Ph.D. / A number of aspects which might influence the buildup of spider mites on cotton were investigated. In contrast to their numbers on cotton treated with some of the insecticides used to control Heliothis arrniaer (Hübner) in field trials, the numbers of spider mites remained very low in the unsprayed cotton. Predators such as especially the pirate bug, Onus thniroborus (Hesse) appear to play an important role in maintaining mites in untreated plots at low population levels. Predator numbers were significantly reduced by some of the insecticides used. It is, however, not yet clear how the predator complex as a whole affects mite numbers ...

In the current studies the effects of administration of 0.1 to 1000 ppb of aldicarb, a carbamate pesticide, on the immune system of C3H mice were investigated. It was observed that aldicarb caused significant immunomodulation of macrophage functions analyzed in a variety of different systems. Initially, it was found that aldicarb decreased the stimulatory functions of the macrophages as studied by decreased capacity to stimulate normal autoreactive TF cells in the SMLR. This decreased stimulatory activity of the macrophages was found not to be due decrease in the expression of class II MHC-antigens (la molecules) nor was it due to the generation of any suppressor macrophages acting to down-regulate the immune response. Further investigations revealed that the decreased stimulatory activity of the macrophages correlated with decreased IL-1 production/signal to the T cells by the macrophages. It was also evident that aldicarb did not affect the T cell functions directly. Thus, T cells from aldicarb-treated mice when studied in the SMLR and AlloMLR or when stimulated with ConA or anti-CD3 mAbs, in the presence of normal macrophages, demonstrated normal responses. In contrast, normal T cells exhibited decreased responsiveness in the presence of aldicarb-treated macrophages. The fact that aldicarb did not affect the T cell functions directly was also evident by the fact that aidicarb-treated T cells could respond normally to stimulation with PMA + Ca²⁺ ionophore, a response which is independent of accessory cells. The aldicarb-treated macrophages also exhibited decreased capacity to process and present the antigen, conalbumin, to the T helper cell clone D10.G4. When the mechanism of aldicarb induced defect was investigated, it was observed that aldicarb-treated macrophages produced decreased amounts of IL-1 which was also confirmed by complete reconstitution of the response following addition of exogenous IL-1. With this in mind, macrophage functions in a number of other systems were examined and demonstrated that aldicarb-treatment also suppressed the macrophage-mediated cytotoxicity of tumor cells, but failed to inhibit the NK cell-mediated cytotoxicity of tumor cells. Together, these studies suggest that aldicarb selectively affects the macrophage but not NK or T ceil functions directly. However, since macrophages play an important role as accessory cells in T cell-mediated responses, it is likely that aldicarb indirectly will also affect the T cell responses. / Master of Science

