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M.Sc. (Aquatic Health) / Previous studies showed that the upper reaches of the Taaibosspruit catchment in the north eastern region of the Free State, contains elevated concentrations of mercury in the sediment. Additional laboratory-based bioaccumulation studies with sediment-dwelling Chironomidae larvae revealed that the mercury was in a bioavailable form, but it was uncertain to what extent this mercury was in the methylated organic form. Therefore, a study to assess the extent of inorganic- and methylmercury contamination in the water and sediment, as well as the uptake of this pollutant by aquatic and aquatic associated biota was undertaken. This study tested the hypothesis that bioaccumulation of mercury occurs in the different trophic levels of the upper reaches of the Taaibosspruit ecosystem. The objectives of this study were to determine the spatial and temporal physico-chemical characteristics of the water and sediment of the Taaibosspruit system; to determine the extent of mercury distribution within the water and sediment of the Taaibosspruit system; to determine the degree of bioaccumulation and possible biomagnification of mercury by the biota from the different trophic levels of the ecosystem in the Taaibosspruit system; to determine the distribution of cadmium, chromium, copper, lead, manganese and uranium within the water and sediment of this system, as well as the degree of bioaccumulation of these metals by invertebrates and fish; and to determine whether the aquatic macroinvertebrate community structures change along the mercury pollution gradient in the Taaibosspruit system. Sampling was done bi-annually at five sites during late high- and late low flow periods. The reference site is situated on a tributary of the Taaibosspruit, which is not directly influenced by discharges from the industrial complex. In situ water quality parameters were determined at each site and the following samples were collected: water, sediment, aquatic rnacroinvertebrates, vertebrates (fish and amphibian species), as well as noninvasive tissue from birds (feathers). The physico-chemical characteristics of the water were determined by measuring the chlorophyll-a concentrations, nutrient levels and suspended solid content. The sediment was analysed for organic content, water content, as well as sediment particle size distribution. The methylmercury concentrations in the water, sediment and biotic components were determined through cold vapour atomic fluorescence spectrometry, whilst inorganic mercury, cadmium, chromium, copper, lead, manganese and uranium concentrations in these samples were determined through inductively coupled plasma mass spectrometry. All analyses were subjected to appropriate quality analysis and quality control procedures. The relationship between environmental mercury exposure and biological effects was determined using changes in the macroinvertebrate community assemblage structure as the biological endpoint. The invertebrate samples were retained for enumeration and lower taxonomic identification in the laboratory. Data were subjected to the appropriate univariate statistical analysis (oneway ANOVA and regression), as well as multivariate statistical analyses (redundancy and principal component) to study spatial and temporal trends in mercury distribution and associated biological responses. This study showed that elevated concentrations of some nutrients and other dissolved solids were present in the system and that the water quality from the upper catchment of the Taaibosspruit (near the industries) had an impact on the downstream sites. The highest mercury concentrations were found at the sites closer to the industrial complex. These concentrations were higher than the concentrations from the reference site and when compared to international mercury guidelines, it was found to be too high. Comparison of the mercury concentrations in the water and sediment with environmental parameters also revealed that some of these parameters significantly influenced the environmental mercury concentrations. It was found that the biota has bioaccumulated mercury, which has resulted in the biomagnification of methylmercury by the biota from the different trophic levels of the ecosystem from this system. The results of the other six metals showed that the highest concentrations were found at the sites closer to the industries, with very high cadmium and copper concentrations in the water at the site where wastewater from the industries enters the system. These high concentrations in the water, suspended particles and sediment resulted in the accumulation of the metals by the biota present in this system. The sediment-dwelling Chironomidae-larvae bioaccumulated these metals to higher levels than the other biota from this system. The response of aquatic macroinvertebrates to high mercury exposure was dearly visible, as the sites where the environmental mercury concentrations were the highest, had low species richness. The environmental variables also influenced the community structure of the macroinvertebrates by reducing the number of different families at the more impacted sites. The results have shown that some of the fauna in the Taaibosspruit system were reduced in favour of the more tolerant families, namely Chironomidae and Oligochaeta. Thus, it was found that Taaibosspruit system is impacted upon by the surrounding industries, namely by enhancing the bioaccumulation of mercury, and influencing the macroinvertebrate community structure.

