For the protection of the ecological Reserve in South Africa, the proposed introduction of compulsory toxicity testing in the licensing of effluent discharges necessitates the development of whole effluent toxicity testing. The elucidation of the effects of effluent on the local indigenous populations of organisms is essential before hazard and risk assessment can be undertaken. The limpet Burnupia stenochorias, prevalent in the Eastern Cape of South Africa, was chosen to represent the freshwater molluscs as a potential toxicity indicator. Using potassium dichromate (as a reference toxicant) and a textile whole effluent, the suitability of B. stenochorias was assessed under both acute and chronic toxicity conditions in the laboratory. In support of the toxicity studies, aspects of the biology of B. stenochorias were investigated under both natural and laboratory conditions. Using Principal Component and Discriminant Function Analyses, the relative shell morphometrics of three feral populations of B. stenochorias were found to vary. Length was shown to adequately represent growth of the shell, although the inclusion of width measurements is more statistically preferable. Two of the feral populations, one in impacted water, were studied weekly for 52 weeks to assess natural population dynamics. Based on the Von Bertalanffy Growth Equation, estimates of growth and longevity were made for this species, with growth highly seasonal. Age is not easily discerned from shell size. Egg laying occurred all year round, with early summer (peak egg lay), mid summer (a second, smaller peak in egg lay), and winter (limited presence of eggs) phases. In toxicity testing, consideration is given to the choice of the test organism based on age and sexual development. Consequently, the sexual development of B. stenochorias relative to shell length was determined with the aid of histological examinations of transverse sections of limpets, of all sizes, collected over one year. Limpets less than 3mm shell length were found to be immature in the development of the oocytes and spermatozoa, and were later chosen for acute toxicity tests. A laboratory diet was developed, for both culturing and maintaining of the limpets during toxicity tests; however, the diet requires optimisation. Under laboratory conditions, growth was linear, and individual fecundity highly variable. Successful methods for the collection of limpets from naturally occurring populations, and their acclimation to the laboratory were developed. Three B. stenochorias populations, representing different hydrological and water quality conditions, were compared to a laboratory population (maintained for three years) in their responses to the textile whole effluent and potassium dichromate. Under acute conditions, variability of mortality between limpet populations and between seasons was consistent with acceptable international standards. However, seasonal differences between feral limpets were apparent, with early summer limpets significantly more susceptible to both potassium dichromate and textile effluent than winter limpets. Although mortality occurred within the effluent at all concentrations, no 96 hour LC₅₀ values were obtained. The chronic toxicity effects of the textile whole effluent were assessed over the entire life cycle of B. stenochorias, based on survival, growth and reproductive effects. Lower concentrations of effluent (# 10%) gave greater variability of responses and toxicity than higher concentrations, with a 43 day LC₅₀ of 3.9% effluent. The No Observed Effect Concentrations for survival (over 43 days) were calculated in consecutive years as 0.1% and 1% effluent. Survival is considered a useful tool for determining toxicity endpoints using B. stenochorias. Limpet growth remained linear in effluent, with an apparent stimulation of growth at the 3-10% effluent concentration, confusing the toxicity and variability assessments. The possible addition of nutrients from the effluent points to either a potential inadequacy of the food quality provided in the chronic assessment, or the presence in the effluent of growth stimulants. Growth was also found to be too variable to allow adequate statistical conclusions about the toxicity of the effluent, although it is suggested that growth may be useful in the assessment of single compounds. Despite large individual variability in fecundity, statistical differences were discernible between effluent concentrations. The application of fecundity of B. stenochorias in hazard assessment therefore warrants further investigation. It was concluded that an assessment of textile whole effluent toxicity to B. stenochorias over an entire life cycle, and an F1 generation, is unnecessary. The development of the bucket/plastic bag method for both acute and chronic toxicity assessment of B. stenochorias was useful. In the final assessment of the usefulness of B. stenochorias as a toxicity indicator, toxicity endpoints were compared with those of the standard laboratory organism Daphnia pulex. Both in acute and chronic toxicity, B. stenochorias was found to be more sensitive. B. stenochorias is therefore considered valuable as a South African freshwater molluscan ecotoxicological indicator, with a place in hazard assessment, although further development and research is necessary before the limpet can be effectively used.

