Thesis (MSc) -- University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Shrews are excellent bioindicators of environmental heavy metal pollution. This is due to their diets and the high rate of consumption at which these small mammals consume their prey. Shrews feed mostly on earthworms, and earthworms accumulate high levels of heavy metals in their bodies. Biomarkers measure responses to environmental pollution in the bodies of exposed organisms. Cellular biomarkers give an indication of responses to pollutants at levels of pollutant exposure that are not yet lethal to the organism. This study was prompted by concern that the number of shrews in the Stellenbosch region was declining. For the present study, shrews (Myosorex varius and Crocidura jlavescens) from the Stellenbosch region were sampled. The levels of Cd and Pb in their bodies were analysed in order to ascertain whether or not these two heavy metals, which occur in the environment as a result of various anthropogenic activities, are present in the food chain of the shrews. In the laboratory, shrews were fed live control and metal exposed earthworms from laboratory cultures. The earthworms had been exposed to either Cd or Pb in these cultures over a number of generations. This was performed to determine whether the presence of the metals in the body of the shrews, had resulted from metal accumulation from the earthworms which were fed to the shrews. Cellular biomarkers were used to determine the cellular response to the metals and membrane integrity and DNA integrity were investigated. This was done by exposing the cells of the shrews and ofBALB/c mice to metal salt solutions of Cd, Pb and Zn in vitro. Membrane integrity was tested by cell leaching techniques viz. the LDH assay and the trypan blue assay. DNA integrity was determined by using the comet assay as a biomarker of exposure. The results of the study show that the shrews in the immediate Stellenbosch region are exposed to lower levels of Cd and even lower levels of Pb than shrews from a site 16km out of the town of Stellenbosch. The cellular responses induced by metal exposure of laboratory mice show that the metals are indeed cytotoxic and genotoxic to the cells of small mammals. Cd and Pb were found to be more cytotoxic than Zn, while Zn was more genotoxic than either CqorPb. / AFRIKAANSE OPSOMMING: Skeerbekke is uitstekende bioindikatore van swaarmetaalbesoedeling in die omgewing. Dit is as gevolg van hul dieët en die hoë koers waarteen hierdie klein soogdiere hul prooi verorber. Skeerbekke voed meestalop erdwurms, en erdwurms akkumuleer hoë vlakke van swaarmetale in hul liggaam. Biomerkers meet response as gevolg van omgewingsbesoedeling in die liggame van blootgestelde organismes. Sellulêre biomerkers gee 'n aanduiding van response op besoedelingstowwe by vlakke wat nog nie dodelik is vir die organisme nie. Die studie is aangespoor deur die bekommernis dat die getal van skeerbekkies in Stellenbosch besig is om te daal. In die studie is skeerbekke (Myosorex varius en Crocidura flavescens) van die Stellenbosch omgewing gevang en die vlakke van Cd en Pb in hul liggame gemeet om vas te stelof die twee swaarmetale, wat in die omgewing beland as gevolg van verskeie antropogeniese aktiwiteite, teenwoordig is in die voedselketting van die skeerbekke. Die skeerbekke is in die laboratorium met lewende kontrole en metaalblootgestelde erdwurms uit kulture in die laboratorium gevoer. Die erdwurms is aan Cd en Pb oor baie generasies blootgestel in hierdie kulture. Dit is gedoen om vas te stelof die teenwoordigheid van die metale in die liggame van die skeerbekke 'n oorsaak is van metaalakkumuleering as gevolg van die erdwurms waarmee hulle gevoer is. Sellulêre biomerkers is gebruik om sellulêre respons op die metale op die membraanintegriteit en DNA-integriteit vas te stel. Dit is gedoen deur die selle van skeerbekke so wel as BALB/c muise in vitro bloot te stel aan metaalsoutoplossings van Cd, Pb en Zn. Membraanintegriteit is getoets deur selmembraan permeabiliteit tegnieke nl. die LDH en tripaan blou toetse. DNA-integriteit is getoets deur die komeettoets te gebruik. Die resultate van die studie dui daarop dat die skeerbekke van Stellenbosch aan laer vlakke van Cd, en nóg laer vlakke van Pb blootgestel is as die skeerbekke van 'n perseel 16km buite die dorp. Die sellulêre response geïndusseer deur blootstelling aan metale by die laboratoriummuise het aangetoon dat die metale inderdaad skadelik is vir die selmembraan en die DNA van die selle van die muise. Cd en Pb is meer toksies vir die selmembraan as Zn, terwyl Zn weer meer toksies is vir die DNA-integriteit as Cd en Pb.