Mercury is a pervasive environmental contaminant which is globally distributed in freshwater ecosystems. In order to assess the risk that mercury and methylmercury pose to public health through consumption and trophic level transfer, it is first necessary to understand the interactions and uptake that occurs between benthic organisms and mercury impacted sediments. Delineation of these interactions currently rely on correlating measurements of bulk sediment concentrations with bioaccumulation of either total mercury or methylmercury. However, it has been proposed that porewater concentrations, rather than sediment concentrations, should be used to predict uptake and bioavailability. Diffusive gradient in thin films (DGTs) have been proposed as a viable technique for porewater measurements to assess the bioavailable fractions of mercury. DGTs were compared to traditional bulk solid sampling to assess their capabilities for the prediction of total and methylmercury bioaccumulation in benthic oligochaetes, T. tubifex. DGTs performed similarly to the bulk solids sampling approach in respect to their correlation with mercury bioaccumulation in the sediment matrix studied. Bioturbation was shown to impact redox profiles in the sediment which led to a decrease in porewater methylmercury concentrations in the uppermost surficial sediment depths. These results indicate that monitoring tools such as DGTs are necessary to better understand the fate of mercury at field scale contaminated sites. / text

Bioconcentration factor testing serves as the most valuable surrogate for the assessment of bioaccumulation. The assessment of potentially harmful chemicals is crucial to not only the health of aquatic environments, but to humans as well. Chemicals that possess the ability to persist in the environment or that have the potential to bioaccumulate, pose a greater risk to organisms that are exposed to these chemicals. The Organization for Economic Cooperation and Development Guideline 305 outlines specific protocols to run an accurate and reliable aquatic flow-through test. However, since its adoption in 1996, very few changes have been made to accommodate the endeavor to lowering the amount of test species to run one of these said tests. Running an aquatic flow-through test, according to 305, takes much time and money as well as numerous amounts of fish. Such burdens can be eliminated through simple modifications to the standard protocols. In this study, we propose an abbreviated study design for aquatic bioconcentration testing which effectively alleviates the burdens of running a flow-through test. Four chemicals were used individually to evaluate the usefulness of the proposed shortened design; 4-Nonyphenol, Chlorpyrifos, Musk Xylene, and DDT. The study consisted of exposing Cyprinus carpio for 7 days followed by 7 days of depuration, for a total of a 14-day study. Our results for each of the four compounds are consistent with literature values, thus, demonstrating that BCFk can be accurately predicted in an abbreviated in vivo test.

Inúmeros pesticidas são usados na agricultura, para controle de pragas e ervas daninhas. Dentre eles destaca-se o herbicida atrazina, intensivamente utilizado nas culturas de cana-de-açúcar, milho e sorgo, que ocupam extensas áreas no estado de São Paulo. Grande parcela do herbicida, que é aplicado na agricultura, entra em contato com o solo, podendo ser lixiviado, atingindo as águas superficiais. Neste sentido, alguns animais como, por exemplo, moluscos bivalves, podem ser utilizados como monitores biológicos do ambiente aquático e auxiliar no estudo da ecotoxicologia. Considerando o risco de contaminação do ambiente aquático pela atrazina, propõe-se, no presente trabalho, desenvolver uma metodologia de análise daquele herbicida nos tecidos nas espécies de bivalves límnicos Anodontites trapesialis (LAMARCK, 1819) e Corbicula fluminea (Muller, 1789), validar esse método e, finalmente, verificar se ocorre a bioacumulação do herbicida nas partes moles dessas duas espécies. Como técnica de extração utilizou-se a extração líquido- líquido e como técnica de análise, a cromatografia líquida de alta eficiência (CLAE). / Large amount of pesticides have been used for the control of agriculture pests and weeds. Particularly important among herbicides is atrazine, extensively employed in cultures of sugar cane, corn and sorghum, that occupies an extensive area in São Paulo state. Large portions of atrazine, applied in the agricultural fields, leaches from the soil to surface water systems. In this way, some organisms such as fresh- water mollusks bivalves, can be used as biological monitors of aquatic environments, contributing for ecotoxicology studies. Considering the existence of risk of contamination by atrazine of the aquatic environment, the purpose of this work was, (i) to develop a method for the analysis of atrazine in the fresh- water bivalves species Anodontites trapesialis (LAMARCK, 1819) and Corbicula fluminea (MULLER, 1789), (ii) to validate such method and, (iii) to detect if these organisms can bioaccumulate atrazine in their tissues. This method involved a simple liquid-liquid extraction procedure, followed by high- performance liquid chromatographic analysis (HPLC).