Made available in DSpace on 2014-10-09T12:33:07Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:17Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:33:07Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:17Z (GMT). No. of bitstreams: 0 / Triclosan é um composto orgânico de baixa solubilidade que vem sendo utilizado em formulações de cremes dentais e faciais, xampu, sabonetes, embalagens de gêneros alimentícios e diversos tipos de materiais, tais como, adesivos, brinquedos, sapatos, selantes, tintas, colchão, roupas, pisos, toldos e rejuntes. O amplo uso deste composto deve-se à grande eficácia contra bactérias Gram negativas e Gram positivas. Por seu extenso uso, evidências da presença de Triclosan têm sido frequentemente relatadas em efluentes urbanos e industriais, águas superficiais e sedimentos de ambientes dulcícolas, estuarinos e marinhos, como também em organismos aquáticos como algas, peixes e mamíferos. Neste contexto, o presente estudo avaliou a toxicidade aguda e crônica de Triclosan para diferentes invertebrados marinhos de águas tropicais. Para tanto, ensaios de toxicidade aguda foram realizados com o copépodo Nitokra sp (mortalidade) e com o ouriço-do-mar Lytechinus variegatus (taxa de fertilização). Para a avaliação do efeito crônico, ensaios de toxicidade de curta duração (desenvolvimento embriolarval) foram realizados com o ouriço-do-mar L. variegatus e Perna perna. Além desses métodos, o ensaio do Tempo de Retenção do Corante Vermelho Neutro foi empregado com a finalidade de se avaliar os efeitos do Triclosan sobre a estabilidade da membrana lisossômica de hemócitos de P. perna. Na avaliação do efeito agudo, o valor médio da CL(I)50;96h encontrada para o copépodo foi de 0,20 mg.L-1 enquanto que o valor médio da CI(I)50;1h para ouriço-do-mar foi de 0,28 mg.L-1. Já na avaliação do efeito crônico, o valor médio da CI(I)50;24h para ouriço-do-mar foi de 0,14 mg.L-1 e para o molusco bivalve a média da CI(I)50;48h, foi de 0,13 mg.L-1. O efeito na estabilidade da membrana lisossômica de hemócitos de P. perna ocorreu em concentrações a partir de 12 ng.L-1. Estes resultados evidenciam o risco ecológico da introdução contínua desse composto em ambientes marinhos, e devem ser considerados para identificação de concentrações seguras e futura regulação do bactericida Triclosan na legislação ambiental nacional e internacional. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Organophosphorus (OP) pesticide toxicity is a global health problem. Respiratory failure due to neuromuscular transmission dysfunction accounts for about 300,000 deaths annually in rural Asia. However, the clinical manifestation is complex, and described in terms of acute, intermediate, and chronic syndromes. The underlying mechanism of toxicity is still unclear. OP pesticides contain inhibitors of acetylcholinesterase (AChE), for example dimethoate, emulsified in an organic solvent, typically cyclohexanone. A hypothesized mechanism is initial excitotoxicity through inhibition of acetylcholinesterase followed by failure of neuromuscular synaptic transmission. I tested this electrophysiologically in vitro by measuring properties of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in isolated sciatic nerve/flexor digitorum brevis muscles from mice, bathed in HEPES-buffered mammalian physiological saline (MPS). Muscle action potentials were abolished with μ-conotoxin (2μM). First, we tested the effects of plasma taken from Göttingen minipigs instilled orally (isofluorane anaesthesia) with a formulated pesticide (2.5ml/kg) whose active ingredient is dimethoate dissolved in cyclohexanone. This plasma abolished evoked synaptic transmission and increased spontaneous MEPP frequency within 60-180 minutes of bath application. However plasma from minipigs instilled with dimethoate alone produced no failure of transmission. Plasma contained either pesticide or dimethoate significantly increased the half decay time of EPPs. However, pesticide-plasma also contained the metabolites omethoate (100μM) and cyclohexanol (5 mM). We found that bath application of omethoate alone caused a potent dose-dependent increase in EPP decay time. Cyclohexanol (5 mM) also increased EPP