The Arctic has long been considered to be a pristine environment, far from population centers and pollution sources. The detection of synthetic organochlorine compounds in various elements of the Arctic food web has confirmed the global dispersion of pollutants, particularly of persistent compounds such as organochlorines and heavy metals. Levels of heavy metals, although elevated, appear to be naturally so throughout much of the Arctic. Arctic pollution is both a humanitarian and ecological concern. Many arctic coastal communities depend heavily on marine mammal fat for sustenance, and are therefore potentially exposed to high levels of organochlorines and some metals. From an ecological perspective, the structure of arctic food webs, the importance of lipid mobilization for winter survival, and the adaptive physiologies of arctic organisms may result in an enhanced response to contaminant exposure. This thesis assesses the exposure and effect of organochlorine and heavy metal exposure in inland freshwater ecosystems of Arctic Alaska, and evaluates the physiological response of arctic grayling to experimental polychlorinated biphenyl (PCB) exposure. / Graduation date: 1995

The work in this thesis demonstrates whether resins are better than conventional soil extractants at predicting plant metal concentrations, and whether low-cost soil treatments can effectively reduce metal content and concentrations in lettuce leaves. / Researchers have shown that ion exchange resins can simulate root metal uptake behaviour by acting as ion sinks. Chemical extractants on the other hand, generally do not behave in this manner. Thus, we would expect resins (in either bead or membrane form) to predict plant metal concentrations better than conventional extractants. For the first study, a procedure using anion exchange resin membranes (AEM) treated with either EDTA or DTPA chelators was chosen, since previous studies showed this method to be effective at predicting plant metal uptake, and practical for routine laboratory use. In addition, several conventional extractants were selected for comparison. / For the second part of the thesis, a study was undertaken to test the effectiveness of various food processing byproducts to stabilize metal contaminants in soils, and compare the results with those of a Na-based aluminosilicate (zeolite). (Abstract shortened by UMI.)

No description available.

Ph.D. (Zoology) / Histomorphology as a tool to assess the health status of five economically important fish species in the Okavango Delta Panhandle, Botswana the Okavango Delta, and an inland freshwater wetland of national and international importance was designated by the Ramsar Convention in 1996. This site is of global importance because of a wide diversity of fish species found in this freshwater ecosystem. The Okavango Delta is also one of the last 'near pristine' sites on the African continent and therefore serves as an ideal reference site to describe the normal histomorphology of selected organs in sentinel fish species which could be used as reference for future toxicological studies. The aim of this study was to assess the health status of five economically important fish species namely, Clarias gariepinus (Burchell, 1822), Clarias ngamensis (Castelnau, 1861), Serranochromis robustus jallae (Gunther, 1864), Serranochromis angusticeps (Boulenger, 1907) and Hydrocynus vittatus (Castelnau, 1861) sampled from the Okavango Delta Panhandle, by describing the normal histomorphology of the liver, gills and gonads of the sentinel species. The sentinel fish species are of significant importance because they form an integral part of the small-scale commercial, subsistence and recreational fisheries in this ecosystem. In the past few decades, fish-health research has focused almost exclusively on histopathological alterations resulting from pollution caused by exposure to chemicals, parasitesand pathogens. Limited attention has been devoted to the normal histomorphology and health status of fish species in various aquatic ecosystems including the Okavango Delta Panhandle. Also, histopathological evaluations noted only the presence of abnormalities without quantitative assessment of the extent of the abnormalities within each tissue section. The absence of baseline normal histomorphology and numerical data however, makes it difficult to establish cause-and-affect relationships between pathology and contamination patterns in various fish species. It is imperative therefore, that the normal histomorphology of organs in fish species occurring in this region should be known so that histopathological conditions maybe accurately diagnosed…

