective infiltrative surface area. This is due to physical obstruction and the migration of fines entrained in the gravel, into the soil matrix. The surface application of effluent is coming into increasing use with the advent of aerobic treatment systems. This has the advantage that treatment is undertaken on the upper soil horizons, which is chemically and biologically the most effective in effluent renovation. Numerous research studies have demonstrated the feasibility of this practice. However the overriding criteria is the quality of the effluent. It has to be of exceptionally good quality in order to ensure that there are no resulting public health impacts due to aerosol drift. This essentially is the main issue of concern, due to the unreliability of the effluent quality from aerobic systems. Secondly, it has also been found that most householders do not take adequate care in the operation of spray irrigation systems or in the maintenance of the irrigation area. Under these circumstances surface disposal of effluent should be approached with caution and would require appropriate householder education and stringent compliance requirements. However despite all this, the efficiency with which the process is undertaken will ultimately rest with the individual householder and this is where most concern rests. Greywater too should require similar considerations. Surface irrigation of greywater is currently being permitted in a number of local authority jurisdictions in Queensland. Considering the fact that greywater constitutes the largest fraction of the total wastewater generated in a household, it could be considered to be a potential resource. Unfortunately in most circumstances the only pretreatment that is required to be undertaken prior to reuse is the removal of oil and grease. This is an issue of concern as greywater can considered to be a weak to medium sewage as it contains primary pollutants such as BOD material and nutrients and may also include microbial contamination. Therefore its use for surface irrigation can pose a potential health risk. This is further compounded by the fact that most householders are unaware of the potential adverse impacts of indiscriminate greywater reuse. As in the case of blackwater effluent reuse, there have been suggestions that greywater should also be subjected to stringent guidelines. Under these circumstances the surface application of any wastewater requires careful consideration. The other option available for the disposal effluent is the use of evaporation systems. The use of evapotranspiration systems has been covered in this report. Research has shown that these systems are susceptible to a number of factors and in particular to climatic conditions. As such their applicability is location specific. Also the design of systems based solely on evapotranspiration is questionable. In order to ensure more reliability, the systems should be designed to include soil absorption. The successful use of these systems for intermittent usage has been noted in literature. Taking into consideration the issues discussed above, subsurface disposal of effluent is the safest under most conditions. This is provided the facility has been designed to accommodate site conditions. The main problem associated with subsurface disposal is the formation of a clogging mat on the infiltrative surfaces. Due to the formation of the clogging mat, the capacity of the soil to handle effluent is no longer governed by the soil’s hydraulic conductivity as measured by the percolation test, but rather by the infiltration rate through the clogged zone. The characteristics of the clogging mat have been shown to be influenced by various soil and effluent characteristics. Secondly, the mechanisms of clogging mat formation have been found to be influenced by various physical, chemical and biological processes. Biological clogging is the most common process taking place and occurs due to bacterial growth or its by-products reducing the soil pore diameters. Biological clogging is generally associated with anaerobic conditions. The formation of the clogging mat provides significant benefits. It acts as an efficient filter for the removal of microorganisms. Also as the clogging mat increases the hydraulic impedance to flow, unsaturated flow conditions will occur below the mat. This permits greater contact between effluent and soil particles thereby enhancing the purification process. This is particularly important in the case of highly permeable soils. However the adverse impacts of the clogging mat formation cannot be ignored as they can lead to significant reduction in the infiltration rate. This in fact is the most common cause of soil absorption systems failure. As the formation of the clogging mat is inevitable, it is important to ensure that it does not impede effluent infiltration beyond tolerable limits. Various strategies have been investigated to either control