Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Tesis presentada a la Universidad de Chile para optar al grado de Doctor en Ciencias Farmacéuticas / La planta Matico, Buddleja globosa Hope, Budlejaceae es una especie nativa en Chile de la cual se usan sus hojas por sus múltiples propiedades terapéuticas. Las investigaciones más recientes han permitido demostrar que el extracto etanólico (EMATst) obtenido a partir de sus hojas y estandarizado en su principal componente (verbascósido) presentó efectos antiinflamatorio, analgésico y cicatrizante. Este compuesto ha sido identificado en distintas especies vegetales y se le ha demostrado mediante ensayos in vitro efecto estrogénico y antiestrogénico dependiente de la concentración. En esta tesis se estudió la propiedad estrogénica y el posible efecto disruptor endocrino (DE) del EMATst y del verbascósido usando distintos modelos experimentales tanto in vivo como in vitro. Esta investigación se inició con la recolección de las hojas de matico en el mes de enero de 2008, en el campus Antumapu de la Universidad de Chile, con el objetivo de obtener el EMATst que fue elaborado por extracción sucesiva del material vegetal con solventes de polaridad creciente (hexano, diclorometano y etanol). La cuantificación del componente mayoritario (verbascósido) del extracto etanólico se realizó por CLAE-DAD y se expresó en ácido cafeíco. Posteriormente a partir del EMATst por sucesivas columnas cromatográficas de silicagel 60 y Sephadex LH20, se aisló el verbascósido. Tanto EMATst como verbascósido fueron utilizados para realizar los trabajos experimentales con el fin de determinar la alteración del ciclo estral de ratas de laboratorio despu es de la administración subcutánea de dos diferentes dosis de EMATst; posible unión competitiva de EMATst y verbacósido al receptor estrogénico (RE) mediante un ensayo in vitro usando citosol de útero de rata como fuente de RE; toxicidad aguda de Daphnia magna mediante la determinación de la concentración letal 50 (CL50) a las 24 h de exposición; toxicidad crónica y efecto DE mediante la determinación de la sobrevivencia, frecuencia de muda y reproducción de dos generaciones a los 14 días de exposición. Nuestros resultados permitieron demostrar que el EMATst en dosis alta (10-5 M) provocó una alteración en el ciclo estral de ratas, lo que podrá atribuirse a un efecto antiestrogénico. Tanto EMATst como verbascósido se unieron levemente de modo competitivo al RE comparado con estradiol. Ambos presentaron una baja toxicidad aguda relativa respecto del t oxico de referencia (dicromato de potasio) y 17 ß-estradiol en D. magna. Los resultados de toxicidad crónica en D. magna, muestran que se produce una disminución de la reproducción en ambas generaciones dependiente de la concentración y más marcada aún en la segunda generación y el efecto DE de ambas muestras se manifestó porque se alteró la reproducción / Matico plant, Buddleja globosa Hope, Budlejaceae is a native species in Chile which used their leaves of its multiples therapeutic properties. More recent investigations have demonstrated that the ethanol extract (EMATst) obtained from leaves and with an standardized main component (verbascoside) presented anti-in ammatory, analgesic and scarring e ects. This compound has been identi ed in di erent plant species and has shown through in vitro assays concentration dependent estrogenic and antiestrogenic e ects. In this thesis we studied the estrogenic property and possible endocrine disruptor (ED) e ect of EMATst and verbascoside using di erent experimental models both in vivo and in vitro. This investigation began with collecting matico leaves in the month of January 2008, in the Antumapu campus of the University of Chile, in order to obtain EMATst which was prepared by the successive extraction of plant material with solvents of increasing polarity (hexane, dichloromethane and ethanol). Quanti cation of the major component (verbascoside) of the ethanol extract was performed by HPLC-DAD and expressed as ca eic acid. Subsequently, verbascoside was isolated from EMATst by successive silica gel 60 and Sephadex LH20 chromatographic columns. Both EMATst and verbascoside were used to perform experimental work to determine the alteration of the estrous cycle of rats after subcutaneous administration of two di erent doses of EMATst; verbacoside and EMATst possible competitive binding to the estrogen receptor (ER) though an in vitro assay using the cytosolic fraction of a rat uterus as ER source; acute toxicity of Daphnia magna by determining the lethal concentration 50 (LC50) after 24 hours of exposure; chronic toxicity and DE e ect by determining the survival, molt frequency and reproduction of two generations at 14 days of exposure. Our results show that the EMATst at a high-dose (10-5 M) caused an alteration in

