Wing-keung Liu. / Thesis (M.Ph.)--Chinese University of Hongkong. / Bibliography: leaves 96-106.

Nanoparticle toxicity has become a major topic of interest due to the inevitable exposure of these nanomaterials to both humans and the environment. Nanotechnology is a rapidly growing industry with diverse material resources and an extensive market for commercialization and introduction of nanomaterials into consumer products. The problem with this flourishing technology is that it has far outgrown research based on the safety and toxicity of the nanomaterials, which in bulk are generally nontoxic. The need for research in determining the toxic effects on cells and the implications it may have on the environment have grown but the different techniques, cell systems and nanoparticles employed are generally to diverse and conflicting in overall results that determination toxicity is nearly impossible. The need for a universal technique to study the interaction of nanoparticles with cells and decouple the molecular effects (chemical properties) from the“nanospecific" effects (including size, concentration, surface charge, functionality and polarity) is apparent. It is additionally necessary to determine the mechanisms associated with nanoparticle-induced cytotoxicity in order to better understand the problems posed to both human and environmental health and then develop new safer nanoparticles. Therefore, the focus of this study is to determine the nano-specific (physical) properties, including size and functionalization that cause toxicity, specifically through interaction with a cell membrane. A supported lipid bilayer (SLB) composed of L-α-phosphatidylcholine (egg PC) was used as a model cell membrane to test the effects of 2, 5, 10 and 40 nm gold nanoparticles (AuNPs). Given the imminent exposure of nanoparticles to the environment it is important to determine how nanoparticles would behave in the presence of natural organic matter or polymers which are naturally present in environmental systems. Poly(methacrylic acid) (PMA) can be used to represent the polymers normally found in the environment. AuNPs were diluted in PMA in order to simulate fundamental environmental conditions. Analysis was done using a quartz crystal microbalance with dissipation (QCM-D), which measures the frequency (f) and dissipation (D) changes directly associated with mass and conformation changes of the SLB. Different overtones for f and D allow for theoretical interpretation of changes correlated to different layers of the membrane. The 2 and 5 nm particles were found to interact strongly with the lipid bilayer by adsorbing to and/or partially/completely penetrating into the lipid bilayer presumably due to a hydrophobic coating caused by PMA adsorption to the NP surface. The penetration caused a much more rigid membrane due to higher lipid packing caused by nanoparticle addition. The 10 and 40 nm particles interaction with the bilayer were not affected by the presence of PMA. Both AuNP sizes removed mass from the membrane with losses similar in de-ionized water and PMA solution. Removal of membrane mass (lipids/hydration) caused a more flexible membrane. It was determine that sized is the limiting factor for nanoparticle solubilization into the membrane. It can be concluded from the results that size coupled with natural organic matter affects the cytotoxicity of the nanoparticles to the membrane. A study was done with 12 nm functionalized AuNPs in the presence of humic acid, a well-known and more complex and realistic model for natural organic matter. A PC lipid bilayer was used to simulate a model cell membrane and QCM-D techniques were utilized in the determination of toxicity and mechanistic interaction of nanoparticles with a lipid bilayer. Functionalized AuNPs were shown to decrease the rigidity of the lipid bilayer by increasing the dissipation and decreasing the mass associated with the adsorbed film (SLB). The presence of humic acid stabilized the nanoparticles and provided increased electrostatic repulsion which resulted in decreased mass losses from the membrane and much smaller decreases in membrane rigidity. It was concluded that presence of humic acid reduces the effects of functionalized nanoparticle interaction with a lipid bilayer. These results may mean that natural organic matter has the ability to reduce the cytotoxic effects of nanoparticles released into the environment. Overall, the QCM-D was found to provide valuable information regarding the possible toxic properties and mechanisms in which different gold nanoparticle interact with a supported lipid bilayer under environmental conditions. The information provided by the studies performed has shed much light on the interaction of gold nanoparticles with a supported lipid bilayer in the presence of model natural organic matter. The experiments done in this study are the first steps towards developing an assay with the ability to determine the toxic physical properties and mechanisms by which nanoparticles interact with lipid bilayers will greatly aid in development of non-toxic nano-materials. The technology and techniques used in this study will greatly improve the field by solidifying one technique to use in the quantitative approach studying nanoparticle/cell interactions. The use of AFM techniques in conjunction with the QCM-D would be highly beneficial by facilitating better understanding of the exact mechanisms by which nanoparticles induce cytotoxicity.