clogging mat formation or to remediate its severity. Intermittent dosing of effluent is one such strategy that has attracted considerable attention. Research conclusions with regard to short duration time intervals are contradictory. It has been claimed that the intermittent rest periods would result in the aerobic decomposition of the clogging mat leading to a subsequent increase in the infiltration rate. Contrary to this, it has also been claimed that short duration rest periods are insufficient to completely decompose the clogging mat, and the intermediate by-products that form as a result of aerobic processes would in fact lead to even more severe clogging. It has been further recommended that the rest periods should be much longer and should be in the range of about six months. This entails the provision of a second and alternating seepage bed. The other concepts that have been investigated are the design of the bed to meet the equilibrium infiltration rate that would eventuate after clogging mat formation; improved geometry such as the use of seepage trenches instead of beds; serial instead of parallel effluent distribution and low pressure dosing of effluent. The use of physical measures such as oxidation with hydrogen peroxide and replacement of the infiltration surface have been shown to be only of short-term benefit. Another issue of importance is the degree of pretreatment that should be provided to the effluent prior to subsurface application and the influence exerted by pollutant loadings on the clogging mat formation. Laboratory studies have shown that the total mass loadings of BOD and suspended solids are important factors in the formation of the clogging mat. It has also been found that the nature of the suspended solids is also an important factor. The finer particles from extended aeration systems when compared to those from septic tanks will penetrate deeper into the soil and hence will ultimately cause a more dense clogging mat. However the importance of improved pretreatment in clogging mat formation may need to be qualified in view of other research studies. It has also shown that effluent quality may be a factor in the case of highly permeable soils but this may not be the case with fine structured soils. The ultimate test of onsite sewage treatment system efficiency rests with the final disposal of effluent. The implication of system failure as evidenced from the surface ponding of effluent or the seepage of contaminants into the groundwater can be very serious as it can lead to environmental and public health impacts. Significant microbial contamination of surface and groundwater has been attributed to septic tank effluent. There are a number of documented instances of septic tank related waterborne disease outbreaks affecting large numbers of people. In a recent incident, the local authority was found liable for an outbreak of viral hepatitis A and not the individual septic tank owners as no action had been taken to remedy septic tank failure. This illustrates the responsibility placed on local authorities in terms of ensuring the proper operation of onsite sewage treatment systems. Even a properly functioning soil absorption system is only capable of removing phosphorus and microorganisms. The nitrogen remaining after plant uptake will not be retained in the soil column, but will instead gradually seep into the groundwater as nitrate. Conditions for nitrogen removal by denitrification are not generally present in a soil absorption bed. Dilution by groundwater is the only treatment available for reducing the nitrogen concentration to specified levels. Therefore based on subsurface conditions, this essentially entails a maximum allowable concentration of septic tanks in a given area. Unfortunately nitrogen is not the only wastewater pollutant of concern. Relatively long survival times and travel distances have been noted for microorganisms originating from soil absorption systems. This is likely to happen if saturated conditions persist under the soil absorption bed or due to surface runoff of effluent as a result of system failure. Soils have a finite capacity for the removal of phosphorus. Once this capacity is exceeded, phosphorus too will seep into the groundwater. The relatively high mobility of phosphorus in sandy soils have been noted in the literature. These issues have serious implications in the design and siting of soil absorption systems. It is not only important to ensure that the system design is based on subsurface conditions but also the density of these systems in given areas is a critical issue. This essentially involves the adoption of a land capability approach to determine the limitations of an individual site for onsite sewage disposal. The most limiting factor at a particular site would determine the overall capability classification for that site which would also dictate the type of effluent disposal method to be adopted.

