Dissertations / Theses on the topic 'Marine bacteria – Effect of water pollution on'

Cu is one of the most toxic elements that affect fish populations when the fish are exposed to concentrations exceeding their tolerance. To investigate the effects of elementary Cu on aspects of bioconcentration, histology and behaviour, O. mossambicus were exposed to 0 and 0.75 ± 0.20 mg/l of Cu for 96 hours (short-term study), and 0, 0.11 ± 0.02, 0.29 ± 0.02, and 0.47 ± 0.04 mg/l of Cu for 64 days (longterm study) under controlled conditions in the laboratory. For the long-term study fish were sampled for gills, liver, and kidney Cu accumulation analysis after 1, 32 and 64 days of exposure and after 1, 2, 4, 16, 32, and 64 days for gills, liver and spleen histology analysis. Cu accumulation was concentration-duration dependent with the highest accumulation capacity in the liver. A multifactor linear model was developed for the relationship between exposure dose, exposure duration and Cu accumulation in the organs with the liver model: Log L = 3.35 + 0.85W + 0.31T (r² = 0.892) giving a better fit than the gills: G = −35.09 + 10.58W + 17.58T (r² = 0.632). Where L = Cu accumulation values in the liver, G = Cu accumulation values in the gills (both in μg/g dry mass); W = exposure dose in water (mg/l); and T = exposure time (days). Using this model Cu accumulation in organs can be estimated when exposure concentration and duration is known. This model should be tested under different conditions to determine the potential of the model in monitoring Cu toxicity in the environment. Lesions were observed in the liver, gills and spleen in all Cu treatments at all exposure concentration and exposure durations. However, the incidence and the degree of alteration was related to the concentration of Cu and duration of exposure. The sequential appearance of lesions in the order of, hepatic vacuolar degeneration, fatty degeneration and necrosis indicated a gradual increase in liver damage with larger duration of exposure time and increasing Cu concentration. The initial lesions in the gills were manifested as hypertrophy and hyperplasia of the gill epithelium causing increase in the thickness of the secondary lamellae, mucous cell hypertrophy and proliferation, mucous hypersecretion, proliferation of eosinophilic granule cells and hyperplasia of interlamellar cells. With increase in exposure time, necrosis of the eosinophilic granule cells, lamellar oedema, epithelial desquamation and increase in severity of lamellar hyperplasia were observed. These lesions indicated an initial defence mechanism of the fish against Cu toxicity followed by advanced histological changes that were related to Cu concentration and duration of exposure. Changes in the spleen were haemosiderosis, increase in the white pulp and macrophage centres, reduction in the red pulp, and necrosis suggesting that fish exposed to environmentally relevant levels of Cu may be histopathologically altered leading to anaemia and immunosuppression. Regression analysis was used to quantify the relationship between the total activity of the fish, and duration of exposure. There was a gradual decline in fish activity related to Cu concentration and duration of exposure before introducing food into the tanks. There was a constant activity after introducing food in the tanks at the control and 0.11 ± 0.02 mg/l Cu exposure levels irrespective of exposure time. Analysis of covariance (ANCOVA) was used to test for the difference in slopes between treatments. There was no significant difference (p > 0.05) between slopes of the control and 0.11 ± 0.02 mg/l Cu, and between 0.29 ± 0.02 and 47 ± 0.04 mg/l Cu before and after introducing food in the tanks. The slopes of both the control and 0.11 ± 0.02 mg/l Cu were significantly different from those of 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu (p < 0.05). There were significant differences in the mean opercular movements per minute between treatments (p < 0.05). There was hyperventilation at 0.11 ± 0.02 mg/l Cu i.e. 87 ± 18 opercular movements per minute (mean ± standard deviation) and hypoventilation at 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu i.e. 37 ± 34 and 13 ± 6 opercular movements per minute compared to the control. Hypo- and hyperventilation were related to the lesser and greater gill damage, respectively. In conclusion Cu accumulation and effects on histology of the liver, gills and were related to the concentration of Cu in the water and duration of exposure showing a gradual increase in incidence and intensity with larger duration of exposure time and increasing Cu concentration. The fish were initially able to homeostatically regulate and detoxify Cu. However, as the exposure continued, the homeostatic mechanism appears to have failed to cope with the increasing metal burden causing advanced histological changes.