decay time but it also decreased both the excitability of axons and MEPP amplitude. In combination, omethoate and cyclohexanol produced greater disruption of neuromuscular transmission than either dimethoate or cyclohexanone, alone or in combination and this was particularly evident in isometric tension recordings, in which prolonged after-contraction and slow relaxation were observed during and immediately following tetanic stiumuation in the presence of omethoate and cyclohexanol. Voltage-clamp recordings of endplate currents (EPC) partially supported the EPP observations. Surprisingly, cyclohexanol-treated preparations showed no significant increase in EPC and MEPC decay time. However, there was some evidence of activity-dependent decline in MEPC amplitude in cyclohexanol while quantal content in these preparations showed evidence of an increase suggesting a homeostatic response in evoked transmitter release with cyclohexanol treatment. Analysis of presynaptic currents in cyclohexanol treated preparations also revealed preliminary evidence of sensitivity to cyclohexanol compared to control preparations. Finally, I tested the effects NMJ transmission of 24hr exposure to OP pesticide and its metabolites using a novel organ culture system, utilising a mouse mutant (WldS) with a slow nerve degeneration phenotype. After incubation of 24 hrs with MPS + pesticides and metabolites, these muscles showed significant reduction in function (response to nerve stimuli with EPP/action potential ± MEPPs) compared to control cultures. Together, the data indicate that failure of neuromuscular transmission by pesticide-plasma cannot be explained solely by dimethoate-mediated inhibition of acetylcholinesterase. Rather, a combination of metabolic breakdown products exerts potent, harmful presynaptic and postsynaptic effects. Either blocking the metabolic conversion of the constituents of OP pesticides, or transiently blocking their effects on receptors may therefore be an effective strategy for treatment of OP pesticide toxicity.

M.Sc. (Aquatic Health) / Sediments can contain a variety of organic and inorganic contaminants. These contaminants accumulate, resulting in extremely high concentrations even once the overlying water concentrations are at or below acceptable water quality guidelines. Any changes in the physical parameters of the overlying water can cause these pollutants to be released back into solution. Accumulated contaminants can be released at even higher concentrations than previously detected. In recent years, sediment contamination has highlighted the need to monitor these previously overlooked pollutant sources that have accumulated in aquatic ecosystems. South Africa does not currently have standardised methods to assess sediment toxicity. Although international methods exist, they are largely untested in South Africa and the organisms needed to conduct these tests are not readily available. Over the years numerous culture methods have been develop globally for culturing organism to be used for water and sediment toxicity tests. In South Africa, the focus has mainly been on culturing organisms for water toxicity testing. Sediment toxicity testing with indigenous organism however, was not developed. Established international culture methods from the United States Environmental Protection Agency, the Organisation for Economic Cooperation and Development, and Environment Canada were taken into consideration when developing the laboratory culture method for two(2) of the selected organisms (Chironomus spp. & Hydra sp.) from this study. A preliminary culture method was also developed for the third selected organism, Melanoides tuberculata (gastropod). The organisms cultured in this study were selected based on their extent of contact with the substrate, ease of handling, availability, culture maintenance as well as their reproductive cycle. The Hydra, Chironomids and M. tuberculata cultures were successfully breeding under laboratory conditions and remained stable. The Chironomus sp. and M. tuberculata maintain contact with the sediment making them suitable as ecologically relevant organisms for use in whole sediment toxicity testing in South Africa.