Pak-chung Leung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 151-158). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.iv / List of Figures --- p.viii / List of Tables --- p.ix / List of Abbreviations --- p.xiv / Chapter / Chapter 1. --- General Introduction --- p.1 / Chapter 2. --- Literature Review: Greening process in higher plants --- p.4 / Chapter 3. --- Greening Bioassay in Excised Cucumber Cotyledons / Introduction --- p.13 / Materials and Methods / Plant material --- p.14 / Measurement of Ch1 synthesizing activity in excised cotyledons of different ages --- p.14 / Measurement of Ch1 accumulation in excised cotyledons after various pretreatment periods --- p.15 / Measurement of Ch1 accumulation in excised cotyledons under different light intensities and temperatures --- p.15 / Greening curve --- p.16 / Results and Discussion / Effect of seedling age on Ch1 accumulation --- p.16 / Effect of pretreatment period on Ch1 accumulation --- p.18 / Effects of light intensity and temperature on Ch1 accumulation --- p.18 / Effect of shaking (for oxygen replenishment) on Ch1 accumulation --- p.19 / Greening curve --- p.20 / Greening bioassay in excised cucumber cotyledons --- p.20 / Chapter 4. --- Regulation of Chlorophyll Accumulation in Excised Cucumber Cotyledons / Introduction --- p.27 / Materials and Methods / Materials --- p.28 / Seed germination --- p.28 / Harvesting and pretreatment --- p.28 / Illumination and Ch1 determination --- p.29 / Results / Effect of BA on Ch1 accumulation --- p.29 / Effect of Na2EGTA on Ch1 accumulation --- p.30 / "Effect of Na+, Ca2+ and K+ on Ch1 accumulation" --- p.32 / Effect of duration of pretreatment with Na+ ， Ca2+ and K+ on Ch1 accumulation --- p.34 / Effect of verapamil and Ca ionophore A23187 on Chl accumulation --- p.36 / Effect of TFP on Chl accumulation --- p.37 / Discussion / Effect of BA on Chl accumulation --- p.38 / "Effect of Na+, Ca2+ and K+ on Chl accumulation" --- p.39 / Demonstration of the involvement of Ca2+ in Chl accumulation --- p.43 / Chapter 5. --- The Calcium Effect on Benzyladenine-induced Stimulation of Chlorophyll Accumulation in Excised Cucumber Cotyledons / Introduction --- p.63 / Materials and Methods / Materials --- p.64 / Plant growth and treatment --- p.64 / Pretreatment experiment --- p.64 / Sequence experiment --- p.65 / Results / Pretreatment experiment --- p.65 / Sequence experiment --- p.66 / Discussion --- p.68 / Chapter 6. --- Regulation of 5-Aminolevulinic Acid and Chlorophyll Accumulation in Levulinic Acid-treated Cotyledons / Introduction --- p.80 / Materials and Methods / Materials --- p.81 / Plant growth and treatment --- p.81 / Chl determination and ALA assay --- p.82 / Measurement of ALA and Chl accumulation in LA-treated cotyledons --- p.83 / ALA accumulation curve --- p.83 / Measurement of Chl accumulation in 50 mM NaCl-treated cotyledons --- p.84 / "Measurement of ALA and Chl accumulation in BA-, cations-, Na2EGTA-, verapamil- and TFP-pretreated cotyledons" --- p.85 / Results / Concentrations effect of LA on ALA and Chl accumulation --- p.85 / ALA accumulation curve --- p.86 / Effect of 50 mM NaCl on Chl accumulation --- p.87 / Effect of BA on ALA and Chl accumulation in LA- treated cotyledons --- p.90 / "Effects of Na+, Ca2+ and K+ on ALA and Chl accumulation in LA-treated cotyledons" --- p.91 / "Effects of Na2EGTA, verapamil and TFP on ALA and Chl accumulation in LA-treated cotyledons" --- p.92 / Discussion / Measurement of ALA accumulation in LA-treated cotyledons --- p.93 / Effect of BA on ALA and Chl accumulation in LA-treated cotyledons --- p.96 / "Effects of Na+, Ca2+ and K+ on ALA and Chl accumulation in LA-treated cotyledons" --- p.98 / Demonstration of the involvement of Ca2+ in ALA accumulation --- p.99 / Chapter 7. --- Regulation of 5-Aminolevulinic Acid Accumulation in Isolated Developing Chloroplasts / Introduction --- p.123 / Materials and Methods / Materials --- p.124 / Plant materials --- p.124 / Chloroplast isolation --- p.125 / Incubation conditions --- p.126 / ALA assay --- p.127 / Measurement of ALA accumulation in LA-treated isolated developing chloroplasts --- p.128 / "Measurement of ALA accumulation in BA-, cations-, Na2EGTA-, verapamil- and TFP-treated isolated developing chloroplasts" --- p.128 / Results / Time course study of Chl