Thesis (MPhil)--Stellenbosch University, 2003. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: This assignment is devoted to an in depth analysis of the pro- and the contra-positions in the long-standing and costly debate about the question whether to spray with DDT or not in the fight against malaria. I argue that the dilemma whether or not to spray with DDT is born out of a political agenda, hype, exaggeration and misinformation of the first order. Radical environmentalists appear to insist that DDT is a principal contributor of environmental degradation, and the major cause of death amongst wildlife and humans. Worse still, many Western people seem to be under the impression that mosquitoes cannot cause so much human misery as purported, and that malaria is caused by some kind of plant form of life, or even a virus. The proponents of DDT, on the other hand, appear to be convinced that DDT is a saviour of humankind, and argue that the horrors associated with DDT are exaggerated and baseless, as they are not backed by scientific inquiry. Proponents of DDT also believe that anything that is overused may kill, even ordinary table salt. Inthis assignment, both of these positions are scrutinized. On the basis of an historical overview in Chapter I of the history of the use of DDT, and the emergence of the debate about DDT in the wake of Rachel Carson's Silent Spring (1962), Chapter 2 is devoted to an evaluation of seven basic arguments against the use of DDT, while in Chapter 3 six arguments for the use of DDT are weighed. In Chapter 4 a resolution of the dilemma is proposed in which a case is made for a limited use of DDT only for indoor spraying of huts and houses against malaria mosquitoes until such time as a less dangerous alternative for DDT is found that can be used as effectively in the fight against malaria. As such, this case is informed by the strong moral conviction that we cannot allow poor people of colour to die because of a general ban on the use of DDT. Further research on this ethical debate is encouraged. / AFRIKAANSE OPSOMMING: Hierdie werkstuk is toegespits op 'n in-diepte analise van die pro- en kontra-posisies in die voortslepende, asook duur debat oor die gebruik van DDT al dan nie in die bekamping van Malaria. Ek argumenteer dat die dilemma rondom die vraag of DDT gebruik moet word of nie, aangewakker word deur politieke agendas, sensasie, oordrywing en foutiewe informasie van die eerste orde. Radikale omgewingsgesindes dring oënskynlik daarop aan dat die gebruik van DDT 'n hoof-oorsaak is van die agteruitgang van die omgewing, asook 'n primêre oorsaak van dood onder wild en mense. Erger nog, dit wil voorkom of heelwat Westerse mense onder die indruk is dat muskiete nie werklik soveel menslike lyding kan veroorsaak as wat voorgegee word nie, en dat malaria eerder veroorsaak word deur 'n sekere soort plantvorm van lewe, of selfs deur 'n virus. Die voorstaanders van DDT, aan die ander kant, is klaarblyklik oortuig dat DDT 'n redder van die mensdom is, en argumenteer dat die gruwels wat geassosieer word met DDT 'n grondelose oordrywing is, aangesien dit nie deur wetenskaplike ondersoek gesteun word nie. Voorstaanders van DDT glo verder dat enige stof wat in oormaat gebruik word, die dood kan veroorsaak, selfs gewone tafelsout. In hierdie werkstuk word albei hierdie posisies krities bestudeer en bespreek. Op grond van 'n historiese oorsig in Hoofstuk 1 oor die gebruik van DDT, en die ontstaan van die debat oor DDT na aanleiding van Rachel Carson se Silent Spring (1962), word Hoofstuk: 2 gewy aan 'n evaluasie van sewe basiese argumente teen die gebruik van DDT, terwyl in Hoofstuk 3 ses argumente vir die gebruik van DDT oorweeg word. In Hoofstuk 4 word 'n voorstel gemaak vir die resolusie van die dilemma deur 'n saak uit te maak vir die beperkte gebruik van DDT, nl. slegs vir binneshuise gebruik in hutte en huise teen malaria-muskiete tot tyd en wyl 'n minder gevaarlike alternatief vir DDT gevind word wat net so effektief sal wees in die stryd teen malaria. As sulks word hierdie studie gerugsteun deur die sterk morele oortuiging dat ons nie kan toelaat dat mense van kleur sterf as gevolg van 'n algemene verbod op die gebruik van DDT nie. Verdere navorsing oor hierdie etiese debat word aangemoedig.

Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity

A prospective ecological risk assessment method was developed evaluating the cumulative probabilistic impact of chemical stressors to aquatic organisms. This method was developed in response to the need to evaluate the magnitude, duration and episodic nature of chemical stressors on aquatic communities under environmental exposure scenarios. The method generates a probabilistic expression of the percent of an ecosystem's species at risk from a designated chemical exposure scenario.

Pesticides are among the most pervasive environmental contaminants and they are an important potential risk for human health. Agricultural workers are constantly exposed to pesticide spray, drift and residues in the soil and foliage. Many agricultural pesticides are readily absorbed by the body, through contact with the skin, the respiratory track, the eyes, and the gastrointestinal system. Multiple studies have reported a strong association between pesticide exposure and various health outcomes including cancer. Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects and neurological diseases. The focus of the present translational study is to examine the relationship between human exposure to the organophosphate pesticide azinphos methyl (AZM) and oxidative stress by measuring biomarkers of oxidative stress in biological fluids (i.e., urine, serum) and peripheral blood lymphocytes (PBLs) of agricultural workers. The findings from these field studies will be validated in vitro by examining cultures of human lymphocytes treated with AZM for similar biomarkers of oxidative stress. Since the collection of PBLs from study participants is highly invasive and not suitable for studies involving younger subjects, we also examined buccal cells for biomarkers of oxidative stress (i.e., DNA damage) as a more universal source of human tissue to assess oxidative stress in pesticide exposed individuals. We demonstrated in this study that AZM induces oxidative stress and causes DNA damage in human tissues. Agricultural workers who had been exposed to AZM showed elevated serum levels of lipid peroxides, increased urinary levels of 8-OH-dG, and lymphocytes from these individuals showed increased DNA damage and associated changes in oxidative DNA repair enzymes. Biomarkers of oxidative stress were also elevated in human lymphocytes treated with physiologically relevant concentrations of AZM. In cultures of human lymphocytes, AZM caused a concentration-dependent loss of viability and associated increases in ROS and a reduction in intracellular GSH. We also demonstrated that viable leukocytes from the oral cavity can be readily obtained from humans and these buccal cells can be used to assess DNA damage following exposure to occupational and environmental genotoxicants. We also noted that oral leukocytes are especially sensitive to cryopreservation with DMSO and thus, these cells must be cryoprotected with 5% DMSO to preserve the viability of these cells for subsequent biochemical studies. In summary, these in vivo and in vitro studies demonstrated that AZM induces oxidative stress in a dose-dependent matter and that oral lymphocytes are a good source of human tissue for assessing DNA damage and possibly other biochemical changes. The possible health implications of the variations in these biomarkers of oxidative stress and DNA damage are undetermined. Yet the findings from these studies have provided a strong foundation for determining the mechanism by which pesticide induce oxidative stress, to explore the putative relationship between pesticide-induced oxidative stress and disease (e.g. cancer, neurodegenerative disorders) and determine whether tissue damage in humans is brought about by direct or by indirect action of organophosphate pesticides. / Graduation date: 2010

