Freshly isolated hepatocytes from fed and starved rats were used as a model in the investigation of the mechanisms by which cadmium chloride exerts its toxic effects at the cellular level. Exposure to cadmium chloride resulted in a slight decrease in viability, more pronounced in hepatocytes from starved rats. Morphological changes preceded the increase in membrane permeability. Hepatocytes exhibited a rapid initial uptake of cadmium chloride, followed by a second slower phase. The accumulation of more metal in hepatocytes from starved rats may contribute to their enhanced susceptibility to cadmium chloride. Adverse metabolic effects of cadmium chloride included an increase in the lactate:pyruvate ratio in hepatocytes from fed rats, with a concomitant decrease in the 3-hydroxybutyrate:acetoacetate ratio in hepatocytes from fed and starved rats. Incubation with cadmium chloride resulted in increased glycogenolysis and glycolysis. Decreased rates of gluconeogenesis from lactate and pyruvate reflected the decreased uptake of gluconeogenic precursors. Studies of intracellular lactate concentrations could not resolve whether the decrease in gluconeogenesis was due to an inhibition of lactate transport into the hepatocyte or due to a decrease in its metabolism. Cadmium chloride caused a slight decrease in the basal and pyruvate-stimulated rates of cellular respiration, a marked dose-related decrease with lactate, and no significant effects with succinate. Carbonyl- cyanide-m-chlorophenylhydrazone was less effective in stimulating respiration in hepatocytes incubated with cadmium chloride, this effect being more pronounced with lactate and pyruvate than with succinate. Cadmium chloride had little effect on the uncoupled rates of FADH2 oxidation with succinate suggesting that electron transport from succinate dehydrogenase to cytochrome a/a3 was not impaired. The results from these studies suggest a primary effect of cadmium chloride on mitochondrial function and cellular energy production, resulting in secondary metabolic changes in an attempt to overcome the declining levels of ATP within the cell.

The main aims of this thesis were to establish whether an acute inflammatory response is provoked in rats by the subcutaneous administration of cadmium, and to evaluate the possible role that such a response might play in the alterations in metal homeostasis and the development of anaemia which accompanies the use of this model of cadmium intoxication. An intense local reaction to the subcutaneous administration of cadmium was found. Many of the systemic changes, most notably in haematological parameters and in levels of iron, copper and plasma proteins, mimicked those seen in the acute inflammatory response. Possible causes of the resultant anaemia are discussed and inflammation is implicated as a predominant factor in its development. The results suggest that many of the effects which in previously published work have been attributed to a direct interaction of cadmium with the system under investigation, may in fact be secondary consequences of cadmium-induced inflammation. Comparison of the effects of subcutaneous administration of cadmium and other selected metal salts with changes occurring in two recognised models of acute inflammation revealed marked differences in the local tissue reaction to different substances as well as in the magnitude of various components of the systemic response. The oedematous, necrotic and extensively destructive nature of the cadmium-induced lesion has been highlighted and shown to be partially alleviated by pre- and simultaneous treatment with zinc. Explanations for this protective phenomenon are offered, based on possible target sites of cadmium, particularly in terms of interaction with zinc-dependent processes. With a view to understanding the mechanisms involved in acute cadmium toxicity, luminol-amplified chemiluminescence, which is indicative of free radical formation and the production of reactive oxygen species, was measured directly from intact tissue samples. Inflamed tissue sampled from subcutaneous sites of cadmium administration emitted substantially more chemiluminescence than noninflamed tissue or tissue from sites to which other metals or turpentine was administered. It was demonstrated that intact tissue samples can also be used to assess free radical generation, as detected by chemiluminescence, during in vitro treatment. A pronounced dose-related response was seen with cadmium which could be inhibited by various pretreatment procedures, such as incubation with zinc or certain metal chelators. The significance of these results in relation to the mechanism of toxic action of cadmium as well as to the potential use of this chemiluminescence technique is discussed.