The unique properties of nanomaterials, such as their small size and large surface area-to-volume ratios, have attracted tremendous interest in the scientific community over the last few decades. Thus, the synthesis and characterization of many different types of nanoparticles has been well defined and reported on in the literature. Current research efforts have redirected from the basic study of nanomaterial synthesis and their properties to more application-based studies where the development of functionally active materials is necessary. Today such nanoparticle-based systems exist for a range of biomedical applications including imaging, drug delivery and sensors. The inherent properties of the nanomaterial, although important, aren’t always ideal for specific applications. In order to optimize nanoparticles for biomedical applications it is often desirable to tune their surface properties. Researchers have shown that these surface properties (such as charge, hydrophobicity, or reactivity) play a direct role in the interactions between nanoparticles and biological systems can be altered by attaching molecules to the surface of nanoparticles. In this work, the effects of physicochemical properties of a wide variety of nanoparticles was investigated using in vitro and in vivo models. For example, copper oxide (CuO) nanoparticles were of interest due to their instability in biological media. These nanoparticles undergo dissolution when in an aqueous environment and tend to aggregate. Therefore, the cytotoxicity of two sizes of CuO NPs was evaluated in cultured cells to develop a better understanding of how these propertied effect toxicity outcomes in biological systems. From these studies, it was determined that CuO NPs are cytotoxic to lung cells in a size-dependent manner and that dissolved copper ions contribute to the cytotoxicity however it is not solely responsible for cell death. Moreover, silica nanoparticles are one of the most commonly used nanomaterials because they are easy to synthesize and their properties (such as size, porosity and surface chemistry) can be fine-tuned. Silica nanoparticles can be found in thousands of commercially available products such as toothpastes, cosmetics and detergents and are currently being developed for biomedical applications such as drug delivery and biomedical imaging. Our findings herein indicate that the surface chemistry of silica nanoparticles can have an effect on lung inflammation after exposure. Specifically, amine-modified silica NPs are considered to be less toxic compared to bare silica nanoparticles. Together, these studies provide insight into the role that material properties have on toxicity and allow for a better understanding of their impact on human and environmental health. The final aim of this thesis was to develop surface-modified nanoparticles for drug delivery applications. For this, biodegradable, polymeric NPs were used due to their inert nature and biocompatibility. Furthermore, polymeric NPs are excellent for loading drugs and using them as drug delivery vehicles. In this work, poly (lactic-co-glycolic acid) (PLGA) NPs were loaded with a therapeutic peptide. These NPs were then coated with chitosan (a mucoadhesive polymer) for the treatment of allergic asthma or coated with a small cationic mitochondrial targeting agent for the treatment of ischemia/reperfusion injury. Taken as a whole, this thesis sheds light on the impact of NPs on human health. First by providing useful toxological data for CuO and silica NPs as well as highlighting the potential of surface-modified polymeric NPs to be used in drug delivery-based applications.

This thesis is focused on attestation of ecotoxicity of octachlorstyrene (OCS), persistent organic pollutant (POP), by biological tests of toxicity. The toxicity of OCS was determined by using two kinds of alternative tests applied on fresh water organisms. First of them, Thamnotoxkit, is based on observation of mobility, immobilization or even killing of crustacean Thamnocephalus platyurus. The second one, Daphnotoxkit, applies crustacean Daphnia magna. For purposes of comparison, a standard test of toxicity – so called test of root growth inhibition of Sinapis alba and two fytotests – test of growth inhibition of Lemna minor and test of root growth inhibition of Allium cepa were performed as well.

