This thesis describes the synthesis, characterization, and application of metal-chelating polymers for mass-cytometric bioassays. Mass cytometry is a cell characterization technique in which cells are injected individually into an ICP-MS detector. Signal is provided by staining cell-surface or intracellular antigens with metal-labeled antibodies (Abs). These Abs are labeled through the covalent attachment of metal-chelating polymers which carry multiple copies of a lanthanide isotope. In this work, my first goal was to develop a facile, straightforward synthesis of a new generation of metal-chelating polymers. The synthesis began with reversible addition-fragmentation chain transfer polymerization, and was followed by numerous post-polymerization pendant group transformations to introduce DTPA lanthanide chelators to every repeat unit, and a maleimide at the end of the chain. The second goal was to apply these metal-chelating polymers in bioassay experiments. The DTPA groups were loaded with lanthanide ions, and the maleimide group was used to covalently attach the polymer to an Ab. This goat anti-mouse conjugate was found to carry an average of 2.4 ± 0.3 polymer chains. Then, primary Ab conjugates were prepared and used in an 11-plex mass cytometry assay in the characterization of umbilical cord blood cells. The third goal was to expand the multiplexity of the assay. In current technology, the number of Abs that can be monitored simultaneously is limited to the 31 commercially available, stable lanthanide isotopes. Thus, I had an interest in preparing metal-chelating polymers that could carry other metals in the 100-220 amu range. I synthesized polymers with four different polyaminocarboxylate ligands, and investigated the loading of palladium and platinum ions into these polymers. Polymer-Ab conjugates prepared with palladium- and platinum-loaded polymers gave curious results, in that only dead cells were recognized. The fourth goal was to create dual-purpose Ab tags. My approach was to synthesize polymers similar to those described above, but which also carried two to six fluorescent dyes. Polymer-Ab conjugates prepared with four different dye-labeled polymers gave mixed results. Two of the four conjugates performed well in FACS and mass cytometric assays, but the other two did not. Further experiments are needed to overcome this problem.

This thesis describes the synthesis, characterization, and application of metal-chelating polymers for mass-cytometric bioassays. Mass cytometry is a cell characterization technique in which cells are injected individually into an ICP-MS detector. Signal is provided by staining cell-surface or intracellular antigens with metal-labeled antibodies (Abs). These Abs are labeled through the covalent attachment of metal-chelating polymers which carry multiple copies of a lanthanide isotope. In this work, my first goal was to develop a facile, straightforward synthesis of a new generation of metal-chelating polymers. The synthesis began with reversible addition-fragmentation chain transfer polymerization, and was followed by numerous post-polymerization pendant group transformations to introduce DTPA lanthanide chelators to every repeat unit, and a maleimide at the end of the chain. The second goal was to apply these metal-chelating polymers in bioassay experiments. The DTPA groups were loaded with lanthanide ions, and the maleimide group was used to covalently attach the polymer to an Ab. This goat anti-mouse conjugate was found to carry an average of 2.4 ± 0.3 polymer chains. Then, primary Ab conjugates were prepared and used in an 11-plex mass cytometry assay in the characterization of umbilical cord blood cells. The third goal was to expand the multiplexity of the assay. In current technology, the number of Abs that can be monitored simultaneously is limited to the 31 commercially available, stable lanthanide isotopes. Thus, I had an interest in preparing metal-chelating polymers that could carry other metals in the 100-220 amu range. I synthesized polymers with four different polyaminocarboxylate ligands, and investigated the loading of palladium and platinum ions into these polymers. Polymer-Ab conjugates prepared with palladium- and platinum-loaded polymers gave curious results, in that only dead cells were recognized. The fourth goal was to create dual-purpose Ab tags. My approach was to synthesize polymers similar to those described above, but which also carried two to six fluorescent dyes. Polymer-Ab conjugates prepared with four different dye-labeled polymers gave mixed results. Two of the four conjugates performed well in FACS and mass cytometric assays, but the other two did not. Further experiments are needed to overcome this problem.