A series of acute (96h) toxicity tests were conducted on six species of crayfish inthe family Cambaridae. Toxicity values fell into a sensitive group comprising Orconectesplacidus and Procambarus acutus (LC 50= 0.368mg Cd/L - 0.487mg Cd/L; LC10= 0.048mg Cd/L - 0.092 mg Cd/L) and tolerant group comprising O. juvenilis, O. virilis, P.alleni, and P. clarkii (LC 50= 2.44 mg Cd/L - 3.30 mg Cd/L; LC10= 0.386 mg Cd/L -0.947 mg Cd/L). For juvenile crayfish, the LC50 and LC10 values were as follows: O.juvenilis, 0.060 and 0.014 mg Cd/L; O. placidus, 0.037 and 0.002 mg Cd/L; P. clarkii,0.624 and 0.283 mg Cd/L. Cd exposure decreased molting success highlighting theimportance of this sensitive process. Behavioral responses were assessed in O. placidus,O. virilis, P. acutus, P. alleni, and P. clarkii. The tail-flip predator avoidance behaviorwas significantly reduced by cadmium exposure. In most species tested, the claw raisedefensive behavior was significantly increased by Cd exposure. Between species, as bodymass increased, the tail-flip response frequency decreased, and the claw-raise responseincreased in frequency. P. clarkii was also analyzed for the effect of Cd exposure onheart rate and response to two stimuli. The data indicate that heart rate may be a usefulphysiological marker of Cd toxicity. The major organ systems were dissected fromsurvivors of four adult crayfish toxicity tests (O. juvenilis, O. placidus, P. acutus, P.clarkii) and analyzed for metal content. Cadmium tissue content correlated with Cdexposure. Cadmium accumulated more in the hepatopancreas, gills and green glands ofsensitive species than in tolerant species. Zn showed negative correlations with Cdexposure in the hepatopancreas. Cu increased in green glands, gills, and hemolymph and,in some cases, decreased in the hepatopancreas. Ca, Fe, Mg, and Zn also showedsignificant trends. Zn accumulated in the exposure water over 24h. These data indicatethat Cd may displace Cu and/or Zn in the hepatopancreas and the displaced metal thenmay move into other tissues, especially the gills and green glands, possibly to beexcreted.

Some species of marine phytoplankton are believed to be more tolerant of high concentrations of trace metals than others, but no conclusive test of this hypothesis has been conducted. Eleven species of phytoplankton representing 5 classes were grown in Aquil medium containing Cd$ sp{2+}$ concentrations between 10$ sp{-9.85}$ and 10$ sp{-6.84}$ M. Growth rates and intracellular concentrations of Cd, C, N and S were measured. Cadmium quotas (mol Cd/litre-cell volume) were lower in members of Bacillariophyceae than in Chlorophyceae, Prymnesiophyceae, Dinophyceae and Cyanophyceae (ANOVA, p $<$ 0.001). Cellular C:S molar ratios decreased in phytoplankton grown at high (pCd 7.37-6.84) compared to low Cd (no added Cd), as S/litre-cell volume increased. Similar results were observed for C:N molar ratios. In two species that were examined, C:S ratios decreased as a linear function of increasing Cd concentration. Mean Cd$ sp{2+}$ concentration that reduced growth rate to 50% of maximum (pCd$ sp{50})$ was not significantly different among phytoplankton classes (ANOVA, p $<$ 0.05). When these experimental data were combined with pCd$ sp{50}$s calculated from published sources, Chlorophyceae were found to be the most resistant class (ANOVA, p $<$ 0.01). Cadmium and Cu resistance (pCd$ sp{50}$ and pCu$ sp{50})$ were correlated (r = 0.52, p $<$ 0.05), suggesting co-tolerance of phytoplankton to toxic levels of these metals. Chlorophyceae were most tolerant and Cyanophyceae the least tolerant of Cu (ANOVA, p $<$ 0.01). No significant differences were observed among Bacillariophyceae, Prymnesiophyceae, and Dinophyceae, which were of intermediate sensitivity to both metals. The results confirm the existence of a phylogenetic dependence of resistance to trace metal toxicity in phytoplankton.

No description available.