accumulation in intact greening cotyledons of different ages --- p.129 / Measurement of ALA synthesizing activity in isolated developing chloroplasts incubated in the dark and light --- p.130 / Concentrations effect of LA on ALA accumulation in isolated developing chloroplasts --- p.130 / Effects of BA and the cations on ALA accumulation in isolated developing chloroplasts --- p.131 / "Effect of Na2EGTA, verapamil and TFP on ALA accumulation in isolated developing chloroplasts" --- p.132 / Discussion / Light regulation of ALA synthesizing activity in isolated developing chloroplasts --- p.132 / Regulation of ALA accumulation in isolated developing chloroplasts --- p.134 / Chapter 8. --- Conclusion --- p.147 / References --- p.151

Thesis (M.Sc.(Biochemistry)) -- University of Limpopo, 2019. / Retinoblastoma binding protein 6 (RBBP6) is the protein encoded by the Retinoblastoma Binding Protein 6 (RBBP6) gene that is located in chromosome 16p12.2. There is a growing list of newly discovered RBBP6 hypothetical splice variants but there are only three RBBP6 splice variants that are well documented. RBBP6 has been previously implicated in the regulation of cell cycle and apoptosis but little is known about the expression and regulation of the human RBBP6 splice variants during cell cycle progression and breast cancer development. This study was aimed at determining the expression pattern of RBBP6 alternatively spliced variants during arsenic trioxide-induced cell cycle arrest and apoptosis in breast cancer MCF-7 cells. It was also aimed at determining RBBP6 specific microRNAs and how they are regulated in MCF-7 breast cancer cells. MCF-7 cells were maintained and subjected to arsenic trioxide-induced cell cycle arrest and apoptosis. The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and the Muse™ Count & Viability assays were used to evaluate the effect of arsenic trioxide on the viability of MCF-7 cells. Cell cycle arrest using 11 μM arsenic trioxide and apoptosis using 32 μM arsenic trioxide were analysed using the MUSE® Cell Analyzer, light and fluorescence microscopy. Arsenic triode-induced apoptosis was analysed using the Muse™ Annexin V & Dead Cell Kit, MultiCaspase and MitoPotential assays using the Muse™ MultiCaspase Kit and Muse™ MitoPotential Kit. Arsenic trioxide-induced cell cycle arrest was analysed using the Muse™ Cell Cycle Kit. Semi-quantitative analysis of RBBP6 variants was carried out using the conventional Polymerase Chain Reaction (PCR), while the quantitative analysis was done using the Real-Time Quantitative PCR. The localization of RBBP6 isoforms was done using Immunocytochemistry (ICC). Web based Bioinformatics tools were used to identify RBBP6-specific microRNAs. The MTT results showed that arsenic trioxide decreased the viability of the MCF-7 cells in a dose-dependent manner. The Muse™ Cell Cycle analysis showed that 11 μM of arsenic trioxide induced G2/M cell cycle arrest in MCF-7 cells, while the Muse™ Annexin V & Dead Cell assay showed that 32 μM of arsenic trioxide induced the extrinsic apoptotic pathway in MCF-7 breast cancer cells. Using the conventional PCR, the MCF-7 cells were found to express the RBBP6 variant 1 transcript but lacks the expression of variant 2 and 3 transcripts, contrary to the kidney embryonic Hek 293 cells that exhibited the expression of RBBP6 variant 1, 2 and 3. Additionally, arsenic trioxide downregulated RBBP6 variant 1 in breast cancer cells during cell cycle arrest and apoptosis. The Real-Time PCR confirmed that MCF-7 cells lowly express RBBP6 variant 3. On the other hand, the ICC analysis showed that RBBP6 isoform 1 is localized and highly expressed in MCF-7 breast cancer cells. The Web based Bioinformatics tools showed that RBBP6 variant 1 specific microRNAs are down regulated in MCF-7 breast cancer cells. These results together showed that As2O3 is effective against MCF-7 cells and also regulated the expression of RBBP6 variants, especially, variant 1. This study showed that there are RBBP6 variants that are involved in breast cancer progression and there are those that may be involved in breast cancer suppression. Targeting these RBBP6 variants for therapeutic development is a promising strategy. In conjunction with RBBP6 expression, arsenic trioxide should be further explored as a breast cancer drug.