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Food demands for the ever-increasing human population is increasing the pressure on the agricultural sector to produce more food. In order to satisfy these demands, farmers are turning to chemical biocides for the control of pest species to produce greater crop yields. All pesticides must be toxic or poisonous to the target species they intend to control. Unfortunately, most pesticides are toxic or poisonous to non-target organisms as well, with detrimental effects on their health. Organic farming was developed to enhance the overall health of the farm's natural soilmicrobe- plant-animal biodiversity. No synthetic fertilisers and/or pesticides are used when farming organically. Life in the soil consists of intricate food webs and interactions between the soil dwelling invertebrates. The soil-organisms are divided into three main groups, viz., Micro-organisms (e.g. protozoa, bacteria and fungi) mesofauna (nematodes, Collembola and Acari) and macro-fauna (e.g. millipedes, isopods, insects, molluscs and earthworms). The invertebrates are very susceptible to chemical contamination by chemical biocides in natural and agro-ecosystems. The soil invertebrate communities are responsible for the decomposition of organic material in soil, thereby remineralising the soil. The decomposition processes start with comminution of the large pieces of organic material by meso- and macro-fauna and ends with the micro-fauna and microbial organisms that complete these processes by returning the nutrients in an inorganic form to the soil. The aim of this study was to investigate whether, and to what extent the soil organisms are influenced by different management practices viz., organic management practices versus conventional management practices. A vineyard on the farm Plaisir de Merle, in Simondium, Western Cape was used for the present study. One half of a one hectare vineyard was managed organically and the other half conventionally. Within each vineyard block six different treatments were performed. Three of the treatments were strictly organic and the other three were strictly conventional. Four replicates of each management treatment were performed. The bait-lamina technique was used to assess the feeding activity of the soil organisms exposed to the different management treatments. In addition to the bait-lamina trials in the vineyard itself, bait-lamina tests were performed in microcosm studies with soil from the organically and conventionally managed vineyard blocks under controlled conditions. In order to assess the impact of the various pesticides that are used in the vineyards in the conventional way, on the soil fauna, standard acute toxicity tests and behavioural tests were performed on Eisenia fetida, the compost worm. The bait-lamina tests in the vineyard revealed that the moisture content of the soil plays an important role in the biological activity of soil fauna. The different management treatments did affect the biological activity of the soil fauna, but seasonal changes also proved to be one of the important factors governing biological processes in the soil. The acute toxicity tests showed that the active ingredients (mancozeb, penconazole and trifloxystrobin) of three of the pesticides that were tested in this study, had negatively affected E. fetida at their recommended application concentrations. The remaining two pesticides' active ingredients (glyphosate and N-acetyl salicylic acid) did not affect the earthworms negatively at the recommended application concentrations. The preference behavioural trials showed that E. fetida could detect and avoid contaminated substrates at the LCso-concentrations of the different pesticides. All the earthworms were influenced positively in the preference behaviour experiments. Because of certain limitations of the bait-lamina technique, it was difficult to formulate conclusions on what happens in the soil. A possible explanation for the differences in feeding activity of soil fauna could be attributed to the migration of the soil fauna to more habitable soil horizons during the dry summer conditions, when most of the pesticides are applied. The ecological relevance of the acute toxicity tests conducted need to be investigated further. It is clear that the acute toxicity tests provided important information that should be considered, but care should be taken and the necessary safety factors be determined and considered when doing risk assessment studies. The results of the preference behaviour studies showed that for certain pesticides E. fetida can be a sensitive bioindicator of acute and/or sub-acute lethal toxicity testing but this might not necessarily be the case for other pesticides. The goal of doing laboratory studies is to gain as much information to make reliable extrapolations to field situations from laboratory data. Laboratory-to-field extrapolations are very complicated because of the physico-chemical composition of soil, the unpredictable way pesticides behave within soil and the reaction of soil organisms to the soil and to the chemical biocides that are used. Further studies need to be done in order to fully understand to what extent the soil fauna were affected by the different management practices applied to the vineyard at Plaisir de Merle. / AFRIKAANSE OPSOMMING: Voedsel aanvraag vir die toenemende menslike bevolking plaas groot druk op die landbou sektor om meer kos te produseer. Om aan die voedsel eise te voldoen, gebruik boere al meer chemiese biosiede om pesspesies te bestry. Alle