The increased use of pesticides has resulted in a corresponding increase in concern for the effect they may have on the health of humans and other non-target organisms. The two main areas of concern are the toxicological effects that mixtures of pesticides may have as well as the endocrine disrupting effects. Although the individual pesticides may be present at concentrations below the levels deemed to be detrimental to health, it has been argued that their combined effect may still result in elevated health risks. Another important aspect of pesticide risk assessment requires a consideration of the breakdown products of pesticides and their effect on human health. There has been very little research into the effects of degradation products and this issue should be addressed as these could potentially pose a higher risk than their parent compounds. One of the most important bio-markers available for use is the ubiquitous enzyme acetylcholinesterase (AChE). This enzyme is responsible for one of the most important functions in the body; namely nerve impulse transmission, upon which all life depends. The inhibition of this enzyme indicates toxicity and as a subsequence, a threat to the organism’s well-being. Bioassays have also recently been developed to test chemicals for endocrine disrupting effects. These tests rely on a dose response equivalent to that of the most potent well known estrogen 17-β estradiol. Any chemical that has a measurable response is deemed to display endocrine disrupting effects. This first aim of this study was to investigate the toxicological and endocrine disrupting effects of three organophosphorus pesticides; aldicarb, parathion and demeton-S-methyl, in addition to two breakdown products; aminophenol and p-nitrophenol. Two carbamate pesticides; carbaryl and carbofuran were also analysed. The toxicological effects of mixtures of the parent pesticide compounds were tested to assess if any antagonistic, additive or synergistic effects were observed. This data was then used in conjunction with an artificial neural network to assess if individual pesticides could be distinguished from mixtures of pesticides. A final objective was to sample various Eastern Cape water sources, utilising the enzymatic assay to determine the presence of any of these pesticides in these samples. There were several conclusions drawn from this study. AChE was successfully used as an assay to test the toxicity of the pesticides under investigation, based on their inhibition of this enzyme. An important factor for consideration throughout the study was the need to establish basal and monitor AChE activity (i.e. the need to monitor AChE activity in the absence of any pesticide). This ensured accurate comparison of the results obtained. It was found that demeton-S-methyl was the most potent of these pesticides followed by carbaryl, parathion, aldicarb and finally carbofuran, and that carbofuran could potentiate AChE. The results indicated that pesticide mixtures generally exhibited an additive inhibitory effect on AChE, although at some concentrations of pesticides, synergistic and antagonistic effects were noted. From the data using mixtures of pesticides, a feed forward neural network was created that was successfully able to distinguish individual pesticides from mixtures within its training parameters. None of the pesticides tested displayed endocrine disrupting properties in the Yeast Estrogen Screen (YES), T47D-KBluc and MDA-kb2 bio-assays. Other studies reported mixed results in this regard and thus no final conclusions could be drawn. The Blaauwkrantz River, Kariega River, Sundays River, Swartkops River and Kowie River were all tested for pesticides and although positive results were recorded, conventional methods indicated that there were no pesticides in the rivers. There were, however, trace metals present which are known to inhibit AChE, thus causing a false positive result. These results indicated that AChE can be used as a high throughput initial pre-screening tool, but that it cannot serve as a substitute for more accurate conventional testing methods.