L'objectif général de ce travail est l étude de la toxicité chronique du chlordane chez un crustacé d eau douce afin d évaluer le risque de ce pesticide sur les organismes aquatiques. Pour ce faire, nous avons choisi le cladocère Daphnia magna comme modèle d étude, à cause de son rôle clé dans la chaîne trophique, sa sensibilité et son cycle de vie qui est mis à profit pour étendre le test de reproduction classique utilisant une seule génération saine sur deux générations successives. Le test chronique (21j) a donné une concentration sans effet observée (NOEC) sur tous les paramètres de 0,18 g/L qui constitue une donnée importante dans l évaluation du risque du chlordane pour le milieu aquatique. L exposition de D.magna aux fortes concentrations de chlordane a induit des malformations et l apparition de mâles chez la descendance. L étude de la bioaccumulation a montré le fort potentiel de bioaccumulation chez D.magna avec un facteur de bioaccumulation de 244000 (poids sec) après 40 jours d exposition à la NOEC. L étude de la toxicité du chlordane sur deux générations successives (F0 et F1) a montré que celle-ci augmente avec la concentration et le temps d exposition. Ainsi, la génération F1 est plus sensible que la génération F0 et la population de la génération F1 issue de la 5ème ponte s est montrée plus sensible que la génération F1 issue de la 2ème ponte. L étude de la capacité de récupération des daphnies de la génération F1, après leur transfert dans un milieu propre a montré l absence d une récupération totale aux plus fortes concentrations testées. La comparaison de la sensibilité de D.magna à un autre crustacé C.dubia suite à une exposition chronique au chlordane a souligné l intérêt de l utilisation de ce dernier dans l étude de la toxicité chronique des polluants persistants à condition de prolonger la durée du test de 7 à 14 jours. L ensemble de ces résultats souligne l intérêt du modèle D.magna dans l étude de la toxicité chronique des polluants persistants et de leurs éventuels effets sur les différents processus de reproduction. / This research aimed at studying the toxicity of chlordane in freshwater crustacean in order to evaluate the risk of this pesticide on aquatic organisms. We choice Daphnia magna as model of study, because her key role in trophic chain, her sensitivity and her short life cycle that are exploited for extended the classic reproduction test using only one healthy generation on two successive generations. In the chronic test (21d), a concentration without observed effect (NOEC) on all parameters of 0.18 g/L was obtained. The exposition of D.magna to the strong concentrations of chlordane led the malformations and the apparition of males at the progeny. The study of the bioacumulation showed the high potential of bioaccumulation at D.magna with a factor of bioaccumulation of 244 000 (dry weight) after 40 days of exposition to the NOEC. The study of the toxicity of the chlordane on two successive generations (F0 and F1) showed that this one increases with the concentration and the time of exposition. So, the F1 generation is more sensitive than the F0 generation and the population of the F1 generation 5th brood is more sensitive than the one of the F1 generation 2nd brood. The study of the recovery of the daphnids after their transfer in clean water showed their capacity to restore reproduction only for the low doses. The comparison of the sensitivity of D.magna in another crustacean C.dubia following a chronic exposition to chlordane underlined the interest of the use of C.dubia in the study of the log term toxicity of the persistent pollutants under the condition to prolong the duration of the test from 7 to 14 days.The present work highlights the interest of our model in the study of the chronic toxicity of persistent pollutants and their possible effects on the different processes of reproduction

Lead is an omnipresent pollutant, and its contamination in natural waters is an issue of current regulatory concern throughout the world including Canada. The free divalent ion of lead (Pb2+) is considered to be the most bioavailable and toxic form of lead. Pb2+ is known to be a calcium antagonist in vertebrates including fish. It is believed that lead causes toxicity to freshwater fish primarily by disrupting ionic homeostasis both during acute and chronic waterborne exposure. Lead can also potentially act as a respiratory toxicant since it is known to impair hemoglobin synthesis in both vertebrates. To date, the mechanistic underpinnings of lead accumulation and toxicity in aquatic invertebrates are not well understood, particularly during acute exposure. Therefore, the main objectives of the present study were in two folds: (i) to investigate the mechanisms of waterborne lead uptake, and (ii) to understand the physiological basis of lead toxicity during acute exposure. I used freshwater crustacean, <i>Daphnia magna</i>, as a model freshwater invertebrate species for my study. <i>Daphnia</i> are