Cellulolytic responses of the Asiatic clam, Corbicula fluminea and a snail, Mudalia dilatata, to selected constituents of power plant effluents (i.e., zinc, cadmium, acidic and alkaline pH, individually and paired) were investigated in 30-day exposures. Exposures were conducted in both laboratory and field-oriented artificial streams and then validated in the river receiving system of a power plant. Cellulolytic activity was reduced by laboratory and field exposures to cadmium and zinc at all levels tested from 0.012 to 0.10 mg cadmium/L and generally at 0.025 to 1.0 mg zinc/L. Clams detected acute lethal levels of metal and used valve closure as an avoidance mechanism for 14 days. Snails, however, did not effectively avoid exposures and were more sensitive to acute stress during all exposures. These behavioral responses were corroborated by both cellulolytic activity and metal accumulation. Measurements of cellulolytic activity for both test species in laboratory exposures differed from those in field artificial streams. Reduced enzyme activity in controls by day 30 was attributed to artificially induced stress associated with the laboratory environment. This factor precluded any analysis of laboratory responses for periods of exposure longer than 20 days as well as recovery analysis. Field oriented artificial streams provided a sufficient environment to adequately assess long-term stress and recovery as measured by cellulolytic activity and metal accumulation in both clams and snails. Enzyme activity responded to metal exposure with respect to both degree and duration of exposure. Cadmium and zinc combined exposures caused significantly reduced cellulolytic activity at the same concentration as those for cadmium alone. Reduced enzyme activity caused by cadmium and zinc addition at levels that were not detectable suggested that the cellulolytic index was sensitive to sublethal stressors. This was supported by metal uptake patterns in clams and snails. Cellulolytic activity responded to zinc addition at alkaline and acidic pH in a manner that supported pH optima for cellulases and bioavailability of metals. Effects seen in macroinvertebrate assemblages (diversity, richness, and similarity) were compared with cellulolytic activity of caged Corbicula from a site specific power plant discharge. Enzyme activity inhibition was the most sensitive indicator measured. Reductions in cellulolytic activity at stations monitored for total zine content were consistent with effects seen at comparable exposures to zine in field-located artificial streams. A zine concentration of 0.05 mg/L consistently caused the first significant reductions in cellulolytic activity. This concentration is comparable to the U.S. Environmental Protection Agency's Water Quality Criteria value (0.047 mg/L zinc) for protection of aquatic life. / Ph. D.

Thesis (PhD (Botany and Zoology)) -- University of Stellenbosch, 2006. / Metals are natural elements of the earth crust usually present at low concentrations in all soils. Although many metals such as cobalt, copper, iron and zinc are essential to living organisms, at elevated concentrations most metals are toxic to organisms living in and on soils. Elevated concentrations of metals are caused either by anthropogenic deposition following remobilisation from the earth crust or are of natural origin. Ultramafic soils do not only pose unfavourable living conditions such as drought and poor organic content, these soils are also characterized by extremely high concentrations of a range of metals known to be toxic under normal circumstances. Ultramafic soils are of high ecological importance as a high proportion of endemic organisms, especially plants, live on these soils. As it is known that earthworms do occur in ultramafic soils, the aims of the present study were to investigate the abilities of earthworms to survive in these soils and the influences of elevated chromium, cobalt, copper, manganese and nickel levels. For the evaluation of the metal background conditions, soils originating from ultramafic rocks of the Barberton Greenstone Belt, Mpumalanga, South Africa were collected and different fractions representing different levels of bioavailability were analyzed for arsenic, chromium, cobalt, copper, manganese and nickel. To assess the mobile, readily available metal fraction, i.e. Ca2+- exchangeable metal cations, a 0.01 mol/L CaCl2 extraction was performed. To investigate the mobilisable metal fraction, representing the amount of easily remobilisable complexed and carbonated metal ions, a DTPA (di-ethylene-triamine-pentaacetic acid) extraction was conducted. In relation to non-ultramafic or anthropogenic contaminated soils, a far lower proportion of metals were extractable by the above mentioned extraction methods. To investigate the availability and effects of these metals on earthworms, two ecophysiologically different species were employed. Aporrectodea caliginosa and Eisenia fetida were long-term exposed to the ultramafic soils collected at the Barberton region and a control soil from a location at Stellenbosch with a known history of no anthropogenic metal contamination. The responses to the ecological stress originating in the ultramafic soils were measured on different levels of earthworm organisation. As endpoints affecting population development, cocoon production, fecundity and viability were evaluated. On individual level, growth, metal body burden and