The major objectives of this research were to examine the bioaccumulation parameters [depuration rates (kd), half life (t1/2) and biomagnification factor (BMF)] of individual isomers of hexabromocyclododecane (HBCD, C12H18Br6) in fish and to test the hypothesis of in vivo bioisomerization. This was done by exposing three groups of juvenile rainbow trout (Oncorhynchus Mykiss) to food fortified with known concentrations of an individual diastereoisomer (α, β, γ) for 56 days (uptake phase) followed by 112 days (depuration phase) of unfortified food. A fourth group of fish were exposed to unfortified food for the duration of the experiment. Fish (n=4) from all four aquaria were sacrificed on days 0, 7, 14, 56, 63, 70, 112 and 168 and muscle tissue was extracted and analyzed for diastereoisomer concentrations by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS). Bioaccumulation of the γ diastereoisomer was linear during the uptake phase while the α and β diastereoisomers were found to increase exponentially with respective doubling times of 14.1 and 20.5 days. Both the β and γ diastereoisomers followed first order depuration kinetics with calculated t1/2’s of 94 ± 25 and 84 ± 51 (± 1 × standard error) days, respectively. The BMF for the α diastereoisomer (BMF = 4.1) was one and a half times greater than the β-diastereoisomer (BMF = 2.6) and about one fifth larger than the γ-diastereoisomer (BMF = 3.6). The large BMF for the α diastereoisomer is consistent with this diastereoisomer dominating higher trophic level organisms in wildlife. Although the BMF of the β diastereoisomer suggests that it will biomagnify, because it is present in small quantities in commercial mixtures it is rarely detected in environmental samples. Results from these studies also provide evidence of bioisomerization of the β and γ diastereoisomers. Most importantly, the α diastereoisomer which was recalcitrant to bioisomerization by juvenile rainbow trout in this study and known to be the dominant diastereosiomer in fish, was bioformed from both the β and γ diastereoisomers. To our knowledge, this is the first report of bioisomerization of a halogenated organic pollutant in biota. / May 2006

Abstract The objective of this study is to investigate the heavy metal pollution in our environment by sampling and analyzing the concentration of heavy metal in seawater, sediment, and aquatic organisms from the seaside along the Chang-hua Coastal Industrial Park. The Chang-hua Coastal Industrial Park, located by the west coast of Chung-hua county in middle Taiwan, is a large-scale and multi-functional industrial estate with industrial production, residence and leisure possibilities. Hsienhsi District is situated at the northern part of the Industrial Park. Lukang District is at the southern part. In the middle of it, it is Lunwei District. In the Chung-hua Coastal Industrial Park, there are the second most electroplate and metal coating factories in Taiwan. Metal waste from the process of electroplating and coasting could easily pollute the tidal flat in Chang-hua Coastal Industrial Park. In order to prevent potential pollution taking place, this study took seawater, segment, and the aquatic organisms from the coast of the Chung-hua Coastal Industrial Park to monitoring the concentration level of copper(Cu), zinc(Zn), nickel(Ni), lead(Pb), chromium(Cr) and cadmium(Cd) in each index. The bioconcentration factor (BCF) indicates distinctions of accumulation in various matrixes. Especially from BCFW and BCFS demonstrate that soldier crab is a better index to illustrate the accumulation of Pb and Cr. Muricidae is a better index for showing Cd accumulating concentration. Furthermore, saccostreamordax is the best index for illustrating accumulative level of Ni. Oyster is better in showing accumulating of Cu and Zn. Though each organism shows distinct accumulation of particular metal, they can still illustrate the concentration of other selected metals to image the pollution of the environment. The comparison of metal accumulating levels in seawater, segment, and aquatic organisms from each test site demonstrated: the heavy metal concentration at site2 was much higher than at the other sites. The second most heavy metal concentration was at site6 and site8. Matrixes at site4 contained the lowest heavy metal concentration. The result was thought to be caused by current capacities of factories in three districts of Chang-hua Coastal Industrial Park. Site2 was at Hsienhsi Districtin, in which contained about 58% of factories in residence. In Lukang District, it reached the highest-77%-where was site 6 and site 8. In terms of Site4, it situated at Lunwei District, where was Lunwei Harbor Reservation with least development following by least capacity of factories. Furthermore, without a sound environmental-protection biological sewage treatment device, residues and chemical waste produced from various highly contaminant industries would bring about this concentration comparison result as well.

No description available.