Organochlorines are ubiquitous pollutants of the marine environment. These lipid-soluble and highly persistent compounds are found in detectable amounts in almost all marine organisms, and accumulate in the lipid tissues of marine animals. This bioaccumulation leads to biomagnification of these contaminants in higher trophic levels. Near the top of many marine food chains are found the marine mammals, in whose blubber high levels of organochlorine residues have been measured. The most commonly occurring of these pollutants in these animals are the polychlorinated biphenyls (PCB's) and p,p-DDE, a metabolite of the insecticide DDT. These substances have been shown to cause disruptions in the endocrine, immune, and reproductive systems, and are passed from mother to offspring through the placenta and by lactation. Presence and levels of residues of these compounds are, therefore, monitored in marine mammals to provide an indication of the health of a given population and the environment in which they live. Such monitoring is generally done with the use of gas chromatography (GC). High performance liquid chromatography (HPLC) is little used due to the poor ultraviolet (UV) absorbance properties of many of the organochlorines. PCB's and p,p-DDE do absorb UV well enough at concentrations usually encountered in marine mammals to permit the use of HPLC for detection and semiquantification of these substances. A method was developed for the screening of blubber of marine mammals for total PCB's and p,p-DDE using HPLC. The method was applied to the detection and approximation of levels of these two organochlorines in marine mammals from the east and west coasts of the United States. Geographical differences in levels of the two pollutants were found, indicating differences in primary feeding ranges. Evidence of placental transfer of these two organochlorines was also found. Especially high residue levels were found in the blubber of stranded killer whales, indicating that acquisition of high pollutant burdens is still a problem in these top predators. It was concluded that HPLC can be used to screen marine mammals for PCB's and p,p-DDE, and that residue levels determined can be useful in investigating species range, pollutant burdens, and health of populations.

Growth of mussels under laboratory conditions was examined under various food regimes in different water types and temperatures. Growth was less than would be useful as an indicator and comparisons with field exposures were of minimal value. The effects of organophosphates on bivalves were examined via toxicity tests, tissue concentration, and by controlling exposure through the use of physical constraints. Upper temperature tolerance of both bivalve species was examined with respect to different acclimation temperatures and organophosphate exposures. Deviations from control exposures occurred at some temperatures. Copper effectively lowered the mean heat coma temperatures of C. fluminea at some concentrations, however, chlorine exposures did not alter heat coma temperature.