tissue distribution were investigated. As endpoints on subcellular level, the membrane integrity was assessed by the neutral red retention assay, the mitochondrial activity was measured by the MTT colorimetric assay and as a biomarker for the DNA integrity, the comet assay was performed. Focussing on manganese and nickel, the uptake by E. fetida of these metals was investigated with the exclusion of soil related properties using an artificial aqueous medium to draw comparisons to the uptake of these metals in natural soils. The possible development of resistance towards nickel was tested by exposing pre-exposed (for more than 10 generations) E. fetida specimens to ultramafic soils with concentrations of more than 4000 mg/kg nickel. The results showed that, except on the endpoint survival, which was less sensitive than all other bioassays, significant responses to the ultramafic challenge were observed in all earthworm bioassays and on all levels of organisation. The sensitivity of the responses of the earthworms towards the ultramafic conditions was not predictable by the level of organisation. The two species showed different strategies of metal elimination. In A. caliginosa, metals such as nickel, manganese and chromium were transported to the posterior section and the posterior section was subsequently pushed off by autotomization. In E. fetida, metals such as chromium and nickel were sequestered in storage compartments in the coelomic cells or fluid. Other metals, such as cobalt, were not taken up at elevated concentrations. Although an increased accumulation of nickel was observed in E. fetida specimens pre-exposed to nickel, development of resistance or cross resistance was not observed in this species. In contrast, pre-exposed specimen exposed to elevated concentrations of nickel showed a higher sensitivity in terms of survival, indicating the absence of acclimatisation or even genetic adaptation. A comparison of the two species employed indicated that A. caliginosa was less suited for the assessment of the ultramafic soils due to the high individual variation in metal body burden, the mass loss observed and the slow reproduction rate even in the control soils. This happened despite the fact that A. caliginosa was a soil dwelling species supposed to be better adapted to the soil substrate than the litter dwelling E. fetida. The toxicity of the ultramafic soils was not necessarily related to total or environmentally available amounts of the selected metals. Thus, it can be speculated that either these soils contained unidentified toxicants with resulting interactions between toxicants playing an important role or earthworms were able to remobilize metals occurring in these soils. As the singular application of an ecotoxicological endpoint did not give reliable results, especially seen over the duration of the exposures, it can be concluded that, when studying soils with such a complex composition, the utilisation of endpoints addressing different levels of organisation is necessary for the assessment of toxic stress emerging from these ultramafic soils.

Splenic and lymph node lymphocytes from Balb/C mice were activated in vitro by the heavy metal cations, Zn⁺⁺ and Hg⁺⁺, as noted by the several-fold increases in ³H-thymidine incorporation at 144 hours of culture. Optimal mitogenic concentrations of Zn⁺⁺ and Hg⁺⁺ were 200 μM and 10 μM, respectively. Data from experiments in which three different methods were used to enrich for either T or B splenic lymphocytes, i.e. cell passage over nylon wool columns, use of athymic Nu/Nu mouse spleen cells, or cell lysis with monoclonal anti-Thy-1 antibody plus complement, suggested that Zn⁺⁺ and Hg⁺⁺ were mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron followed by cell passage through nylon wool eliminated the lymphocyte responses to Zn⁺⁺ and to Hg⁺⁺. Moreover, addition of these macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn⁺⁺ and Hg⁺⁺ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce gamma interferon. Furthermore, Zn⁺⁺ induced low levels of natural killer activity in spleen cells. In contrast to spleen and lymph node cells, thymocytes and bone marrow lymphocytes did not respond to either cation under standard culture conditions. However, when cultured in the presence of E. coli-derived lipopolysaccharide (LPS) and 2-mercaptoethanol for 144 hours, thymocytes were activated by Zn⁺⁺ (200 μM) but not by Hg⁺⁺. Quantities of LPS as low as 1.0 ng/ml satisfied this culture requirement. Purified interleukin 1 could not replace the helping activity mediated by LPS. Thymocyte subpopulation studies showed that Zn⁺⁺ activated enriched peanut lectin receptor-negative mature thymocytes, but LPS was required for the response. Spleen cells from mice, intraperitoneally injected with ZnCl₂ for 7 to 14 days, were not activated in vivo as assessed by ³H-thymidine incorporation in vitro, nor did they display enhanced responses to T-cell or B-cell mitogens. However, zinc administration had negative effects by decreasing spleen cell numbers by 31% and thymic weight by 59%. A theoretical model is presented in which Zn⁺⁺ and Hg⁺⁺ may mediate their stimulating effects in vitro by altering histocompatibility "self" structures on the surface of lymphocytes and macrophages via interactions with sulfhydryl groups on these structures to which T lymphocytes with receptors for "altered self" structures respond with proliferation or cytotoxicity.