Agricultural runoff is a major non-point source of phosphorus (P) and nitrogen (N) in aquatic environments and often contributes to coastal eutrophication. Chemical extraction techniques that measure P and N in runoff and sediments have provided little information on biologically available P and N, particularly when soils/sediments move from an agricultural setting to freshwater, estuarine and marine systems. The aims of this thesis were to a) develop algal bioassay techniques for assessing bioavailability of P in marine and freshwater sediments, b) apply algal bioassays to determine the role of suspended sediments as P and N sources c) examine how sediment type and sediment load affect P bioavailability and identify the chemical analysis of soil P that best reflects bioavailable P, and d) determine nutrient bioavailability of agricultural soil runoff in estuarine and coastal marine ecosystems. An accurate and repeatable sediment bioassay was developed using non-axenic uni-algal cultures of the euryhaline diatom Skeletonema costatum and freshwater chlorophyte Selenastrum capricornutum. Bioassays were conducted in 250 mL Erlenmeyer flasks, with 100 mg L-1 of simulated sediment and 100 mL of modified growth media, which were continuously shaken. Suspended sediments were simulated from 7 major soil types of the Maroochy River catchment in Australia, a coastal catchment influenced by agriculture. Simulated sediments were used as the sole P source in replicate batch 7-14 d bioassays in which changes in in vivo Chl a fluorescence were measured. Maximum algal biomass in bioassays was significantly correlated to total sediment P content of the added sediment in marine and freshwater. Bioassay methodology was adapted to assess N as well as P bioavailability in simulated suspended sediments using Skeletonema costatum. S. costatum biomass was significantly influenced by sediment type and correlated with the Total P content of the sediment. Conversely, S. costatum response did not correlate to total nitrogen (TN), ammonium (NH4+), nitrate (NO3-) and easily mineralisable N content of the sediment. Sediment type did significantly influence response, suggesting that sorption/desorption properties of the sediment influenced N bioavailability. Suspended sediments appear to be a significant source of bioavailable P as S. costatum biomass was enhanced by their addition. Conversely, the lack of stimulation by sediments when used as a N source implies that suspended sediments are not an immediate source of bioavailable N, although they do contribute to the sediment nutrient pool once deposited. The influence of sediment type and sediment load on phosphorus (P) bioavailability was compared using Skeletonema costatum bioassays. The amount of sediment and associated P load influenced the maximum algal biomass reached, with low sediment loads (&lt 100 mg L-1) capable of stimulating high algal biomass. Maximum algal biomass correlated with sediment iron-oxide extractable inorganic P (FeO-P) and inorganic P (Pi) extracted by sodium bicarbonate for all sediment types. Total P (TP) did not discriminate at low TP concentrations and organic P (Po) extracted by sodium bicarbonate only correlated with algal biomass when bioavailable P was low. Thus FeO-P and Pi are the most representative analyses of bioavailable P. FeO-P can also be used to accurately measure rate of sediment P desorption, which significantly influences algal growth rate. Nutrient bioavailability of agricultural soil runoff was investigated in the Maroochy River catchment in southeast Queensland Australia, a coastal catchment influenced by agriculture. Suspended sediments, river and estuarine sediments and deposited sediment in the near-shore coastal zone were collected and analysed for nutrient bioavailability using chemical analyses and S. costatum bioassays. Suspended sediments in the Maroochy River, which consisted of silt and clay sized particles, had elevated FeO-P and TP concentrations comparable to fertilised soil. Similarly the deposited sediment sampled offshore to the river mouth had elevated total P, FeO-P and total N concentrations which were much greater than the underlying marine sediment. The deposited offshore sediment contained mainly silt sized particles and appeared to be terrigenous in origin as it was more similar in composition (TP, FeO-P, TN, total carbon, total aluminium, total iron, total silica) to estuarine suspended sediments and terrestrial soils, than the underlying marine sediment. This study demonstrated that fine silt sized particles, rich in bioavailable P and N, are being transported and deposited offshore during erosion events. These findings highlight the importance of effective land management and erosion control measures that reduce the suspended sediment load. Although agricultural practices are continually improving to reduce soil erosion, practices need to encompass a broader approach that emphasizes the link between land and sea. Effective management must consider agricultural productivity as well as the environmental impact or ‘algal bloom potential’ that a soil may have when eroded into a waterway. Soils high in bioavailable nutrients must be targeted and managed effectively, particularly in areas prone to erosion.