Aluminium (Al) and silver (Ag), through human activities, are present in the environment at concentrations sufficient to cause toxicity. The aim of this study was to administer Al and Ag to the short lived model organism Drosophila melanogaster, so that developmental toxicity and potential ameliorative interventions could be examined over a compressed timescale relative to mammalian models. Aluminium was administered to Drosophila in food as either the chloride salt or citrate complex at concentrations of 1, 10 and 100 mM and various developmental parameters were assessed. The lowest concentration to delay pupation relative to the control was 10 mM but this depended upon the food in which it was administered. Higher whole body tissue levels of Al were seen following Al citrate administration compared to AlCl3, but Al citrate was less toxic as this did not did not impair larval viability at 100 mM; 100 mM AlCl3 resulted in 100% mortality. Eclosion success was significantly impaired with either form of Al at 10 mM, but no difference was seen between the forms of Al. When Drosophila were fed AlCl3 over their entire lifespan, a small but significant reduction in the lifespan of male flies was seen. No behavioural toxicity could be demonstrated. Existing studies have demonstrated significant tissue Al concentrations and toxicity whereas these have been minimal in this study. It is suggested that these differences may have a genetic component, with food composition exerting an influence also. Silver, either as AgNO3 or Ag nanoparticles (AgNPs) was administered in concentrations up to 500 micromolar and 10 mM, respectively. Either form of Ag, at 50 micromolar was sufficient to significantly retard pupation rate, although pupation or eclosion success was not impaired until 100 micromolar. The concentration-response relationship for AgNO3 was steep with pupation success dropping to nearly zero by 300 micromolar; Drosophila in this study were far more sensitive to AgNO3 than those in other reports. Animals exposed to AgNPs were still able to pupate at 500 micromolar, but these pupae were almost all non-viable when exposed to 400 micromolar AgNPs. At 1 mM and above, AgNPs, however, showed reduced toxicity compared to lower concentrations. The reasons for this are unclear. Both forms of Ag caused de-pigmentation in adults after larval exposure that may be explainable by inhibition of polyphenol oxidase enzymes by Ag (I) ions. The de-pigmentation was preventable by pre-loading larvae with Cu. Ascorbate prevented the de-pigmentation caused by AgNPs but not AgNO3 suggesting that AgNP toxicity is due to Ag (I) ion release. Oxidation of AgNPs was found to be greatly accelerated by Fe (III) and Cu (II) ions in the presence of Cl- ions. Although some of the results here conflict with the literature, developmental toxicity has been observed here, for both Al and Ag, and the variability across studies may provide an opportunity for dissecting the mechanisms behind Al and Ag toxicity through identification of the traits that confer sensitivity or resistance.

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A sediment toxicity test using the freshwater oligochaetes Lumbriculus variegatus and Tubifex tubifex was performed. We evaluated acute and chronic toxicity affects of copper and cadmium on reproduction in both species and the bioaccumulation of both metals by L. variegatus using artificial sediment. L. variegatus bioconcentrated copper 22‐fold and cadmium 16‐fold after a 14‐day exposure to spiked artificial sediments with 0.02% organic content. The EC50 for T. tubifex varied depending upon endpoint from 2.7 to 2.8 mg/L for cadmium and from 8.4 to 8.9 mg/L for copper. The EC50 for L. variegatus was 2.2 mg/L for cadmium and 3.9 mg/L for copper. Based on these results, L. variegatus appears to be more sensitive to metal toxicity in artificial sediments than T. tubifex.

Standard toxicity tests often require high costs for maintaining healthy cultures, so few test species are employed in routine ecotoxicological analysis. An alternative is the "battery of tests" approach involving using rapid toxicity tests for screening. Rapid-screening toxicity tests must display organism sensitivity, similarity in responses to other test organisms, relevancy to many circumstances, and repeatability. Protozoa are ideal candidates for rapid-screening bioassays. They are cosmopolitan, play important roles in ecosystems, and have high reproductive rates. Many protozoa can form a resting stage (cyst) that remains viable during adverse conditions, eliminating the need for maintaining continuous cultures for testing. The main objective of this research was to evaluate the soil ciliate, Colpoda inflata (Stokes), as a bioassay organism in rapid-screening tests by determining its sensitivity to a variety of model compounds. These tests were based on the principle that exposure to a toxic compound would negatively affect population growth. To test for sensitivity, C. inflata was exposed to different levels of dissolved organic carbon in test media for each compound tested. C. inflata was expected to be more sensitive to toxicants in an inorganic medium than in media with high organic carbon content. Data were analyzed by determining the median tolerance limit for inhibition of population growth (IG50) relative to controls. IG50 values of the eight model compounds tested varied considerably. C. inflata growth was not significantly affected by 2,4-D or malathion. C. inflata showed differences in sensitivity between organic and inorganic media for the toxic metals tested and the order of toxicity corresponded to those found in standard tests. A significant difference occurred between the test media and the pesticide PCP, where growth was not inhibited in the organic medium; in the inorganic medium the IG50 was 0.269 mg/L. No significant effect of test media was found for ammonia or SDS. Compared to several published toxicity results, C. inflata proved more sensitive overall than other rapid-screening tests and many standard acute toxicity tests. Results of this study show that this rapid-screening toxicity test is sensitive, repeatable, and provides information similar to traditional standard toxicity tests.