by Wong Pik-kwan. / Thesis submitted in: September 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 116-139). / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENT --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF TABLES --- p.viii / LIST OF FIGURES --- p.ix / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER TWO --- LITERATURE REVIEW / Chapter 2.1 --- Introduction --- p.4 / Chapter 2.2 --- Heavy metals --- p.6 / Chapter 2.3 --- Mechanisms of metal toxicity --- p.9 / Chapter 2.4 --- Toxic effects of metals on marine organisms --- p.10 / Chapter 2.4.1 --- Cadmium --- p.10 / Chapter 2.4.2 --- Chromium --- p.10 / Chapter 2.4.3 --- Copper --- p.11 / Chapter 2.4.4 --- Lead --- p.12 / Chapter 2.4.5 --- Nickel --- p.12 / Chapter 2.4.6 --- Zinc --- p.13 / Chapter 2.5 --- Metal uptake and elimination in marine organisms --- p.14 / Chapter 2.5.1 --- Uptake of metals --- p.14 / Chapter 2.5.2 --- Elimination of metals --- p.15 / Chapter 2.5.3 --- Metal detoxification system in fish --- p.16 / Chapter 2.6 --- Heavy metals in marine fish --- p.17 / Chapter 2.7 --- Bioaccumulation --- p.20 / Chapter 2.7.1 --- Models of metal accumulation --- p.21 / Chapter 2.7.2 --- Compartment model --- p.21 / Chapter 2.7.3 --- Physiologically based pharmacokinetic (PB-PK) model --- p.22 / Chapter 2.8 --- The influence of environmental factors on bioaccumulation of metals --- p.23 / Chapter 2.8.1 --- Temperature --- p.23 / Chapter 2.8.2 --- Salinity --- p.23 / Chapter 2.8.3 --- Organic matter --- p.24 / Chapter 2.8.4 --- pH --- p.25 / Chapter 2.8.5 --- Chelators and surfactants --- p.25 / Chapter 2.8.6 --- Other metals --- p.26 / Chapter 2.9 --- Biological effects of heavy metals on man --- p.26 / Chapter 2.10 --- The use of biological indicator organisms for metal pollution --- p.28 / Chapter CHAPTER THREE --- HEAVY METAL CONCENTRATIONS IN CULTURED MARINE FISH IN HONG KONG / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.36 / Chapter 3.2.1 --- Sampling --- p.36 / Chapter 3.2.2 --- Water analysis --- p.36 / Chapter 3.2.3 --- Sediment analysis --- p.39 / Chapter 3.2.4 --- Mussel analysis --- p.40 / Chapter 3.2.5 --- Fish analysis --- p.40 / Chapter 3.2.6 --- Quality control and statistical analysis --- p.41 / Chapter 3.3 --- Results --- p.42 / Chapter 3.3.1 --- Seawater --- p.42 / Chapter 3.3.2 --- Sediment --- p.46 / Chapter 3.3.3 --- Mussel --- p.46 / Chapter 3.3.4 --- Fish --- p.50 / Chapter 3.4 --- Conclusion --- p.67 / Chapter 3.4.1 --- "Metal concentration in seawater, sediment, green mussel and fish" --- p.67 / Chapter 3.4.2 --- Accumulation of heavy metals in different tissues of cultured fish --- p.69 / Chapter 3.4.3 --- Relationship between the body weight and metal accumulation --- p.71 / Chapter 3.4.4 --- Heavy metal pollution in fish culture sites --- p.72 / Chapter 3.4.5 --- Selection of fish culture site --- p.72 / Chapter CHAPTER FOUR --- ACUTE AND SHORT-TERM EFFECTS OF COPPER(II) IONS ON SPARUS SARBA / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Experimental animals --- p.79 / Chapter 4.2.2 --- Determination of the 96 hour median lethal concentrations --- p.19 / Chapter 4.2.3 --- Determination of growth rate --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- Determination of the 96 hour median lethal concentrations --- p.82 / Chapter 4.3.2 --- Determination of growth rate --- p.82 / Chapter 4.3.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.82 / Chapter 4.4 --- Conclusion --- p.91 / Chapter 4.4.1 --- Determination of the 96 hour median lethal concentrations --- p.91 / Chapter 4.4.2 --- Determination of growth rate --- p.93 / Chapter 4.4.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.94 / Chapter CHAPTER FIVE --- ACCUMULATION AND ELIMINATION OF COPPER(II) IONS TO SPARUS SARBA / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Materials and methods --- p.98 / Chapter 5.2.1 --- Experimental animals --- p.98 / Chapter 5.2.2 --- Uptake and elimination of Cu ion in S. sarba during continuous exposure to waterborne Cu --- p.98 / Chapter 5.3 --- Results --- p.100 / Chapter 5.4 --- Conclusion --- p.108 / Chapter CHAPTER SIX --- GENERAL CONCLUSION --- p.112 / CHAPTER SEVEN REFERENCES --- p.116