pestisiede moet toksies of giftig wees vir die teiken organisme waarvoor dit bestem is. Ongelukkig is die meeste pestisiede ook toksies of giftig vir nie-teiken organismes, wat tot nadelige effekte op hul gesonheid kan lei. Organiese boerdery is ontwikkel om die algehele gesondheid van die plaas se natuurlike grond-mikrobe-plant-dier biodiversiteit te bevorder. Geen sintetiese bemestingstowwe en/of pestisiede mag gebruik word wanneer daar organies geboer word nie. Die lewe in die grond bestaan uit ingewikkelde voedselwebbe en interaksies tussen die grondlewende invertebrate. Die grond invertebrate word verdeel in drie hoof groepe, nl. mikro-organismes (bv. Protozoa, bakterieë en fungi) meso fauna (Nematoda, Collembole en Acari) en makrofauna (bv. Millipoda, Isopoda, Insecta, Mollusca en erdwurms). Die Invertebrata is die mees vatbaarste vir chemiese kontaminasie deur chemiese biosiedes in natuurlike en landbou ekosisteme. Die grond invertebraat gemeenskappe is verantwoordelik vir die afbreek van alle organiese materiaal in die grond en dus vir remineralisering van die grond. Die afbreekproses begin by die komminusie van groter stukke organiese materiaal deur die meso- en makrofauna en eindig met die mikrofauna en mikrobes wat die prosesse voltooi deur die nutriente terug te plaas in die vorm van anorganiese produkte in die grond. Die doel van hierdie studie was om te ondersoek of, en tot watter mate, grond organismes geraak word deur verskillende grondbestuurspraktyke, nl. Organiese grondbestuurspraktyke teenoor die konvensionele grondbestuurspraktyke. 'n Wingerd op die plaas Plaisir de Merle, in Simondium, Wes-Kaap, was gebruik vir die huidige studie. Een helfte van 'n een hektaar wingerd is organies bestuur en die ander helfte is op die konvensionele manier bestuur. Op elk van die twee wingerd blokke is ses verskillende behandelings toegepas. Drie van die behandelings was streng organies en die ander drie was streng konvensioneel van aard. Vier replikate van elke behandeling is toegepas op elk van die twee wingerdblokke. Die bait-lamina metode is gebruik om die voedingsaktiwiteit van die grond organismes te asses seer. As toevoeging tot die bait-lamina proewe in die wingerd self, is bait-lamina toetse ook in mikro-kosmosse in die laboratorium gedoen met grond afkomstig vanaf die twee wingerdblokke. Om die impak van die verskillende pestisiede op die grondorganismes te ondersoek, is standaard akute toksisteitstoestse en gedragstoetse uitgevoer met die komposerdwurm, Eisenia fetida. Die bait-lamina resultate in die wingerd het getoon dat die voginhoud van die grond die belangrikste rol speel wat die biologiese aktiwiteit van die grondorganismes beïnvloed. Die verskillende behandelings het die biologiese aktiwiteit van die grond fauna geaffekteer, maar seisoenale veranderings is ook uitgesonder as een van die bepalende faktore wat die biologiese prosesse in die grond stuur. Die akute toksisiteitstoetse het getoon dat die aktiewe bestandeie van drie van die pestisiede (mancozeb, penconazole en trifloxystrobin), E. fetida negatief beïnvloed het teen die aanbeveelde konsentrasies wat toegedien is. Die aktiewe bestandeie van die ander twee pestiede (glyphosate en N-asetiel sallisiel suur) het nie die erdwurms nadelig beïnvloed teen die aanbeveelde konsentrasies wat toegedien is nie. Die gedragsproewe het getoon dat E. fetida die LCso-konsentrasies van al die verskillende pestisiede kan waarneem en vermy. Al die erdwurms is positief beïnvloed in die gedragseksperimente met die verskillende pestisiede. Omdat die bait-laminametode sekere beperkings het, was dit moeilik om tot gevolgtrekkings te kom oor wat presies in die grond gebeur. 'n Moontlike verklaring vir die verskillende voedingsaktiwiteite van die grond fauna kan toegereken word aan die migrasie van die grondorganismes na meer leefbare grondhorisonne gedurende die droë somer toestande, wat toevallig met die spuit van die meeste pestisiede ooreenstem. Die ekologiese relevansie van die akute toksisiteitstoetse wat uitgevoer is, moet meer deeglik ondersoek word. Die belangrikheid van die akute toksisteitstoetse is duidelik en het waardevolle informasie gelewer, maar sorg moet geneem word, en die nodige veiligheids faktore moet bepaal word en in ag geneem word, wanneer riskobepalingstudies gedoen word. Die gedragsproewe het getoon dat vir sekere pestisiede E. fetida 'n sensitiewe bioindikator van akute en/of sub-akute letale toksisiteits toetse kan wees, maar nie noodwendig vir ander pestisiede nie. Die doel van laboratoriumstudies is om so veel as moontlik inligting te versamelom vertroubare ekstrapolasie te kan maak na situasies in die veld vanaf laboratorium data. Laboratorium-na-veld ekstrapolasies is dikwels baie gekompliseerd as gevolg van die fisies-chemiese samestelling van die grond, die onvoorspelbare manier waarop chemiese pestisiede met die grond reageer en die reaksie van die grondorganismes op chemiese biosiede in die grond. Verdere studies moet gedoen word om so deeglik maanlik die mate van die impak wat die verskillende bestuurspraktyke op die grond fauna het, te verstaan op Plaisir de Merle.