Performance evaluation of onsite wastewater treatment : field sampling and preliminary evaluation of results

Background The onsite treatment of sewage and effluent disposal within the premises is widely prevalent in rural and urban fringe areas due to the general unavailability of reticulated wastewater collection systems. Despite the seemingly low technology of the systems, failure is common and in many cases leading to adverse public health and environmental consequences. Therefore it is important that careful consideration is given to the design and location of onsite sewage treatment systems. It requires an understanding of the factors that influence treatment performance. The use of subsurface effluent absorption systems is the most common form of effluent disposal for onsite sewage treatment and particularly for septic tanks. Additionally in the case of septic tanks, a subsurface disposal system is generally an integral component of the sewage treatment process. Therefore location specific factors will play a key role in this context. The project The primary aims of the research project are: • to relate treatment performance of onsite sewage treatment systems to soil conditions at site; • to identify important areas where there is currently a lack of relevant research knowledge and is in need of further investigation. These tasks were undertaken with the objective of facilitating the development of performance based planning and management strategies for onsite sewage treatment. The primary focus of the research project has been on septic tanks. Therefore by implication the investigation has been confined to subsurface soil absorption systems. The design and treatment processes taking place within the septic tank chamber itself did not form a part of the investigation. In the evaluation to be undertaken, the treatment performance of soil absorption systems will be related to the physico-chemical characteristics of the soil. Five broad categories of soil types have been considered for this purpose. The number of systems investigated was based on the proportionate area of urban development within the Brisbane region located on each soil types. In the initial phase of the investigation, though the majority of the systems evaluated were septic tanks, a small number of aerobic wastewater treatment systems (AWTS) were also included. This was primarily to compare the effluent quality of systems employing different generic treatment processes. It is important to note that the number of different types of systems investigated was relatively small. As such this does not permit a statistical analysis to be undertaken of the results obtained. This is an important issue considering the large number of parameters that can influence treatment performance and their wide variability. The report This report is the second in a series of three reports focussing on the performance evaluation of onsite treatment of sewage. The research project was initiated at the request of the Brisbane City Council. The work undertaken included site investigation and testing of sewage effluent and soil samples taken at distances of 1 and 3 m from the effluent disposal area. The project component discussed in the current report formed the basis for the more detailed investigation undertaken subsequently. The outcomes from the initial studies have been discussed, which enabled the identification of factors to be investigated further. Primarily, this report contains the results of the field monitoring program, the initial analysis undertaken and preliminary conclusions. Field study and outcomes Initially commencing with a list of 252 locations in 17 different suburbs, a total of 22 sites in 21 different locations were monitored. These sites were selected based on predetermined criteria. To obtain house owner agreement to participate in the monitoring study was not an easy task. Six of these sites had to be abandoned subsequently due to various reasons. The remaining sites included eight septic systems with subsurface effluent disposal and treating blackwater or combined black and greywater, two sites treating greywater only and six sites with AWTS. In addition to collecting effluent and soil samples from each site, a detailed field investigation including a series of house owner interviews were also undertaken. Significant observations were made during the field investigations. In addition to site specific observations, the general observations include the following: • Most house owners are unaware of the need for regular maintenance. Sludge removal has not been undertaken in any of the septic tanks monitored. Even in the case of aerated wastewater treatment systems, the regular inspections by the supplier is confined only to the treatment system and does not include the effluent disposal system. This is not a satisfactory situation as the investigations revealed. • In the case of separate greywater systems, only one site had a suitably functioning disposal arrangement. The general practice is to employ a garden hose to siphon the greywater for use in surface irrigation of the garden. • In most sites, the soil profile showed significant lateral percolation of effluent. As such, the flow of effluent to surface water bodies is a distinct possibility. • The need to investigate the subsurface condition to a depth greater than what is required for the standard percolation test was clearly evident. On occasion, seemingly permeable soil was found to have an underlying impermeable soil layer or vice versa. The important outcomes from the testing program include the following: • Though effluent treatment is influenced by the physico-chemical characteristics of the soil, it was not possible to distinguish between the treatment performance of different soil types. This leads to the hypothesis that effluent renovation is significantly influenced by the combination of various physico-chemical parameters rather than single parameters. This would make the processes involved strongly site specific. • Generally the improvement in effluent quality appears to take place only within the initial 1 m of travel and without any appreciable improvement thereafter. This relates only to the degree of improvement obtained and does not imply that this quality is satisfactory. This calls into question the value of adopting setback distances from sensitive water bodies. • Use of AWTS for sewage treatment may provide effluent of higher quality suitable for surface disposal. However on the whole, after a 1-3 m of travel through the subsurface, it was not possible to distinguish any significant differences in quality between those originating from septic tanks and AWTS. • In comparison with effluent quality from a conventional wastewater treatment plant, most systems were found to perform satisfactorily with regards to Total Nitrogen. The success rate was much lower in the case of faecal coliforms. However it is important to note that five of the systems exhibited problems with regards to effluent disposal, resulting in surface flow. This could lead to possible contamination of surface water courses. • The ratio of TDS to EC is about 0.42 whilst the optimum recommended value for use of treated effluent for irrigation should be about 0.64. This would mean a higher salt content in the effluent than what is advisable for use in irrigation. A consequence of this would be the accumulation of salts to a concentration harmful to crops or the landscape unless adequate leaching is present. These relatively high EC values are present even in the case of AWTS where surface irrigation of effluent is being undertaken. However it is important to note that this is not an artefact of the treatment process but rather an indication of the quality of the wastewater generated in the household. This clearly indicates the need for further research to evaluate the suitability of various soil types for the surface irrigation of effluent where the TDS/EC ratio is less than 0.64. • Effluent percolating through the subsurface absorption field may travel in the form of dilute pulses. As such the effluent will move through the soil profile forming fronts of elevated parameter levels. • The downward flow of effluent and leaching of the soil profile is evident in the case of podsolic, lithosol and kransozem soils. Lateral flow of effluent is evident in the case of prairie soils. Gleyed podsolic soils indicate poor drainage and ponding of effluent. In the current phase of the research project, a number of chemical indicators such as EC, pH and chloride concentration were employed as indicators to investigate the extent of effluent flow and to understand how soil renovates effluent. The soil profile, especially texture, structure and moisture regime was examined more in an engineering sense to determine the effect of movement of water into and through the soil. However it is not only the physical characteristics, but the chemical characteristics of the soil also play a key role in the effluent renovation process. Therefore in order to understand the complex processes taking place in a subsurface effluent disposal area, it is important that the identified influential parameters are evaluated using soil chemical concepts. Consequently the primary focus of the next phase of the research project will be to identify linkages between various important parameters. The research thus envisaged will help to develop robust criteria for evaluating the performance of subsurface disposal systems.