known to be quite sensitive to metals and widely used as a model species for toxicity assessments. The results of my study suggest that lead inhibits waterborne Ca2+ uptake in <i>Daphnia</i> in a concentration dependent manner, and this inhibition occurs predominantly through a direct competitive interaction. The entry of waterborne Pb2+ in <i>Daphnia</i> likely occurs via both lanthanum-sensitive and verapamil-sensitive epithelial calcium channels. Moreover, my results also indicate that acute waterborne lead exposure severely disrupts both Ca2+ and Na+ uptake from water, which are concomitant with the increase in the lead body burden in <i>Daphnia</i>. Interestingly however, acute exposure to lead does not affect the rate of oxygen consumption in <i>Daphnia</i>, indicating no acute respiratory toxicity of lead. Overall, it appears that lead acts as an ionoregulatory toxicant to <i>Daphnia</i> during acute waterborne exposure.

Lead is an omnipresent pollutant, and its contamination in natural waters is an issue of current regulatory concern throughout the world including Canada. The free divalent ion of lead (Pb2+) is considered to be the most bioavailable and toxic form of lead. Pb2+ is known to be a calcium antagonist in vertebrates including fish. It is believed that lead causes toxicity to freshwater fish primarily by disrupting ionic homeostasis both during acute and chronic waterborne exposure. Lead can also potentially act as a respiratory toxicant since it is known to impair hemoglobin synthesis in both vertebrates. To date, the mechanistic underpinnings of lead accumulation and toxicity in aquatic invertebrates are not well understood, particularly during acute exposure. Therefore, the main objectives of the present study were in two folds: (i) to investigate the mechanisms of waterborne lead uptake, and (ii) to understand the physiological basis of lead toxicity during acute exposure. I used freshwater crustacean, <i>Daphnia magna</i>, as a model freshwater invertebrate species for my study. <i>Daphnia</i> are known to be quite sensitive to metals and widely used as a model species for toxicity assessments. The results of my study suggest that lead inhibits waterborne Ca2+ uptake in <i>Daphnia</i> in a concentration dependent manner, and this inhibition occurs predominantly through a direct competitive interaction. The entry of waterborne Pb2+ in <i>Daphnia</i> likely occurs via both lanthanum-sensitive and verapamil-sensitive epithelial calcium channels. Moreover, my results also indicate that acute waterborne lead exposure severely disrupts both Ca2+ and Na+ uptake from water, which are concomitant with the increase in the lead body burden in <i>Daphnia</i>. Interestingly however, acute exposure to lead does not affect the rate of oxygen consumption in <i>Daphnia</i>, indicating no acute respiratory toxicity of lead. Overall, it appears that lead acts as an ionoregulatory toxicant to <i>Daphnia</i> during acute waterborne exposure.

This research examined the suitability of the biotic ligand model (BLM) approach for assessing environmental risk in surface waters consisting substantially of treated sewage effluent, and the implications of its use within a compliance-based regulatory framework aimed at controlling discharges of metals into the aquatic environment. The results from a series of Daphnia magna acute copper toxicity assays conducted in an undiluted sewage effluent medium demonstrated that BLMs could predict an acute copper toxicity endpoint with suitable accuracy. This finding contributes further support for the use of BLMs for assessing risk and compliance, even in effluent impacted waters. Additional studies, however, also demonstrated that effluent derived organic matter contained a greater concentration of metal complexing ligands per milligram of dissolved organic carbon than organic matter derived from natural sources. This indicates that effluent derived organic matter offers greater protection against the potentially adverse effects from metals than the protection offered by organic matter derived from natural sources. These studies also demonstrated that improvements in the accuracy of BLM forecasts were achievable by taking these differences into account which, from a regulatory perspective, is also desirable since this enhances the environmental relevance of compliance criteria. These findings therefore justify that consideration for the influence of site-specific metal complexation characteristics should included as part of the regulatory framework within which BLMs will be applied. The implication of the BLM approach for Severn Trent Water Limited is that, as currently proposed, the approach will require a reduction in the concentration of copper in the effluent discharges for a small number of wastewater treatment works (9), whereas for zinc, concentration reductions may be required for numerous treatment works (126).