Wild populations of fish are faced with a multitude of stressors, which may include human interaction, toxins, and disease. Benzo(a)pyrene (BaP), a known carcinogen and immunotoxin, has been reported in the stomach contents of juvenile chinook salmon, Oncorhynchus tshawytscha, in urban waterways. We investigated the impact of chronic dietary exposure of environmentally relevant levels of BaP on the immune system and cytochrome P4501A1 (CYP1A1) expression in juvenile chinook salmon. Two experiments were carried out in which juvenile fish were fed food treated with ethanol (control diet), low or high concentrations of BaP, or cortisol. In the first experiment we measured mitogen-stimulated proliferation of splenic leukocytes using flow cytometry and a colorimetric assay using Alamar Blue[superscript TM] Susceptibility to a microsporidian parasite, Loma salmonae, was evaluated in the second experiment by quantification of xenomas in the gills. Hepatic CYP1A1 and plasma cortisol were measured in both experiments. No significant trends were found in leukocyte mitogen activation or plasma cortisol between treatments or days. However, western blot analysis of CYP1A1 concentration in liver revealed interesting patterns of induction: in cortisol fed groups CYP1A1 was <20% of control on all days, groups fed low levels of BaP were 250% of control values on days 8 and 21 then dropped below control values on day 29, and groups fed high levels of BaP had less CYP1A1 than controls on all days. Similar patterns of CYP1A1 levels were found in the second experiment, and diseased control groups showed about a 55% decrease in CYP1A1 concentration when compared with non-diseased control groups. Susceptibility to L. salmonae was significantly higher in groups receiving cortisol. Whereas there was no effect of the high BaP dose, the low BaP dose appeared to increase disease susceptibility. This study supports concerns of stress and toxin induced immune dysfunction in wild populations of fish. / Graduation date: 2004

South Africa is an arid country and its growing population is putting freshwater resources under increasing pressure. Natural salinization of freshwater systems is being exacerbated by anthropogenic influences. The National Water Act (No. 36 of 1998) stipulates the need for an ecological Reserve, that quantity and quality of freshwater needed to protect freshwater ecosystems while allowing sustainable use of freshwater resources. Water guidelines do exist in the form of the South African Water Quality Guidelines (DWAF, 1996) and more recently, Jooste and Rossouw (2002) compiled benchmark values for water quality variables marking the boundaries between ecological health classes in the 4-category classification system. Predominantly international toxicity data were used to compile the guidelines and the benchmark values. In addition, there is a paucity of chronic toxicity data nationally and internationally. This thesis showed that it is statistically possible to derive protective chronic endpoints for salinity from acute toxicity data through extrapolation. The Acute to Chronic Ratio (ACR), Two-Step Linear Regression (LRA) and Multi-Factor Probit Analysis (MPA) extrapolation methods were investigated to derive chronic toxicity data from acute toxicity data. The authors of LRA and MPA recommend associating a time independent LCx value in the range of LC₀¸₀₁ to LC₁₀ with a Predicted No Effect Concentration (PNOEC). In addition to published methods, this thesis studied the possibility of equating a time independent LC₅₀ value and subjected to a safety factor of 5 (LRA LC₅₀/5), to the PNOEC. Extrapolated chronic toxicity data where the toxicants are NaCl and Na₂SO₄ were derived for indigenous South African macroinvertebrates. NaCl and Na₂SO₄ are salts associated with salinisation in South Africa. In addition, a chronic salinity toxicity test protocol for an indigenous South African aquatic macroinvertebrate was designed and chronic toxicity test were performed using NaCl and Na₂SO₄ as toxicants. The experimental chronic toxicity data produced were used to validate results from the acute to chronic extrapolation methods. Extrapolated chronic toxicity data were inputted into Species Sensitivity Distribution curves, and concentrations that were predicted to protect 95 % of species (PC95) were compared to the sub-lethality benchmarks proposed by Jooste and Rossouw (2002) for NaCl and Na₂SO₄. This study concluded that the LRA LC₅₀/5 extrapolation method is the most protective and accurate and proposed that LRA replace the ACR method in future guideline development for inorganic salts.