The Canada-wide Standards for Petroleum Hydrocarbons (PHC CWS) in soils are remedial standards based on four petroleum distillates (Fraction 1 [F1; ECN C6-C10], Fraction 2 [F2; ECN >C10-C16], Fraction 3 [F3; ECN >C16-C34], and Fraction 4 [F4; ECN >C34-C50]). Knowledge gaps regarding petroleum toxicity to soil organisms were identified including concerns that the ecological values for F3 were overly conservative, possibly due to differences in toxicity between the low and high boiling point constituents of this distillate, and unexpected less-than-concentration-additive toxicity of binary mixtures of distillates to earthworms. An understanding of petroleum toxicokinetics with soil organisms was also needed to interpret toxicity results. Toxicity studies with one plant and two invertebrate (earthworm and collembolan) species were conducted with F3 and two subfractions of F3, F3a (ECN >C16-C22) and F3b (ECN >C22-C34), to determine if the toxicities of F3a and F3b were sufficiently different to recommend regulating the two separately. The difference in toxicities between the two was generally within the range of variability noted for the toxicity tests and thus it was not recommended to regulate the two separately. The toxicity data indicated that the exposure duration of standard test methods may be insufficient for determining the toxicity of higher distillate ranges. Toxicokinetic studies conducted with earthworms and F2, F3a, and F3b confirmed that standard test durations generally were not of sufficient duration to attain maximum body residues with F3b and sometimes F3a. Internal exposure scenarios also differed among distillates, with various accumulation curves noted and attributed to differences in loss of distillate from the soil and changes in bioavailability. Aromatics were disproportionally accumulated by earthworms relative to the ratio of aromatics to aliphatics in soil, suggesting that aromatics were the main contributors to earthworm toxicity. Toxicity and toxicokinetic studies with binary combinations of F2, F3a, and/or F3b and earthworms demonstrated that, on a soil concentration basis, toxicity was less-than-additive. Toxicokinetics indicated that this was due to a decrease in the bioavailability of distillates when a second distillate was present presumably as a non-aqueous phase liquid. However, on an internal tissue concentration basis, results were closer in agreement with concentration-addition.

The antibiotic-resistant bacteria seriously threaten public heath. Scientists of both public and private sectors are devoted to develop new drugs to overcome this problem. Marine species are highly diversity and have been shown to be a potential sources to find efficient compounds. The medicine application of the natural products from ocean increases in last decade. The purpose of the project was to isolate antibiotic-producers from sea-bottom mud. Three filament microorganisms from marine environment, Acremonium altematum T1-1, Aspergillus ochraceus N7 and Nocardiopsis dassonvillei F5, can produce bioactivity compounds. All compounds purified using silica chromatographic separation from culture extraction and determination of their structure by NMR spectrum and X-ray diffraction pattern for crystals. A. altematum T1-1 produced 5,6-dihydropenicillic acid (DHPA), A. ochraceus N7 produced penicillic acid (PA), and N. dassonvillei F5 produced iodinin. The structures of DHPA and PA were identified as derivatives of five-member lactone, and iodinin is a derivative compound of phenazine. The optimal growth of DHPA -producer A. altematum T1-1 was in 6% salinity at 30¢J and best DHPA production was in 0% salinity at 25¢J. The optimal growth of PA-producer A. ochraceus N7 was in 6% salinity at 25¢J, and best PA production conditions was below 3% salinity at 25 ¢J. The optimal growth of iodinin-producer N. dassonvillei F5 was in 6% salinity at 30¢J and best iodinin production condition was 1-3% salinity at 25¢J. Only PA has highly inhibited of B. cereus at the MIC of 75 mg/ml. The PA had cytotoxicity against cancer cell lines of P-388, HT-29 and A549 while iodinin inhibited P-388 and A549. The ED50 on the swimming ability of rotifer was 1.03 and 2.5 mg/ml for PA and DHPA, respectively. It revealed that the growth of these two mold can occur in regular marine environment and produce toxin to kill rotifer. The growth of N. dassonvilleiand F5 and iodinin caused the death and virus-invasion of shrimp.

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No description available.