The burden of ticks in semi-arid lands of Ethiopia is not as pronounced as in some more humid areas of the continent. Nevertheless, the increasing recourse to chemicals smuggled by illegal traders has led to serious problems, including poisoning of humans and animals, discontinuous and irrational treatment regimens, tick-resistance to acaricidal products, loss of traditional knowledge and weakening of social structures. In order to encourage a resumption of the long-established ethnoveterinary practices, a survey on plants locally used in tick control was undertaken, and plant species used in other parts of the continent for the same purpose were considered. On these bases, 28 plant species or varieties were collected in the study area: Acacia seyal var. seyal, Adenium somalense, Aloe calidophila, Aloe parvidens, Azadirachta indica, Boscia angustifolia, Calotropis procera, Calpurnia aurea, Cissus quadrangularis, Commiphora erythraea, Cordia africana, Croton macrostachys, Croton megalocarpus, Datura stramonium, Euphorbia candelabrum, Euphorbia tirucalli, Ficus sycomorus, Ficus thonningii, Lantana camara, Maerua triphylla, Ocimum suave, two varieties of Ricinus communis (one with green fruits and another with red ones), Solanum incanum, Solanum somalense, Sterculia rhynchocarpa, Tagetes minuta and Vernonia amygdalina. In general, leaves were collected and used. However, due to the scarcity of foliar material, the whole plant of T. minuta and O. suave, the whole stem of A. somalense and C. quadrangularis, the branches of E. candelabrum and E. tirucalli, the bark in the case of A. seyal, C. erythraea and S. rhynchocarpa, were examined. After drying and grinding, the plant material was extracted with hexane and acetone, and made up to different concentrations to test the relevant repellent and toxic properties on adult Rhipicephalus pulchellus unfed ticks. For every bioassay, four replications, each using ten ticks, were performed. For the repellency bioassays, ticks were placed on a rectangular polystyrene platform stuck in a plastic basin and surrounded by water, in order to prevent them from moving away. Two glass rods, each provided with filter paper at the top and at the base, were inserted at opposite edges of the platform. The two filter papers of one rod were impregnated with the testing solution (i.e. solvent plus extract) at different concentrations while those of the other rod were treated with the pertinent extractant (hexane or acetone). Because of their inherent tendency to climb, most of the ticks settled onto the rods (mainly at the top), and their distribution was different depending on the repellency capacity of the extracts. The relevant data were then converted into repellency indexes using the formula [(Nc - Nt)/(Nc + Nt)] x 100, where Nc refers to the number of ticks on the control rod and Nt to the number of ticks on the test rod (Lwande et al., 1999; Pascual-Villalobos and Robledo, 1998). For the toxicity bioassays, 1 ìl of the extract at different concentrations was placed onto each tick and the mortality or weakening ratio was recorded after 24 hours. Because of the intrinsic toxicity of hexane, only acetone extracts were used for these assays. Due to the efficacy in extracting volatile compounds, hexane extracts had, for 24 plant species, better repellent properties than acetone extracts. Moreover, at a concentration of 10%, four species had negative repellency indexes with hexane extracts and five with acetone ones. At such concentration, these extracts therefore seemed to attract the ticks rather than repel them. At a concentration of 10%, thirteen hexane and five acetone extracts had repellency indexes > 50. At a concentration of 5%, only five hexane extracts and no acetone ones exceeded this value. Finally, only one species had a repellency index > 50 with the hexane extract at a 1% concentration. The plants showing good repellency indexes with at least one of the two solvents were A. calidophila, C. quadrangularis, C. erythraea, C. macrostachys, C. megalocarpus, D. stramonium, L. camara, M. triphylla, O. suave, the two varieties of R. communis and T. minuta. Amomg them, from a practical point of view, it is suitable to concentrate on O. suave, T. minuta and, to a certain extent, A. calidophila. In fact, C. quadrangularis, C. erythraea, C. macrostachys, D. stramonium, M. triphylla and the two varieties of R. communis had good repellent properties using hexane extracts at 10%, but not at 5%. Because trees like C. erythraea, C. macrostachys, C. megalocarpus and M. triphylla are highly valuable in a very dry environment, an excessive exploitation can put them in danger. Since D. stramonium, L. camara and R. communis are toxic plants, their