by Wong On Nei. / Thesis submitted in: October 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 101-116). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xiii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Lethal and sublethal effects of SS on fish --- p.2 / Chapter 1.2.1 --- Biological effects --- p.2 / Chapter 1.2.2 --- Molecular biomarkers --- p.3 / Chapter 1.2.2.1 --- Acetylcholinesterase activity inhibition assay --- p.4 / Chapter 1.2.2.2 --- Induction of cytochrome P450 mRNA --- p.5 / Chapter 1.2.2.3 --- Induction of metallothionein mRNA --- p.6 / Chapter 1.2.3 --- Study on CYP1A and MT expression / induction --- p.8 / Chapter 1.2.3.1 --- Reverse Transcription (RT) --- p.8 / Chapter 1.2.3.2 --- Polymerase chain reaction (PCR) --- p.8 / Chapter 1.3 --- Objectives --- p.11 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.13 / Chapter 2.1 --- Study sites --- p.13 / Chapter 2.2 --- Sediment --- p.15 / Chapter 2.2.1 --- Sediment samples collection --- p.15 / Chapter 2.2.2 --- Sediment handling --- p.15 / Chapter 2.2.3 --- Sediment dry-wet (w/w) ratio measurement --- p.15 / Chapter 2.2.4 --- Heavy metal content analysis --- p.16 / Chapter 2.2.5 --- Organic content analysis --- p.16 / Chapter 2.3 --- Test organism --- p.17 / Chapter 2.4 --- Bioassays --- p.17 / Chapter 2.4.1 --- 10-day exposure treatments --- p.17 / Chapter 2.4.1.1 --- Experimental setup --- p.17 / Chapter 2.4.1.2 --- Procedure --- p.21 / Chapter 2.4.1.3 --- Tissue sample collection --- p.22 / Chapter 2.4.2 --- 30-day exposure treatments --- p.22 / Chapter 2.4.2.1 --- Behavioural observations --- p.23 / Chapter 2.4.2.2 --- Tissue sample collection --- p.23 / Chapter 2.5 --- Molecular Biomarkers --- p.25 / Chapter 2.5.1 --- Tested samples --- p.25 / Chapter 2.5.2 --- Acetylcholinesterase activity inhibition assay --- p.25 / Chapter 2.5.2.1 --- Acetylcholinesterase activity assay --- p.25 / Chapter 2.5.2.2 --- BioRad Bradford assay --- p.26 / Chapter 2.5.2.3 --- Calculation of specific enzyme activity --- p.26 / Chapter 2.5.3 --- Study on CYP1A and MT expression / induction --- p.27 / Chapter 2.5.3.1 --- Gill and liver tissue samples --- p.27 / Chapter 2.5.3.2 --- Preparation of ribonuclease free reagents and apparatus --- p.27 / Chapter 2.5.3.3 --- Isolation of total RNA --- p.27 / Chapter 2.5.3.4 --- Spectrophotometric analyses of DNA and RNA --- p.28 / Chapter 2.5.3.5 --- First strand cDNA synthesis --- p.28 / Chapter 2.5.3.6 --- Cloning and sequencing of CYP1A and MT gene --- p.29 / Chapter 2.5.3.7 --- RT-PCR co-amplification of CYP1A and 18S rRNA --- p.34 / Chapter 2.5.3.8 --- Real-time RT-PCR --- p.36 / Chapter CHAPTER THREE --- RESULTS --- p.39 / Chapter 3.1 --- Sediment chemistry --- p.39 / Chapter 3.1.1 --- Sediment dry-wet (w/w) ratio --- p.39 / Chapter 3.1.2 --- Heavy metal content of sediments --- p.39 / Chapter 3.1.3 --- Levels of total PCBs and PAHs in sediment --- p.39 / Chapter 3.2 --- Monitoring of test conditions --- p.42 / Chapter 3.3 --- Bioassays --- p.42 / Chapter 3.3.1 --- Survivorship --- p.42 / Chapter 3.3.2 --- Growth --- p.46 / Chapter 3.3.3 --- Feeding rate --- p.51 / Chapter 3.3.4 --- Behaviour --- p.54 / Chapter 3.3.5 --- Sediment clogging --- p.59 / Chapter 3.3.6 --- Body lesions --- p.59 / Chapter 3.3.7 --- Abnormal behaviour --- p.59 / Chapter 3.4 --- Molecular biomarkers --- p.63 / Chapter 3.4.1 --- Acetylcholinesterase activity inhibition assay --- p.63 / Chapter 3.4.2 --- Cloning and sequencing of CYP1A and MT gene --- p.66 / Chapter 3.4.3 --- RT-PCR co-amplification of CYP1A and 18S rRNA --- p.73 / Chapter 3.4.4 --- Real-time RT-PCR --- p.77 / Chapter CHAPTER FOUR --- DISCUSSION --- p.84 / Chapter 4.1 --- Sediment chemistry --- p.84 / Chapter 4.2 --- Biological responses --- p.85 / Chapter 4.3 --- Molecular biomarkers --- p.91 / Chapter 4.3.1 --- Acetylcholinesterase activity inhibition assay --- p.91 / Chapter 4.3.2 --- Cloning and sequencing of CYP1A and MT gene --- p.93 / Chapter 4.3.3 --- RT-PCR co-amplification of CYP1A and 18S rRNA --- p.93 / Chapter 4.3.4 --- Real-time RT-PCR --- p.95 / Chapter 4.4 --- Recommendations --- p.99 / Chapter 4.5 --- Conclusions --- p.100 / REFERENCES --- p.101 / APPENDIX --- p.117

