The pharmacokinetics of a range of substituted benzyl -cyclopropane-l-carboxylates topically applied in acetone to adult mustard beetles, Phaedon cochleariae, and in Sirius mineral oil to larval Spodoptera littoralis were investigated with particular reference to tissue binding and distribution. A set of pyrethroids with a wide range of binding properties was applied to adult mustard beetles. The form of the pharmacokinetic profiles was obtained by exhaustive soxhlet extraction of the tissues. Binding varied with the physicochemical properties of the compounds. Two phases of binding were identified; rapid binding to cuticle occured within seconds of topical application, followed by a slower binding which proceeded to a maximum after several hours. When cypermethrin was applied to larvae of S. littoralis, two similar phases were observed; rapid binding which took place over the first hour was followed by a slower binding which continued at a constant rate for up to 72 hours after dosing. Increasing the viscosity of the carrier oil reduced the rate of penetration of cypermethrin into larvae of S. littoralis. The tissue concentration of cypermethrin when equilibrated throughout the larval tissues was related to the ratio of tissue solids to tissue water. This suggests that the distribution of insecticide in the tissue is determined by partition processes. All of the tissues without exception reached steady state within one hour of dosing and the tissue levels maintained thereafter. However, this pattern was not observed for the gut contents, where cypermethrin levels reached a peak after six hours. Thereafter cypermethrin disappeared - from the gut, presumably as material was eliminated or degraded. The gut appears to be the only important site of loss of cypermethrin from Spodoptera larvae. The toxicological implications of these results are discussed.

In this thesis, we examined the effects of the exposures to anthropogenic pollutants on the fish, primarily juvenile chinook salmon, immune system using newly and recently developed immune assays. In addition, we developed a new assay for measuring immunocompetence of fish. In the first chapter, the Alamar Blue assay was developed to quantify the proliferation of chinook salmon (Oncorhynchus tshawytscha) leukocytes. Isolated splenic and pronephric leukocytes were stimulated with different concentration of mitogens (LPS, PWM, and ConA) for various incubation times. Optimum cell culture conditions (cell density, mitogen concentration, and incubation time) for the Alamar Blue assay were evaluated by comparison with flow cytometric analysis. The Alamar Blue dye was non-toxic for leukocytes, and the assay proved to be able to quantify the mitogenic responses using LPS, but PWM and ConA. In the second chapter, we determined the effects and mechanisms by which p,p'- DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha). Isolated salmon splenic and pronephric leucocytes were incubated with different concentrations of p,p'-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p'- DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p'-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leucocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p'-DDE. The effect of p,p'-DDE on leucocytes appeared to vary between developmental stages or season. The mitogenic response of leukocytes of chinook salmon exposed to p,p'-DDE in vivo exhibited a biphasic dose-response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p'-DDE had a significantly lower percentage of Ig+ blasting cells than controls. Our results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression. In the third chapter, we evaluated the direct effects of in vitro exposures to tributyltin (TBT), widely used biocide, on the cell mediated immune system of chinook salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from juvenile chinook salmon were exposed for 6, 24, or 96 hr to a concentration range of 0.03 0.1 mg TBT 1����� in cell cultures. Effects of TBT on cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry. Splenic and pronephric leukocytes in the presence of TBT experienced a concentration-dependent decrease in the viability in cell cultures following the induction of apoptosis. In addition, pronephric lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon LPS stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg 1����� of TBT in the cell cultures. Flow cytometric assay with the fluorescent conjugated monoclonal antibody against salmon surface immunoglobulin was employed for the conclusive identification of B-cell in the chinook salmon leukocytes. Our findings suggest that adverse effects of TBT on the function or development of fish immune systems could lead to an increase in disease susceptibility and its subsequent ecological implications. / Graduation date: 2004