extracts can be a serious threat for both humans and animals. Furthermore, L. camara is one of the worst weeds in the world, making it very inappropriate for lands already subject to the problem of bush encroachment. For all these reasons, T. minuta and O. suave appear to be the most promising plants; moreover, they are very well known in Southern Ethiopia and occur widely all over the area. On the contrary, A. calidophila is limited to just some places and the cultivation of Aloe species needs special attention, so it is not very suitable for people with a nomadic lifestyle. Concerning the toxicity bioassays, C. aurea extracts yielded by far the best results. In fact, all the ticks used had severe movement impairment when put in contact with acetone extracts at the concentrations of 20% and 10%. At a 5% concentration, 85% of the ticks had the same symptoms. In a separate test, a 10% water extract had a similar effect on 30 ticks out of 40, demonstrating the ease of extraction and application of the active compounds. The plant is well known, mainly by the Borana pastoralists, and is resistant to drought. It is also well able to grow in overgrazed areas, and its cultivation does not require special skills. Some of the extracts of other species gave good or fair results in the toxicity bioassays but, apart from S. incanum, only at a very high concentration (20%). Further studies may include isolation and characterization of the active compounds from the best species, setting up of a suitable plan for livestock treatment, and organization of a production and distribution cycle of appropriate phytomedicines in the pertinent pastoral area. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

Anthracnose, incited by Colletotrichum cereale, is a destructive disease of physiologically stressed creeping bentgrass putting greens in Mississippi and Alabama. Anthracnose severity and frequency of occurrence have increased over the past 15 years, and fungicide resistance may have had a role in the increase. In vitro bioassays were performed to evaluate thiophanate methyl and azoxystrobin against C. cereale isolates exposed to the fungicides and baseline isolates that had not been exposed to either fungicide. All isolates collected from creeping bentgrass were uninhibited by both fungicides at discriminatory doses. Partial nucleotide sequences of the â-tubulin 2 (thiophanate methyl) or cytochrome b (azoxystrobin) gene was compared to confirm fungicide resistance. Thiophanate methyl resistance was conferred by either a point mutation from glutamic acid to alanine at position 198, or phenylalanine to tyrosine at position 200. Azoxystrobin resistance was conferred by an amino acid point mutation from glycine to alanine at position 143.

El lufenuron un insecticida del grupo de los benzilureas, el cual interfiere con la síntesis de la quitina que causa una inhibición en la muda de los insectos. Se evaluó el efecto tóxico del lufenuron sobre seis organismos bioindicadores de calidad ambiental. Se desarrollaron los bioensayos con el lufenuron sobre seis organismos bioindicadores de calidad ambiental: Chlorella vulgaris (Beijerinck, 1890), Artemia franciscana (Kellogg, 1906), Daphnia magna (Straus, 1820), Carassius auratus (Linnaeus, 1758), Microorganismos de Suelo y Eisenia foetida (Savigny, 1826). Se utilizó el análisis de varianza (ANDEVA) con prueba de Tukey y con el Probit se calcularon los parámetros ecotoxicológicos. Los resultados son los siguientes, C. vulgaris (CI50<2,46 mg i.a.·L-1 a 96 h), A. franciscana (CL50= 11,41 μg i.a.·L-1 a 48 h), D. magna (CL50= 0,05 μg i.a.·L-1 a 48h), D. magna (NOEC= 0,00005 mg i.a.·L-1 / LOEC= 0,0001 mg i.a.·L-1 para el numero de crías vivas a 21 d), C. auratus (NOEC= 0, 061 mg i.a.·L-1/ LOEC= 0.122 mg i.a.·L-1 para el porcentaje de mortalidad acumulada a 26 d), Microorganismos de Suelo (NOEC= 1820 mg i.a.·g-1 / LOEC> 1820 mg i.a.·g-1) y E. foetida (DL50= 206,31 mg i.a.·Kg-1 a 14 d ; NOEC< 21,2 mg i.a.·Kg-1 / LOEC= 21,2 mg i.a.·Kg-1 a 14 d). Se observó la siguiente secuencia de ecotoxicidad decreciente mediado por los efectos letales y subletales producidos durante los bioensayos con los seis organismos: D. magna > A. franciscana > C. vulgaris > C. auratus > E. foetida > Microorganismos del suelo. Daphnia magna fue el organismo bioindicador más sensible al lufenuron y el más resistente fueron los microorganismos del suelo. El lufenuron presentó mayores porcentajes de mortalidad e inhibición en los organismos acuáticos que en los terrestres. Se concluyó que los seis organismos escogidos para evaluar los posibles daños ambientales del insecticida lufenuron, ayudaron a clarificar que este compuesto daña principalmente la cadena trófica acuática