Thesis (Ph.D.)--Baylor University, 2006. / Includes bibliographical references (p. 93-103).

Metacommunity ecology explicitly incorporates processes at multiple spatial scales to explain the assembly and dynamics of a community. In a metacommunity, local communities interact with one another through the dispersal of individuals across a region. As such, metacommunities are molded by two sets of processes: local and regional. Local factors are those that directly impact a single local community, such as environmental conditions, competition, and predation. On the other hand, regional factors affect communities across a landscape and include mechanisms such as, immigration and emigration. The potential interactions between local and regional factors make metacommunity dynamics a unique body of theory when compared to classic community theory. However, while the direct influence of dispersal on metacommunity dynamics continues to be a well-researched topic, how dispersal interacts with local factors to shape metacommunity dynamics is a more open topic. In particular, one continuing gap in my knowledge is how dispersal interacts with biotic effect how it may affect metacommunities. One type of local biotic process that can directly affect communities is a strong interactive species, i.e., a species that affects community structure and diversity, and to the best of my knowledge, the interaction between dispersal and strongly interactive species has not been directly addressed experimentally. In the following study, I investigated the interaction of dispersal and a strong interactive species on metacommunity diversity and assembly. I chose Daphnia magna as my strong interacting species due to its biological and physical traits. Dispersal is known to create predictable patterns of diversity as it increases in a metacommunity. We made logical predictions based off of my knowledge of these patterns, and my inclinations regarding how dispersal would interact with a strong interacting species. The following predictions were made in relation to the control: 1) Alpha diversity would be the highest during low dispersal as new species would be introduced and maintained above the extinction threshold. I also predicted beta diversity would decrease with increased dispersal due to the homogenization of communities. 2) In the presence of D. magna, beta diversity would only increase during low dispersal due to possible rescue effects. 3) Temporal variability would decrease for the low dispersal treatment and increase for the high dispersal treatment in the absence of D. magna. 4) Temporal variability would overall increase across all treatments in the presence of D. magna. To carry out the study, I assembled outdoor mesocosms using a 2x3x3x4 factorial design (Daphnia Treatment: no addition of D. magna, addition of D. magna; Dispersal Treatment: no dispersal, low dispersal, high dispersal; three buckets were equivalent to one metacommunity; 4 replicates). There was a significant interaction between D. magna and dispersal. Over time, beta diversity decreased as communities became homogenized; however, the no dispersal treatment homogenized at a slower rate compared to the other treatments. In addition, D. magna appeared to create local selection for certain taxa resulting in the increase of Bosmina and Simocephalus while other taxa decreased, for example Streblocerus. This trend was likely due to the feeding and grazing habits of D. magna which is known to outcompete other large zooplankton for larger phytoplankton taxa. Lastly, D. magna directly influenced temporal variability of metacommunities in the experiment. In particular, the low dispersal treatment increased in temporal variability in the presence of D. magna. Again, this result could likely be attributed to D. magna effects selecting for certain taxa, or by the re-introduction of new or dying species with each dispersal through rescue effects. Overall, the results in my study supported majority of my predictions. It is clear that D. magna had an effect on communities as taxa abundances increased and beta diversity in the no dispersal treatment did not decrease as quickly. This result suggests that the introduction of D. magna as an invasive to non-local waters could pose a threat to local community dynamics. It is important to understand how a strong interactive species can affect communities across a landscape as they can greatly alter diversity and composition. Future studies should focus on expanding the dispersal gradient and incorporating a local strong interactive species and non-local strong interactive species to understand how they may change community dynamics. / Master of Science