Risk control in transmission system expansion planning with wind generators

With the advent of large-scale wind farms and their integration into electrical grids, more uncertainties, constraints and objectives must be considered in power system development. It is therefore necessary to introduce risk-control strategies into the planning of transmission systems connected with wind power generators. This paper presents a probability-based multi-objective model equipped with three risk-control strategies. The model is developed to evaluate and enhance the ability of the transmission system to protect against overload risks when wind power is integrated into the power system. The model involves: (i) defining the uncertainties associated with wind power generators with probability measures and calculating the probabilistic power flow with the combined use of cumulants and Gram-Charlier series; (ii) developing three risk-control strategies by specifying the smallest acceptable non-overload probability for each branch and the whole system, and specifying the non-overload margin for all branches in the whole system; (iii) formulating an overload risk index based on the non-overload probability and the non-overload margin defined; and (iv) developing a multi-objective transmission system expansion planning (TSEP) model with the objective functions composed of transmission investment and the overload risk index. The presented work represents a superior risk-control model for TSEP in terms of security, reliability and economy. The transmission expansion planning model with the three risk-control strategies demonstrates its feasibility in the case study using two typical power systems

Nourishing or polluting : redefining the role of waste in the fashion system

Waste is intrinsic to the fashion system. Fashion is predicated on built-in obsolescence, and as such outmoded garments are rapidly discarded to charity shops or landfill. However, the story of fashion is also one of abundance and extravagance in design ideas. Every season there are new design details – prints, embroidery, embellishments, shapes and textures. This excess of ideas is in itself another form of waste, albeit one that is culturally nourishing. The grave of a fashion garment may also be the grave of a season’s research and creativity. This paper compares the tangible waste of the industry with its intangible waste, namely fashion’s creativity and cultural excess. Fashion’s excess and abundance of trends and ideas makes any move to curb the environmental impact difficult. For all practitioners of fashion – whether designers or consumers – the waste and excess inherent in the fashion system is a difficult ethical terrain to negotiate. However, inverting the wasteful phases of the production cycle can help reframe waste from pollution to a source of nourishment for future practice. While creative excesses of designers may be ‘wasted’ after a season, fashion styles and tropes are recycled and reinvented, with the once passé styles and design ideas from previous years revalorized and returned into the fashion system. Similarly, material garments acquire new value through entering or re-entering the second hand or vintage markets. Design processes can utilise pre or post-consumer textile waste, or eliminate waste through design. In these processes, waste becomes the primary source of nourishment for future fashion cycles.

Resource allocation in multiuser cooperative OFDM system using forced cost based decisions

A sub optimal resource allocation algorithm for Orthogonal Frequency Division Multiplexing (OFDM) based cooperative scheme is proposed. The system consists of multiple relays. Subcarrier space is divided into blocks and relays participating in cooperation are allocated specific blocks to be used with a user. To ensure unique subcarrier assignment system is constrained such that same block cannot be used by more than one user. Users are given fair block assignments while no restriction for maximum number of blocks a relay can employ is given. Forced cost based decisions [1] are used for block allocation. Simulation results show that this scheme outperforms a non cooperating scheme with sequential allocation with respect to power usage.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

A decision support system for sustainable urban development : the integrated land use and transportation indexing model

Broad, early definitions of sustainable development have caused confusion and hesitation among local authorities and planning professionals. This confusion has arisen because loosely defined principles of sustainable development have been employed when setting policies and planning projects, and when gauging the efficiencies of these policies in the light of designated sustainability goals. The question of how this theory-rhetoric-practice gap can be filled is the main focus of this chapter. It examines the triple bottom line approach–one of the sustainability accounting approaches widely employed by governmental organisations–and the applicability of this approach to sustainable urban development. The chapter introduces the ‘Integrated Land Use and Transportation Indexing Model’ that incorporates triple bottom line considerations with environmental impact assessment techniques via a geographic, information systemsbased decision support system. This model helps decision-makers in selecting policy options according to their economic, environmental and social impacts. Its main purpose is to provide valuable knowledge about the spatial dimensions of sustainable development, and to provide fine detail outputs on the possible impacts of urban development proposals on sustainability levels. In order to embrace sustainable urban development policy considerations, the model is sensitive to the relationship between urban form, travel patterns and socio-economic attributes. Finally, the model is useful in picturing the holistic state of urban settings in terms of their sustainability levels, and in assessing the degree of compatibility of selected scenarios with the desired sustainable urban future.

Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.