que la terrestre, siendo más sensibles los ecosistemas acuáticos que los terrestres que presentan riesgo bajo.Lufenuron is an insecticide from the group of benzylureas, which interferes with the synthesis of chitin causing inhibition of insect moulting. The toxic effect of lufenuron on six environmental quality bioindicator organisms was evaluated. Bioassays with lufenuron were developed on six environmental quality bioindicators: Chlorella vulgaris (Beijerinck, 1890), Artemia franciscana (Kellogg, 1906), Daphnia magna (Straus, 1820), Carassius auratus (Linnaeus, 1758) and Eisenia foetida (Savigny, 1826). The analysis of variance (ANDEVA) was used with Tukey's test and with the Probit we calculated the ecotoxicological parameters. The results are as follows: C. vulgaris (IC50 <2,46 mg i.a.·L-1 at 96 h), A. franciscana (LC50 = 11,41 μg i.a.·L-1 at 48 h), D. magna LC50 = 0,05 μg i.a.·L-1 at 48 h), D. magna (NOEC = 0,00005 mg i.a.·L-1 / LOEC = 0,0001 mg i.a.·L-1 for the number of live offspring at 21 d), C. auratus (NOEC = 0,061 mg i.a.·L-1 / LOEC = 0.122 mg i.a.·L-1 for the percentage of accumulated Mortality at 26 d), Soil Microorganisms (NOEC = 1820 mg i.a.·g-1/ LOEC> 1820 mg i.a.·g-1) and E. foetida (LD50 = 206,31 mg i.a.·kg-1 at 14 d; NOEC <21,2 mg i.a.·kg-1 / LOEC = 21,2 mg i.a.·kg-1 at 14 d). The following sequence of decreasing ecotoxicity mediated by the lethal and sublethal effects produced during the bioassays with the six organisms was observed: D. magna> A. franciscana> C. vulgaris> C. auratus> E. foetida> Soil microorganisms. Daphnia magna was the most bioindicator organism of lufenuron, and the most resistant soil microorganisms. Lufenuron had higher rates of mortality and inhibition in aquatic organisms than in terrestrial organisms. It was concluded that the six organisms chosen to evaluate the possible environmental damages of the insecticide lufenuron, helped to clarify that this compound damages mainly the trophic chain aquatic that the terrestrial chain, being more sensitive aquatic ecosystems that the terrestrial ones.

A água é de grande importância para a sobrevivência da vida, o rio Corumbataí abastece as cidades de Analândia, Corumbataí, Rio Claro, Piracicaba entre outras. A toxicologia ambiental vem sendo disseminada como ferramenta no monitoramento ambiental, pois através de bioensaios é possível avaliar a qualidade dágua. O objetivo deste estudo foi avaliar a qualidade do corpo hídrico em questão através de testes de toxicidade crônica, aplicação de protocolo rápido, determinação de metais em sedimento em suspensão e a toxicidade aguda de dois herbicidas (ametrina e glifosato) com Ceriodaphnia silvestrii. As coletas foram realizadas mensalmente no período de agosto/2009 à julho de 2010. Para as 12 coletas verificou-se que pelo menos um ou mais pontos apresentavam concentração de Fe acima do valor máximo permitido pelo CONAMA. A oscilação da pontuação do protocolo de avaliação rápida esteve diretamente relacionada com os aspectos visuais e olfativos. No ensaio crônico com Ceriodaphnia silvestrii houve variação na reprodução dos organismos e através de análises estatísticas foi possível observar que o pH, oxigênio dissolvido, condutividade, Zn, Ti e Ni influenciaram na toxicidade da amostra. Os herbicidas ametrina e glifosato apresentaram a CE50 (48h) respectivamente de 0,50 e 4,5 mg L-1. Conclui-se que a toxicidade crônica foi influenciada pelos parâmetros físico-químicos e metais durante os 12 meses de coleta.O protocolo de avaliação rápida complementa os resultados obtidos dos testes de toxicidade crônica. Através dos ensaios de toxicidade agudo verificou-se que a ametrina é nove vezes mais tóxica do que o glifosato / Water is very importance for survive of life, the Corumbataí river provides water for Analândia, Corumbataí, Rio Claro and Piracicaba and others cities. The environmental toxicology has been disseminated how a tool in environmental monitoring, because through the bioassays is possible evaluate the quality of water body. The objective of this study was assess the quality of the water body in focus through chronic tests, rapid assessment protocols, determination of metals in suspended matter and acute toxicity of two herbicides (ametryn and gliphosate) with Ceriodaphnia silvestrii. The water samples were collected monthly between august 2009 to july 2010. For the 12 samples verified at least one or more samples has more iron concentration than maximum value permitted of CONAMA. The oscillation of the rapid assessment protocols scores was directed associated with visual and olfactory aspects. In chronic assays with Ceriodaphnia silvestrii has the variation in the reproduction of the organisms and through the statics analyzes was possible observed that the pH, dissolved oxygen conductivity, Zn, Ti and Ni influenced in the toxicity of sample. The herbicides ametryn and glyphosate showed the EC50 (48h)respectively 0.50 and 4.5 mg L-1. It is conclude that the chronic toxicity was influenced by physics- chemistries parameters and metals during the 12 months. The rapid assessment protocol complete the results from chronic tests of toxicity. Through of acute toxicity assays verified that the ametryn is nine times more toxic than glyphosate.