The ecotoxicological literature reveals that countless researchers worldwide rely upon informally identified commercial earthworm stocks for laboratory bioassays. The primary aim of this study was to investigate laboratory and commercial stocks of Eisenia species used in South Africa in order to confirm their taxonomy, assess their levels of genetic richness and differentiation. To do so, populations of potential Eisenia andrei and Eisenia fetida were purchased/obtained from vermiculturists and laboratories from four provinces of South Africa. DNA barcoding was used to investigate these taxonomic uncertainties. The COI gene was partially amplified and sequenced in selected earthworms from eight local populations (focal groups) and two European laboratory stocks (non-focal groups). Only nine COI haplotypes were identified from the 224 sequences generated. One of these haplotypes was found to belong to the Megascolecidae Perionyx excavatus. The remaining eight haplotypes belonged to the genus Eisenia although only a single Eisenia fetida haplotype, represented by six specimens, was found in one of the European populations. The other seven haplotypes, all occurring in South Africa, were Eisenia andrei. No Eisenia fetida was found in the South African based populations. One of the commercial stocks from South Africa and a laboratory culture from Europe were mixes of E. andrei - P. excavatus and E. andrei – E. fetida respectively. COI haplotype numbers were limited to two to three distinct sequences within each of the local groups. This translated into a haplotype diversity (H) lower than 0.45 in all the populations, which is very low when compared to other such earthworm studies in which COI polymorphism has been investigated. Of all the local populations investigated, only the lone field population included was genetically divergent from the other populations. This was explained by the haplotype distribution across the populations which indicated that this population was the only one not harbouring the haplotype which represented 75% or more of the COI sequences within the local populations. Because research suggests that earthworm populations with limited genetic diversity may suffer inbreeding depression which could affect traits such as reproduction and survival, the secondary aim was to test whether metal-sensitive earthworms were overly present in the populations investigated. To do so, the three most common COI haplotypes identified between the 8 local populations of E. andrei (called Hap1, Hap2 and Hap3) were paired up and exposed to cadmium. A total of six couples were exposed to 0, 25, 50 and 100 mg Cd/kg for 4 weeks at 20ºC. The survival, biomass variation, cocoon production and cocoon hatching success were assessed for all the couples. The results indicated that couple 6 (Hap3xHap3) was the most sensitive for three of the endpoints assessed whereas couple 4 (Hap1 x Hap3) was the least sensitive. Cocoon hatching success could not help differentiate the couples. The analysis of Cd tissue contents revealed that with increasing Cd concentration, Cp6 (Hap3xHap3) could accumulate significantly more Cd than any other couple (p ≤ 0.01). These findings indicate that earthworm populations may carry intrinsically metal-tolerant and metal-sensitive genotypes. In the context of ecotoxicological testing, the present results underline the importance of using genetically diverse populations in laboratory testing as Cp6 (Hap3xHap3) could have suffered from the deleterious effect of inbreeding. Because E. fetida could not be found in the local populations assessed, it is recommended that further earthworm DNA barcoding studies, covering a more representative geographical area of South Africa and including more field populations of Eisenia spp. be conducted. Because of the occurrence of genetic homogeneity in the populations studied, it is suggested that captive breeding initiatives be established using specimens obtained from several geographically distant field and reared populations. Further research investigating patterns of Cd accumulation/excretion kinetics between the Cd-tolerant and Cd-sensitive individuals reported in the present study, should be conducted to help determine whether inbreeding is the sole factor explaining the observed genotypic responses to Cd. Finally, the necessity of a standardised earthworm barcoding protocol that could help both to properly identify laboratory earthworm stocks and to select genetically diverse stocks suitable for laboratory testing, is discussed together with the relevance of the present work to ecotoxicological testing in general. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The ecotoxicological literature reveals that countless researchers worldwide rely upon informally identified commercial earthworm stocks for laboratory bioassays. The primary aim of this study was to investigate laboratory and commercial stocks of Eisenia species used in South Africa in order to confirm their taxonomy, assess their levels of genetic richness and differentiation. To do so, populations of potential Eisenia andrei and Eisenia fetida were purchased/obtained from vermiculturists and laboratories from four provinces of South Africa. DNA barcoding was used to investigate these taxonomic uncertainties. The COI gene was partially amplified and sequenced in selected earthworms from eight local populations (focal groups) and two European laboratory stocks (non-focal groups). Only nine COI haplotypes were identified from the 224 sequences generated. One of these haplotypes was found to belong to the Megascolecidae Perionyx excavatus. The remaining eight haplotypes belonged to the genus Eisenia although only a single Eisenia fetida haplotype, represented by six specimens, was found in one of the European populations. The other seven haplotypes, all occurring in South Africa, were Eisenia andrei. No Eisenia fetida was found in the South African based populations. One of the commercial stocks from South Africa and a laboratory culture from Europe were mixes of E. andrei - P. excavatus and E. andrei – E. fetida respectively. COI haplotype numbers were limited to two to three distinct sequences within each of the local groups. This translated into a haplotype diversity (H) lower than 0.45 in all the populations, which is very low when compared to other such earthworm studies in which COI polymorphism has been investigated. Of all the local populations investigated, only the lone field population included was genetically divergent from the other populations. This was explained by the haplotype distribution across the populations which indicated that this population was the only one not harbouring the haplotype which represented 75% or more of the COI sequences within the local populations. Because research suggests that earthworm populations with limited genetic diversity may suffer inbreeding depression which could affect traits such as reproduction and survival, the secondary aim was to test whether metal-sensitive earthworms were overly present in the populations investigated. To do so, the three most common COI haplotypes identified between the 8 local populations of E. andrei (called Hap1, Hap2 and Hap3) were paired up and exposed to cadmium. A total of six couples were exposed to 0, 25, 50 and 100 mg Cd/kg for 4 weeks at 20ºC. The survival, biomass variation, cocoon production and cocoon hatching success were assessed for all the couples. The results indicated that couple 6 (Hap3xHap3) was the most sensitive for three of the endpoints assessed whereas couple 4 (Hap1 x Hap3) was the least sensitive. Cocoon hatching success could not help differentiate the couples. The analysis of Cd tissue contents revealed that with increasing Cd concentration, Cp6 (Hap3xHap3) could accumulate significantly more Cd than any other couple (p ≤ 0.01). These findings indicate that earthworm populations may carry intrinsically metal-tolerant and metal-sensitive genotypes. In the context of ecotoxicological testing, the present results underline the importance of using genetically diverse populations in laboratory testing as Cp6 (Hap3xHap3) could have suffered from the deleterious effect of inbreeding. Because E. fetida could not be found in the local populations assessed, it is recommended that further earthworm DNA barcoding studies, covering a more representative geographical area of South Africa and including more field populations of Eisenia spp. be conducted. Because of the occurrence of genetic homogeneity in the populations studied, it is suggested that captive breeding initiatives be established using specimens obtained from several geographically distant field and reared populations. Further research investigating patterns of Cd accumulation/excretion kinetics between the Cd-tolerant and Cd-sensitive individuals reported in the present study, should be conducted to help determine whether inbreeding is the sole factor explaining the observed genotypic responses to Cd. Finally, the necessity of a standardised earthworm barcoding protocol that could help both to properly identify laboratory earthworm stocks and to select genetically diverse stocks suitable for laboratory testing, is discussed together with the relevance of the present work to ecotoxicological testing in general. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Made available in DSpace on 2016-06-02T19:29:38Z (GMT). No. of bitstreams: 1 3822.pdf: 3918575 bytes, checksum: d646b2b0a3b661d4dfa2d3c8a1ee8a69 (MD5) Previous issue date: 2011-03-28 / Universidade Federal de Sao Carlos / The Alto Jacaré-Guaçu Basin was evaluated regarding limnological and ecotoxicological aspects in four water and sediment sampling campaigns at 14 points distributed in the area. Toxicity tests with water, in the laboratory and in situ, showed chronic toxicity, with effects on the survival, growth and gill morphology of juveniles of both fish species examined (Danio rerio and Poecilia reticulata). The lowest survival percentages in the laboratory assays were observed for point L, and in the field tests for point J1. The analysis of the gills revealed first-stage alterations in the specimens of both species exposed at all the sample points, and there were second-stage alterations (aneurism and vascular congestion) at various points. The high concentrations of nutrients and metals in the basin s water and sediments reveal a system impacted by human activities in the surrounding region. The analyses of metals quantified Cd, Cr, Cu, Mn and Fe levels in the water above those permitted by the Brazilian National Environmental Council (CONAMA 357/05), and of Cd in the sediments