The decrease of biological diversity from low latitudes near the equator towards high latitudes is one of the most fundamental patterns noted in ecology. These trends have been the subject of numerous terrestrial and marine studies and have caused the proposal of several explanatory hypotheses. No single hypothesis has adequately accounted for these observed trends. Furthermore, large-scale patterns are frequently modified by processes acting at meso- to small-scales. It is imperative to understand the interaction of these processes to elucidate the mechanisms controlling the structure of intertidal assemblages. The main aim of this thesis was to test the influence of multiple processes at a range of spatial scales on biogenic engineers. Biogenic engineers, such as macroalgae, have been reported to be strongly influenced by processes such as grazing, biogeography and upwelling and subsequent effects are expected to be linked to their associated assemblages. I used infaunal assemblages associated with macroalgae as a model system to understand the interactive effects of meso-scale upwelling in conjunction with large-scale factors (regional and biogeographic). Three species of macroalgae, Hypnea spicifera, Champia lumbricalis and Bifurcaria brassicaeformis, inhabiting different but overlapping home ranges were used in this study. Smaller scale, physical attributes and the associated fauna of these algal species were compared. The effects of meso- to large-scale physical gradients on marine organisms have been investigated in many instances and resultant gradients in physical variables observed. This study was undertaken to investigate morphological trends exhibited by Hypnea spicifera and Champia lumbricalis to compare possible similarities between coastlines and to determine if inferred meso- to large-scale processes influence physical trends in a similar manner despite the coastlines being affected by different hydrodynamic processes. The macroalgae were located in two distinctly different upwelling regimes, Hypnea spicifera on the east and south coasts and Champia lumbricalis on the west coast. Upwelling (delivery of nutrients and primary production) was not found to be an important factor in determining the physical characteristics of macroalgae but there were clear patterns linked to latitude. Mean percentage cover of Hypnea spicifera was positively correlated with distance along the east and south coasts. Mean mass and mean surface area of H. spicifera were, however, negatively correlated with distance from Port St Johns. Mean frond length showed a negative trend but this relationship was not statistically significant. On the west coast, there was a significant increase in mean frond length from north to south for Champia lumbricalis. Mean surface area and mean mass of C. lumbricalis exhibited non-significant negative trends from north to south while there was a non-significant positive trend for mean percentage cover. Following the “Productivity Hypothesis”, these patterns from north to south along the coastlines of South Africa could have important implications for biodiversity associated with these algae. Many previous studies have focussed on the effects of upwelling on species (i.e. effects of nutrients and temperature) but have failed to separate this effect from large scale effects such as biogeography and latitudinal gradients. I tested the influence meso-scale upwelling, large-scale biogeographic processes and latitudinal gradients with two different species of macroalgae one on the east and south coasts of South Africa and the other on the west coast. Hypnea spicifera inhabits the east and south coasts of South Africa spanning two biogeographic provinces and is generally affected by relatively weak upwelling, whereas Champia lumbricalis inhabits the west coast spanning one biogeographic province which is subjected to intense persistent upwelling year round. Within the east and west coast biogeographic provinces there are, however, regions with both upwelling and non-upwelling. On the east and south coasts, entire assemblages differed significantly among the three Regions (St Lucia, Port Alfred and Knysna, while assemblages between upwelling/non-upwelling areas were not different. Assemblages on the different shores differed significantly from each other. There were no significant effects of region or upwelling for the number of individuals of Crustacea or Polychaeta, while Mollusca showed a significant effect of region. The number of individuals of other taxa showed a significant interaction of region and upwelling. Region had a significant effect on number of species of molluscs and other taxa, while there was no effect for either region or upwelling for the Crustacea or Polychaeta. In general different factors were shown to be important (region and shore) while upwelling was rarely important. On the west coast assemblages associated with C. lumbricalis were not influenced by region or upwelling but there were significant differences between shores. Region, upwelling ad shore did not affect number of individuals. There was a significant interaction of region and upwelling for the number of species of crustaceans, while numbers of species of Mollusca, Polychaeta and other taxa showed no effects. In general, regional factors strongly influenced most organisms, while upwelling played a minor role. Hypotheses about differences between upwelling and non-upwelling regions in terms of species abundances and composition due to the input of cold nutrient rich water were not supported. A link between the effects of larger-scale biogeographic factors and their influence on habitat forming taxa and the resultant effects on associated infauna would be a likely explanation for the patterns observed in this study. Diversity of assemblages is known to be influenced by the structural complexity of a habitat, increasing complexity increases the amount of available niches therefore potentially increasing the number of species found within that habitat. Three species of macroalgae, Hypnea spicifera, Champia lumbricalis and Bifurcaria brassicaeformis were selected on the basis of being structurally similar and having their distributional ranges overlap in order to elucidate the effects of structure and macroalgal species on associated assemblages. Hypnea spicifera is different in terms of surface area and biomass from Champia lumbricalis and Bifurcaria brassicaeformis. Assemblages of species found on the three rhodophytes showed both shore and the species of alga had significant effects on composition. Only numbers of individuals of molluscs were affected by the species of alga. Crustacea and Polychaeta showed an interaction of the species of alga with shore. In the case of this study, it is likely that the three species of macroalgae mitigate biological stressors such as predation and physical stressors such as wave exposure and desiccation. In general, meso-scale upwelling is suggested to be marginalised when considering the structuring of assemblages associated with macroalgae, while large scale biogeography has more of an influence. Within shores, assemblages were also strongly influenced by smaller-scale factors such as differences in the structure and species of alga. This study indicates the importance of investigating patterns across a range of spatial scales to gain a comprehensive understanding of factors influencing intertidal organisms.

Thesis (Ph. D.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2000.<br>Includes bibliographical references (p. 207-219).<br>Processes affecting organic carbon distribution and composition can control the speciation of organic contaminants such as polychlorinated biphenyls (PCBs) and ultimately determine their residence time in a particular environment. In marine systems, the microbial loop influences organic carbon dynamics by recycling a significant fraction of dissolved and particulate organic matter. The goal of this thesis was to understand how these recycling processes affect chlorobiphenyl (CB) cycling in marine systems by monitoring CB dynamics among organic carbon pools represented by dissolved organic matter, bacterial prey and phagotrophic protozoan grazers. Initially, I studied the extent to which a protozoan grazer (Uronema sp.-10[micro]m ciliate) equilibrated with aqueous PCBs within 2-3 hours. Initial calculations predicted rapid equilibration via passive diffusion. Experimentally, no difference in equilibration time was noted between grazing and non-grazing protozoa, indicating that diffusion was the primary uptake pathway for these organisms. The results were extended to determine the transition size of an organism where the rates of diffusive and ingested uptake are equivalent (100-500[micro]m). Disassociation rate constants were estimated for complexes of CB congeners and dissolved organic carbon (DOC). CB-DOC complexes enhanced the diffusive uptake rate constant for Tenax resin and, by inference, protozoan grazers. In the second phase of this work, concentrations of surfactants, organic carbon and cells were monitored over time in protozoan cultures. The effects of bacterial growth substrate and protozoan species were examined. Surfactants increased during protozoan exponential growth while total DOC concentrations decreased. Production of<br>(cont.) surface-active material in ciliate cultures was significantly higher than in flagellate cultures, and all protozoan cultures were higher than the bacterial control. Common headspace vessels were then used to compare and contrast the affinity of protozoan and bacterial culture filtrates (<0.2[micro]m) for PCBs relative to a seawater control. Affinities were normalized to bulk DOC and surfactant concentrations to determine underlying relationships among these parameters. Values of equilibrium partition coefficients (K[oc]) ranged from 10⁴·⁶ in Vineyard Sound seawater to 10⁵·⁴ and 10⁵·⁵ in protist cultures, indicating that "grazer-enhanced" DOM was a better sorbent for PCBs than DOM in bacterial controls and Vineyard Sound seawater.<br>by Elizabeth Belle Kujawinski.<br>Ph.D.

Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment

Contaminants such as pharmaceuticals are of increasing concern due to their ubiquitous use and persistence in surface waters worldwide. Limited attention has been paid to the effects of pharmaceuticals on marine life, despite widespread detection of these contaminants in the marine environment. Of the existing studies, the majority assess the negative effects of pharmaceuticals over an exposure period of 30 days or less and focus on cellular and subcellular biomarkers. Longer studies are required to determine if chronic contaminant exposure poses risks to marine life at environmentally relevant concentrations. Also scarce in the literature is examination of whole organism effects to identify potential community-level consequences. Two long-term studies with the antidepressant pharmaceutical, fluoxetine (the active constituent in Prozac®) were conducted to determine whether nominal concentrations detected in estuarine and coastal environments affect organism health and interactions. First, we measured whole organism metrics in the California mussel, Mytilus californianus over a period of 107 days. Specifically, we measured algal clearance rates, growth, and condition indices for both reproductive and overall health. We found that fluoxetine negatively affects all measured characteristics, however many effects are mediated by length of exposure. Perhaps the most notable result was that mussels spiked with fluoxetine cleared less algae after 30 days of exposure. Reduced growth and condition indices likely are a consequence of improper nutrition among fluoxetine-treated mussels. Any level of fluoxetine significantly affected the gonadosomatic index after 47 days. The results from this study on mussels fill an important data gap, highlighting organism-level effects of chronic exposure periods; such data more explicitly identify the impacts of pharmaceuticals and other contaminants on marine communities and ecosystems. Fluoxetine has also been documented to affect the behavior of fish and invertebrates, including freshwater and marine bivalves, crustaceans, and fish. Given that other crustaceans exhibited increased activity levels under fluoxetine exposure, we hypothesized that this would subject them to greater predation risk. In our second exposure study, we assessed whether a similar range of fluoxetine concentrations used in the mussel study altered the risk behavior of the Oregon mud crab, Hemigrapsus oregonensis, in response to a common predator, the red rock crab, Cancer productus. We conducted this study for 60 days, conducting day and night behavioral trials (with and without predators) four times a week. We found that crabs exposed to any amount of fluoxetine (3 or 30 ng/L) had increased activity levels relative to controls; however behaviors of 3 ng/L-spiked crabs were not always significantly different from controls. Among control crabs, day and night trials yielded similar results, where a clear response to the addition of the predator was observed. Crabs dosed with fluoxetine exhibited more foraging and active behaviors in the presence of the predator. Additionally, crabs spiked with fluoxetine at 30 ng/L had the greatest risk of mortality either by predation by red rock crabs or due to more aggressive behaviors among conspecifics. The results of this study shed light on a particularly unexplored area of contaminants research: how do psychoactive pharmaceuticals affect animal behavior when exposed to the low concentrations persisting in the aquatic environment for a prolonged period of time?

Substantial hunting pressure and habitat destruction caused by oil extraction has critically endangered the Amazonian manatee in Ecuador. The current population status is unknown because an effective method to observe them in the wild has yet to be developed. This study explored whether the Amazonian manatee persists or has been extirpated in the eastern Ecuadorian Amazon utilizing side-scan sonar to increase odds of detection. Spatial differences in probability of detection were quantified if manatees were observed. The level of chemical contamination was determined and compared spatially and temporally against historical data. Data were collected using opportunistic transect surveys and grab sampling of surface water in Yasuni National Park, Lagartococha, and Cuyabeno Wildlife Reserve. Surveys confirmed that the manatee population is extant. Manatees were encountered more often in Cuyabeno Wildlife Reserve than in Lagartococha and Yasuni. Side-scan sonar detected more manatees than previously reported in 1996-1999. Side-scan sonar is a viable method for detection of manatees in the Ecuadorian Amazon system and resulted in greater detection as a function of effort. All future population studies should incorporate side-scan sonar. Lead, arsenic, mercury, polynuclear aromatic hydrocarbons [PAHs], and volatile organic compounds [VOCs] were not detected in the waters of the study region. High total petroleum hydrocarbon [TPH] levels were measured in 7 samples from Yasuni National Park. The concentrations of TPH were higher in Yasuni National Park than in Lagartococha and Cuyabeno. TPHs were detected only in the study region with a recent oil spill; there was no evidence that TPHs were higher near oil production wells and pipelines. The concentrations of TPH were significantly different than those measured in 1998 (z =3.01710, p=0.0026). A dedicated study should be performed to develop a protocol for monitoring persistent oil contaminants in the Ecuadorian Amazon and determine their sink.