Orientador: Manoel Victor Franco Lemos / Banca: Edgar Augusto de Toledo Picoli / Banca: Fernando Hercos Valicente / Banca: Jaime Maia dos Santos / Banca: Flávia Maria de Souza Carvalho / Resumo: A produção de alimentos é prejudicada devido a incidência de diversas pragas e doenças. A fim de poder controlá-las, utilizam-se uma grande quantidade e variedade de produtos químicos. Em muitos casos, esses produtos incidem sobre uma vasta gama de organismos, fato este que causa um grande impacto no meio ambiente. A fim de minimizar os efeitos negativos do uso de produtos químicos com grande espectro de ação, há grande empenho no desenvolvimento de produtos cujos alvos sejam específicos. Neste sentido, o uso do controle biológico tem se apresentado como uma alternativa bastante interessante. Dentre os vários sistemas utilizados no controle biológico de pragas, a bactéria Bacillus thuringiensis é a opção de maior destaque. No entanto, inúmeros casos de resistência têm surgido ao longo destes últimos anos, devido aos produtos codificados pelos genes com ação entomopatogênica estarem sendo utilizados de maneira descontrolada. Desse modo, a busca par novas proteínas Cry é tarefa importante dentro da estratégia de controle biológico de pragas agrícolas. Com o intuito de se identificar novas genes cry par meio de análises de PCR Múltiplas, seguida de análise por RFLP, identificaram-se 3 isolados de B. thuringiensis cujos fragmentos de genes analisados diferiram entre si e das linhagens padrão conhecidas até o momento. Ao analisar essas sequências gênicas depositadas no GenBank, verificou-se que dois desses fragmentos de DNA pertencentes aos isolados 113 e 133 possuíam alta similaridade com os genes efetivos contra nematoides, uma terceira sequência caracterizada a partir do isolado 123 não foi similar a nenhuma outra sequência depositada no GenBank atualmente. Os isolados cuja sequência de aminoácidos de suas proteínas Cry predita "in silica" mostraram-se similares a genes cry... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Food production is strongly harmed by a large amount of plant pests and diseases. Aiming to control these problems, a large quantity and variety of chemical compounds have been used. In many cases, these products cause a severe environmental impact over a wide range of non-target organisms. In order to minimize the negative effects of using chemical products with great action spectrum, there is a great effort to develop products of which targets are specific. Therein, the biological control has been presenting itself as a very interesting alternative. Among the various systems used in biological control, the bacteria Bacillus thuringiensis is the option pinpointed as the best notability. However, some resistance towards its use as a biological controlling agent have been reported recently due to the uncontrolled use of products decoded by genes of entomopathogenic action. Thus, the search of new Cry proteins is an important task inside the strategy of biological control of field pests. In order to overcome these situations efforts on the isolation and characterization of new B. thuringiensis isolates using molecular techniques such as PCR and RFLP were adopted and three B. thurigiensis isolates were compared among them and were considered different among the known and described standard type strains up to the present time. When part of the obtained gene sequences were compared to those deposited on the GeneBank, it was possible to observe that for two of them, isolate 113 and 133, a high degree of similarity with sequences described as active against nematodes; the third isolate, 123, had no similarity to any deposited sequence up to date. The isolates, which aminoacid sequence of their Cry proteins "in silico" were similar to cry genes earlier deposited on GenBank, had their respective gene fully characterized. To this, several... (Complete abstract click electronic access below) / Doutor