Sibutramine hydrochloride monohydrate is a weight loss agent banned from most global markets due to reports of serious adverse events including death. It is classified as a serotonin-norepinephrine reuptake inhibitor and acts within the central nervous system by inhibiting the reuptake of these neurotransmitters essentially influencing satiety and also energy expenditure. Despite its ban, sibutramine is still available for use in some countries and is often an unlisted ingredient in herbal or natural weight loss products. This leads to the oblivious and unintentional consumption of this compound which is of great concern especially if used by pregnant women. The aim of this study was to investigate the possible toxic and teratogenic effects of sibutramine by using a Spraque Dawley rat and an in ovo chick embryo model. The Sprague Dawley rat was used as in vivo model and animals were divided into three experimental groups, each consisting of 12 animals (6 males, 6 females). Rats were administered sibutramine over a 28 day period according to their assigned experimental groups namely control (C; sterile H2O), Low dose (LD; 1.32 mg/kg) and High Dose (HD; 13.2 mg/kg). On the day of termination blood was collected for various analyses which included biochemical tests for liver and kidney function, hormonal changes as well as the investigation of coagulation profiles. Brain, heart, lung, kidney and liver tissue of each animal was harvested for investigation of tissue and cellular structure. Rats were weighed daily and this data suggested that sibutramine was well tolerated by all animals, with only the female rats in the HD group showing a significant 8.2% decrease in their rate of weight gain. Biochemical data of liver and kidney function in all groups were normal. Thyroid hormone levels were comparable to control values though cortisol levels were lowered in the HD female group, a finding which correlates with the observed weight loss. Investigations of the ultrastructural morphology of the platelets and fibrin networks revealed differences between the experimental groups that are consistent with changes associated with a procoagulable state. Brain, kidney and liver tissue morphology appeared normal upon investigation, the latter confirming the biochemical findings. Examination of cardiac tissue revealed a slight increase in collagen deposition between the muscle fibers and ultrastructural analysis of lung tissue showed thickening of the alveolar walls, decreased intra-alveolar space and drastic increases in collagen deposition in the sibutramine-exposed groups. These findings were dosage dependant. The in ovo model was implemented with ephedrine, a known teratogen, as positive control. Three control groups were included to ensure the efficacy of the method. Eight experimental groups, each containing sixteen eggs, were exposed to four different concentrations of each drug (i.e. 0.5, 2.5, 5.0, 10.0 μmol). The brain, heart, liver and kidneys were harvested on embryonic day thirteen for morphological analysis. Macroscopic evaluation revealed that both drugs caused congenital abnormalities which included ventral wall and limb defects as well as growth retardation and increased mortality. Sibutramine was found to have a greater teratogenic potential than ephedrine. Histological investigation of kidney and brain samples of embryos revealed no morphological differences between the various experimental groups. Livers and hearts of embryos exposed were severely affected by both compounds in a dose dependant manner. Replacement of myocardium with adipose and connective tissue was observed in cardiac tissue, which is characteristic of muscular dystrophy. Severe liver steatosis was also evident. In conclusion, results from this animal-based study show that, at concentrations which are not toxic to the liver or kidneys, sibutramine administration led to increased coagulation, moderate cardiac and excessive lung fibrosis within the Sprague Dawley rat model. This indicates significant toxicity with the cardiac and respiratory system being more susceptible targets. In addition sibutramine was shown to possess greater teratogenic effects than ephedrine. Both drugs caused cardiac dystrophy and liver steatosis resulting in extensive liver and heart damage. Understanding the underlying mechanisms involved in the pathogenesis of these findings strongly emphasize an important area for future research. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Anatomy / MSc / Unrestricted

Among the vast diversity of plants in the mid-South to which grazing livestock are exposed, there is a need to address certain species that are potentially harmful to livestock health and production. Field and greenhouse experiments were conducted on endophyte-infected tall fescue [Schedonorus arundinaceus (Schreb.) Dumort = Lolium arundinaceum (Schreb.) Darbysh.], a cool-season perennial forage, and perilla mint, (Perilla frutescens (L.) Britton) an herbaceous annual, to determine management techniques and control measures for healthier pasture and livestock systems. When seedheads reached maturity, spring and autumn glyphosate applications at 1.68 kg ae ha-1, coupled with autumn tillage and summer and winter cover crops effectively reduced tall fescue coverage to < 27% by 10 months after initial treatment (MAT) following a single year of management. If seedhead maturity is inhibited prior to first glyphosate application, tall fescue was reduced to <1% coverage 10 MAT. Despite seedhead suppression, at least 78% increase in coverage occurred in 24 months from remnant populations suggesting two years of renovation is required. Forage soybeans proved to be a valuable cover crop that maintained nutritive quality and mean dry matter yields of 5487 kg ha-1 across two years. Tall fescue seedheads were suppressed below 14% coverage (> 68% visual control) by nicosulfuron + metsulfuron (20 + 5 and 40 +11 g ai ha-1), imazapic (26 and 53 g ai ha-1), and sulfosulfuron (53 g ai ha-1) at 90 DAT. Reduction in forage heights may be expected, but was not consistently reduced when compared to untreated plots across locations. To control perilla mint, postemergence applications of aminocyclopyrachlor blends, glyphosate, picloram + 2,4-D, aminopyralid + 2,4-D, and 2,4-D alone provided superior control when applied prior to the plant’s reproductive growth stage. Picloram + 2,4-D also provided inield soil residual activity and the most effective preemergence control, followed by aminocyclopyrachlor + chlorsulfuron, pendimethalin, and aminopyralid + 2,4-D for at least 141 DAT. Seed from weedy populations in north Mississippi tend to germinate in a range of night/day soil temperatures from 10/15 C to 25/30 C. Therefore, effective preemergence treatments should be applied by mid- to late- February in this region.