Chan Kwan-ling.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 91-105).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>Acknowledgements --- p.v<br>Contents --- p.vi<br>List of figures and tables --- p.ix<br>Chapter Chapter 1 --- Introduction<br>Chapter 1.1 --- Literature review --- p.1<br>Chapter 1.1.1 --- The effect of pollutants on the genetic materials of aquatic organisms --- p.1<br>Chapter 1.1.1.1 --- Response of individual to genotoxicants --- p.1<br>Chapter 1.1.1.2 --- Effects of genotoxicants on population structure --- p.3<br>Chapter 1.1.2 --- Application of genetic markers in monitoring water pollution --- p.3<br>Chapter 1.1.2.1 --- DNA adduct --- p.4<br>Chapter 1.1.2.2 --- Sister chromatid exchange (SCE) test --- p.5<br>Chapter 1.1.2.3 --- Micronucleus --- p.6<br>Chapter 1.1.2.4 --- DNA strand breaks --- p.7<br>Chapter 1.1.3 --- Single-cell gel electrophoresis (comet) assay --- p.9<br>Chapter 1.1.4 --- Test organisms for comet assay --- p.12<br>Chapter 1.2 --- Objective of the present study --- p.13<br>Chapter Chapter 2 --- Genotoxicity of pollutants on Hyale crassicornis<br>Chapter 2.1 --- Introduction --- p.22<br>Chapter 2.2 --- Materials and methods --- p.24<br>Chapter 2.2.1 --- Sampling of amphipods --- p.24<br>Chapter 2.2.2 --- Acclimation --- p.24<br>Chapter 2.2.3 --- Acute toxicity test --- p.26<br>Chapter 2.2.4 --- The effect of test duration on DNA damage --- p.27<br>Chapter 2.2.5 --- Effect of toxicants on DNA damage --- p.28<br>Chapter 2.2.6 --- Comet assay --- p.29<br>Chapter 2.2.7 --- Chemicals --- p.34<br>Chapter 2.2.8 --- Data analysis --- p.34<br>Chapter 2.3 --- Results --- p.34<br>Chapter 2.4 --- Discussion --- p.47<br>Chapter Chapter 3 --- Genotoxicity of hydrogen peroxide on different tissue types of Metapenaeus ensis<br>Chapter 3.1 --- Introduction --- p.54<br>Chapter 3.2 --- Materials and Methods --- p.57<br>Chapter 3.2.1 --- Collection and acclimation of shrimps --- p.57<br>Chapter 3.2.2 --- Incubation --- p.59<br>Chapter 3.2.3 --- Comet Assay --- p.60<br>Chapter 3.2.4 --- Chemicals --- p.61<br>Chapter 3.2.5 --- Data analysis --- p.61<br>Chapter 3.3 --- Results --- p.61<br>Chapter 3.4 --- Discussion --- p.67<br>Chapter Chapter 4 --- Genotoxicity of wastewater on Hyale crassicornis<br>Chapter 4.1 --- Introduction --- p.71<br>Chapter 4.2 --- Materials and Methods --- p.72<br>Chapter 4.2.1 --- Collection of wastewater samples --- p.72<br>Chapter 4.2.2 --- Metal content analysis --- p.73<br>Chapter 4.2.3 --- Genotoxic effect of wastewater samples on Hyale crassicornis --- p.74<br>Chapter 4.2.4 --- Chemicals --- p.76<br>Chapter 4.2.5 --- Statistical analysis --- p.77<br>Chapter 4.3 --- Results --- p.77<br>Chapter 4.3.1 --- Metals content in water samples --- p.77<br>Chapter 4.3.2 --- DNA damage --- p.79<br>Chapter 4.4 --- Discussion --- p.79<br>Chapter Chapter 5 --- Conclusions --- p.89<br>References --- p.91