South Africa is one of the most important mining countries in the world, hosting the world's largest reserves of platinum group metals (PGMs). Even though mining is clearly an important activity in South Africa, contributing approximately US$ 7.4 billion annually to the countries' gross domestic product (GDP), the costs to the environment are not insignificant. One of the most severe environmental aspects associated with mining is the storage of mineral waste on tailings storage facilities due to their impacts on air quality, ground water quality, aesthetics and land use. It is also unknown whether the environmental effects of tailings storage facilities increase or decrease over time. The aim of this study was to determine the ecotoxicity of platinum tailings storage facilities of different ages by means of soil physical and chemical analysis, earthworm ecotoxicological studies, dehydrogenase activity and soil mesofauna studies. Samples were obtained from three platinum tailings storage facilities of different ages of which two were already rehabilitated while the third was still operational at the time this study was performed. The latter was used as a negative control for the purpose of the study. Soil samples were physically and chemically analysed. Earthworm ecotoxicological studies were conducted to determine changes in biomass, reproduction, mortality, neutral red retention times and tissue metal concentrations. Dehydrogenase activity was determined before the introduction of earthworms and manure, after introductions of manure and after introductions of earthworms and manure. Soil mesofauna were extracted and identified in order to determine species richness, diversity, abundance and functional grouping. Soil chemical analysis indicated that concentrations of certain heavy metals, especially chrome (Cr), present in platinum tailings materials could have a potential effect on microorganisms, microbial processes and earthworms. Earthworm ecotoxicological results indicated that earthworms that bioaccumulated higher levels of heavy metals showed poor hatchability of cocoons. Dehydrogenase activity indicated that earthworms play a significant role in increasing the number and biomass of soil microbes because significant increases in dehydrogenase activity were noticed after the addition of earthworms to platinum tailings materials. Results from the earthworm ecotoxicological studies, dehydrogenase activity, and soil mesofauna composition indicated that environmental impacts of tailings storage facilities did not increase with age, but is more likely to be an indication of the rehabilitation measures administered to the different tailings storage facilities. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

This dissertation characterizes the influences of water chemistry on the acute toxicity of lead (Pb) to two of the long-standing sentinel test organisms commonly employed by the United States Environmental Protection Agency (USEPA), the fathead minnow (Pimephales promelas) and daphnid (Ceriodaphnia dubia), for parameterization of an acute Pb Biotic Ligand Model (BLM). In addition, a toxicogenomic approach was employed to identify genes that might serve as molecular markers of Pb exposure and long-term effects, as well as provide new insights as to the underlying toxic mechanisms of chronic Pb exposure in P. promelas. The endpoints of growth, reproduction, Pb accumulation, prey capture ability, and swimming performance of P. promelas were examined to assess the influences of water chemistry during chronic Pb exposures and to potentially link microarray-identified genes to outcomes of ecological significance. Importantly, this work revealed that calcium does not protect against acute toxicity to C. dubia or chronic Pb accumulation by P. promelas, indicating that current hardness-based regulations are inappropriate and provide further support for the need for alternative approaches to setting environmental regulations for Pb. The findings reported herein should facilitate the arrival of such an approach in the form of a new acute Pb BLM. However, different responses with respect to the influences of water chemistry on the acute toxicity of Pb were exhibited by these species suggesting that development of separate BLMs for P. promelas and C. dubia should be considered to ensure adequate protection for both species. Furthermore, the influences of water chemistry were found to be inconsistent during acute and chronic Pb exposures to P. promelas and thus caution against inferring chronic effects from acute exposures. A number of Pb-responsive genes were identified that exhibited a strong potential for serving as robust indicators of Pb exposure and accumulation in P. promelas. While these genes also provided insight as to the likely toxic mechanisms of Pb, additional work will be necessary to firmly link these genes to chronic outcomes of ecological relevance in the context of ambient water chemistry.