above the threshold levels permitted both by national (CONAMA 344/04) and international regulations (CCME, 2003). Simple synthetic sediments were prepared composed of sand, clay and organic matter (obtained from the aquatic macrophyte Scirpus cubensis, at concentrations of 0, 0.25, 0.5 and 1%) to assess the toxicity of Cd. The synthetic sediments were viable for the growth and survival of Ceriodaphnia silvestrii, Chironomus xanthus and Danio rerio, and were suitable for use in spiked experiments. The evaluation of the acute toxicity of the sediments fortified with Cd indicated mean 48-h;EC50 for C. silvestrii of 3.09mg.Kg-1 in the sediments with 0% OM and 2.98mg.Kg-1 in the sediments with 1% OM. The mean 96-h;LC50 for C. xanthus was 18.67 mg.Kg-1 in the sediments with 0% OM and nearly four times lower in those with 1% OM (4.93 mg.Kg-1). A similar pattern was noted for D. rerio, a higher 48-h;LC50 in the sediments with 0% OM (114.88 mg.Kg-1) than in the sediments with 1% OM (75.31 mg.Kg-1). In subsequent experiments, the sediments were also spiked with 0.6mg.Kg-1 of Cd, a concentration considered safe for protection of aquatic life according to the applicable national and international regulations. In these tests, the survival of C. silvestrii was impaired in the Cd spiked sediments, so it was not possible to assess the effects on reproduction. There were also effects on the survival, biomass, mentum morphology and Cd concentration in the tissues of C. xanthus. For D. rerio, the survival and growth were compromised in the sediments with higher percentage of organic matter. Besides this, the organisms showed impaired gill morphology and higher Cd concentration in tissue in function of exposure to sediments spiked with the metal. Based on the results, it can be concluded that C. silvestrii was the most sensitive species to the sediments fortified with Cd, followed by C. xanthus and D. rerio, the least sensitive. These effects on the iv organisms at concentrations considered safe for the biota suggest more conservative thresholds of this metal should be established for tropical aquatic habitats. / A bacia do Alto Jacaré-Guaçu foi avaliada quanto aos aspectos limnológicos e ecotoxicológicos em quatro campanhas de amostragem de água e sedimento em 14 pontos distribuídos na área. Testes de toxicidade com água, tanto em laboratório como in situ, apresentaram toxicidade crônica com efeitos na sobrevivência, crescimento e morfologia de brânquias de juvenis de ambas as espécies de peixes avaliadas (Danio rerio e Poecilia reticulata). As menores porcentagens de sobrevivência nos testes em laboratório foram observadas no ponto L e, nos testes in situ, em J1. A análise das brânquias mostrou alterações de primeiro estágio nos organismos expostos em todos os pontos avaliados, e em diversos pontos foram verificadas alterações de segundo estágio (aneurisma e congestão vascular). As elevadas concentrações de nutrientes e metais na água e sedimentos da Bacia do Alto Jacaré-Guaçu revelam um sistema impactado pelas atividades humanas que se desenvolvem em seu entorno. As análises de metais quantificaram Cd, Cr, Cu, Mn e Fe acima da CONAMA 357/05 na água e Cd acima dos valores estabelecidos pela legislação nacional (CONAMA 344/04) e internacional (CCME, 2003) nos sedimentos. Foram desenvolvidos sedimentos sintéticos simples compostos de areia, argila e matéria orgânica (obtida a partir da macrófita aquática Scirpus cubensis, nas concentrações de 0; 0,25; 0,5 e 1%) para avaliar a toxicidade do Cd. Os sedimentos sintéticos se apresentaram viáveis para o crescimento e sobrevivência de Ceriodaphnia silvestrii, Chironomus xanthus e Danio rerio, sendo adequados para uso em experimentos de fortificação. A avaliação da toxicidade aguda dos sedimentos fortificados com Cd, indicaram CE50;48h média para C. silvestrii de 3,09mg.Kg-1 nos sedimentos sem MO e 2,98mg.Kg-1 nos sedimentos com 1% de MO. A CL50;96h média para C. xanthus foi de 18,67mg.Kg-1 nos sedimentos sem MO e, cerca de 4 vezes menor nos sedimentos com 1% de MO, 4,93mg.Kg-1. Para D. rerio também foi notada tendência semelhante, CL50;48h maior nos sedimentos sem MO, 114,88mg.Kg-1 e menor nos sedimentos com 1% de MO, 75,31mg.Kg-1. Os sedimentos também foram fortificados com 0,6mg.Kg-1 de Cd, concentração considerada segura para a proteção da vida aquática segundo legislações nacionais e internacionais, em experimentos posteriores. C. silvestrii teve a sobrevivência comprometida nos sedimentos fortificados com Cd, de modo que não foi possível avaliar os efeitos na reprodução. C. xanthus também apresentou efeitos na sobrevivência, biomassa, morfologia de mento e concentração de Cd nos tecidos. D. rerio teve a sobrevivência e crescimento comprometidos nos sedimentos com maior porcentagem de matéria orgânica. Além disso, os organismos apresentaram a morfologia de brânquias comprometida e concentração de Cd em ii seus tecidos elevada em função da exposição aos sedimentos fortificados com o metal. Com base nos resultados foi possível concluir que C. silvestrii foi a espécie mais sensível aos sedimentos fortificados com Cd, seguido de C. xanthus e, a menos sensível, D. rerio. Tais efeitos observados nos organismos em concentrações consideradas seguras para a biota sugerem a avaliação mais cuidadosa dos valores para espécies e climas tropicais.