Predation is considered a powerful ecological force influencing community structure, diversity, and abundance. Paleoecology offers a unique perspective, allowing us to examine ecological processes such as predation over evolutionary timescales. The three studies summarized below include two case studies testing the role of predation in evolution and one method-oriented project, which explores new tools with which to examine predator-prey interactions in the fossil record. Considering the importance of community interactions in the current global biodiversity crisis, understanding ecological and evolutionary dimensions of predation is critical to conservation biology and ecology, as predators are thought to play a vital role in maintaining ecosystem health and biodiversity. Predation has been dismissed as a causal mechanism for some major prey groups. For example, the evolutionary decline and cryptic or antitropical distribution of brachiopods is often explained as due to the potentially low energetic value and suspected non-palatability or toxicity of brachiopod tissues. Here we demonstrate that multiple invertebrate marine predators (crustaceans, echinoderms, and gastropods) are willing and able to consume brachiopods, and that predation pressure on the living brachiopod population may be consequential. Examination of the fossil record is consistent with this interpretation: evidence for drilling and repair of brachiopod shells is found throughout the fossil record in multiple orders. This suggests that although brachiopods may be unwanted prey in the presence of energetically more desirable targets, they do appear to be edible and are subject to intense predator-prey interactions. Limpets are important prey for some crab species, yet little is known about the role of durophagy in the evolution of the limpet shell. Feeding trials using three common species of Pacific Northwest limpets (Lottia digitalis, L. pelta and Tectura scutum) were conducted to assess how different shell morphologies affect mortality and handling time. We predicted that large size, shell ornament, and low-spires would result in either increased survivorship, and/or longer handling times. Contrary to our expectation that ridges resist predation, individuals with smooth morphologies experienced significantly lower mortality, as did those with low-spires. As species possessing high-spires and ridges typically occur high in the intertidal where predation risk due to crabs is relatively low, these morphologies are likely adaptions to physical factors such as thermal stress. One of the major caveats of using gastropod drill holes to assess predator-prey interactions in both the modern and the ancient is the correct identification of drill holes of predatory origin. By examining known predatory drill holes using environmental scanning electron microscopy, we aim to refine the development of a novel technique for augmenting their identification, and to explore the relationship between predator body size, predatory radula dentition, and radular microrasping marks observed on the shells of prey organisms. Electron micrographs were used to measure the spacing of microrasping marks produced by the radula, and the intercusp spacing of the radula dentition. A relationship between predator body size and microrasping marks makes it possible to infer predator size from these microtraces in both modern and fossil specimens, augmenting our ability to examine predator-prey interactions throughout the history of this important ecological interaction. Proxies for predation intensity such as predation traces or antipredatory morphologies provide an invaluable method to examine predation in both modern communities, and the fossil record. Our understanding of the importance of predation in regulating biodiversity and in evolution will continue to grow with the development of new methodologies, and a comprehensive understanding of predatory defenses. / Ph. D.