Thesis (MScAgric (Zoology))--University of Stellenbosch, 1996. / A number of studies on the general physico-chemical character of the Eerste River, Western Cape, had previously been done, but the problem of heavy metal pollution had, by 1993, not been addressed. A study was therefore undertaken from 1993-1995 in order to investigate these aspects. Since several researchers have shown that freshwater crabs accumulate certain heavy metals in their bodies and may therefore be used as monitors of environmental heavy metal pollution, the present study concentrated mainly on metal concentrations (Mn, Zn, Cu, Pb and Cd) in the local freshwater crab species, Potamonautes perlatus, and its possible use as biomonitor in the Eerste River. Two localities in the Eerste River were chosen,. in order to make comparisons, namely a relatively uncontaminated site in the Assegaaibosch Nature Reserve, Jonkershoek, and a visibly polluted site downstream from Stellenbosch, behind Stellenbosch Farmers' Winery (SFW). Crabs, water and sediment samples were collected seasonally at both localities, and metal concentrations thereof determined by atomic absorption spectrophotometry. The results showed that the Eerste River down to the SFW locality is still relatively unpolluted in terms of heavy metals. It was, however, clear that runoff from the Stellenbosch municipal, industrial and agricultural areas do have an influence on other physico-chemical features of the river. The concentrations of heavy metals in whole crabs, tissues and carapace showed that Zn concentration was well regulated in P. perlatus from both localities, Mn and Cu were accumulated in individuals from SFW, and Pb and Cd accumulated in both populations. Compartmentalization of heavy metals was shown to occur in P. perlatus: the carapace was found to be the most important storage site for Mn, Zn and Pb, the carapace and gonads equally important for Cd storage, and the digestive gland the most important site for Cu storage. Whereas gender was shown, generally, to be of little importance in heavy metal uptake in P. perlatus, crab body size and seasonality were both shown to influence heavy metal uptake to some extent. However, only summer peaks in whole crab, carapace and tissue manganese concentrations were shown to correlate with peaks in environmental Mn concentrations. It was concluded that P. perlatus would possibly only be a Suitable monitor of environmental Mn, Pb and Cd pollution, although there is no guarantee that the crab body would accurately reflect environmental concentrations. It was also ascertained that, since a study of the sperm ultrastructure of P. perlatus showed a significantly larger number of abnormal spermatozoa in male crabs from ,SFW, and since these observed differences could possibly be related to heavy metal exposure, the sperm of this species might be a more reliable indicator of heavy metal pollution. It was finally concluded that more intensive research need to be undertaken on various aspects, especially the use of the spermatozoon as indicator of environmental heavy metal pollution, and that the results of the present study could serve as a basis for future studies.