The contamination of heavy metals in the environment is a looming concern worldwide. Egeria densa (Planch) (Submerged aquatic plant) from two ponds: Site A with co-ordinates (32º 48’22.04”S; 26°48’58.79” E) and Site B with co-ordinates (32°48’33.25”S; 26°48’33.25”S) in Alice (Eastern Cape) was evaluated for its ability to absorb heavy metals, phytochemical constituents, antimicrobial activity and ultra-structure using standard analytic procedures. Cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), and zinc (Zn) were measured in water, sediments and plant. The concentrations of these metal elements were determined with use of Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES). In sediments, the heavy metals (mg/kg) decreased in the order of their average concentration as follows: Fe (40.320) > Zn (1.259) > Pb (0.564) > Mn (0.186) > Cu (0.037) in Pond 1 whereas in Pond 2 Fe (61.527) > Cd (0.999) > Mn (0.648) > Pb (0.586) > Zn (0.156) > Cu (0.045). The highest concentration of Fe was detected in both sites and Cu being the least. The concentrations of the metals in the plants sample (from Pond 1) were found in order of Mn > Pb > Cu > Fe whereas cadmium and zinc were not detected, while the concentration in Pond 2 decreases in order of Zn > Mn > Pb > Cd > Fe > Cu. In the water samples, concentrations of heavy metals (mg/L) decreased in the order of their average concentrations as follows: Pb (35.36) > Fe (3.07) > Mn (0.238) > Cu (0.104), both cadmium and zinc were below the limit of detection in Pond 1, whereas in Pond 2 the concentrations decreased as follows: Pb (13.033) >Fe (1.69) > Cu (0.270) > Mn (0.248) > Cd (0.004) and Zinc was not detected. Phytochemical analyses of the plant extracts revealed the presence of phenols, flavonoids, proanthocyanidin, flavonols, saponins, alkaloid and tannins in all the extracts (water, acetone and n-hexane). Both acetone and water extracts, showed high concentration of proanthocyanidin, while tannin was the lowest in acetone extract. Antimicrobial evaluation using, Gram positive (Staphylococcus aureus, Bacillus pumilus, Bacillus cereus, Streptococcus pyogenes, Enterococcus faecalis) and Gram negative (Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Serratia marcescens) bacteria showed negative results for all the strain, except Streptococcus pyogenes which was inhibited at MIC of 0.1 mg/ml. Scanning electron microscopy (SEM) of ultra-structure of Egeria densa, showed that certain bacteria attached to the leaf, However more work has to be done on E. densa to verify the mechanism by which it accumulates heavy metals. The study shows that E. densa has a potential of accumulating heavy metals especial Manganese in plant.

Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ;cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Listeria monocytogenes is a zoonotic foodborne pathogen, transmissible from the natural agricultural environment to animals and humans. In recent years, the pig production industry has experienced a series of monetary losses as a result of the L. monocytogenes outbreak which threatened the economy of South Africa. This outbreak also had a detrimental effect on the health system of the country. In South Africa however, there is limited information regarding the genomic diversity of L. monocytogenes. Therefore, an overview of the genomic diversity of L. monocytogenes strains circulating at different levels of the pork production chain needs to be determined so as to be able to identify routes of contamination of the pathogen and thus improve meat safety. This study was aimed to determine the antimicrobial resistance patterns and population structure of L. monocytogenes isolated from pig farms in South Africa. Based on wholegenome sequence analysis, 77 isolates of L. monocytogenes were differentiated into four molecular serogroups with IIa (45.5%) being the most prevalent followed by IIc (26.0%), IVb (22.1%) and IIb (6.5%). Overall, 11 clonal complexes (CCs) were identified in this study, with the predominance being observed from; CC204 (23.4%), CC1 (19.5%) and CC2 (16.9%). Genetic elements associated with biocide, antimicrobial and heavy metal resistance were noted in 24.7 %, 48% and 11.7% of the isolates, respectively. Listeria pathogenicity island 1 and 3 that harbored clusters of virulence genes were present in 38.8% of the isolates. Five different plasmids were found in 68.9% of the isolates. This study has given baseline data on the genomic diversity of L. monocytogenes strains that are associated with biocides, heavy metal and antibiotics resistance genes. The data again demonstrated the genotypes of L. monocytogenes that are prone to contaminate the farm environment and possibly cause diseases in animals and humans. / Life and Consumer Sciences / M. Sc. (Life Sciences)