Date of Award
Doctor of Philosophy (PhD)
Dr. B.E. McCarry
A methodology for the extraction, clean-up and compound class fractionation of complex environmental mixtures was developed and tested using standard reference materials and sediment from Hamilton Harbour. Samples were extracted using a Soxhlet apparatus or an ultrasonication apparatus and the resulting organic solvent extracts were fractionated into compound classes using an alumina/Sepahadex LH20 clean-up procedure and high performance liquid chromatographic techniques.
In the next phase of the study, sediment samples, sediment trap samples, and air particulate material from the Hamilton Harbour area of western Lake Ontario were fractionated using the developed methodology. These samples were chosen with the aim of evaluating the contributions of a variety of sources to chemical and genotoxic contamination in the harbour. The resulting fractions were analyzed by chromatographic techniques and tested for genotoxicity using Ames Salmonella/microsome assay with TA98-like and TA100-like bacterial strains modified by the inclusion of genes for the activating enzymes nitroreductase and O-acetyltransferase. These data were used to construct chemical and biological profiles of the samples and to identify specific compounds and compound classes responsible for mutagenic activity observed in the sample extracts.
The majority of the mutagenic activity displayed by a Randle Reef sediment sample extract was found to be present in the fraction containing the polycyclic aromatic hydrocarbons (PAH). Extracts of the PAH-containing fraction displayed dramatically higher responses with the TA100-type strains with metabolic activation. The PAH fraction was further fractionated and analysed to identify the compound(s) responsible for the mutagenic activity. The biological activity of this PAH-containing fraction was found to co-elute with compounds of molecular mass 252, 276, 278, and 302 amu.
In contrast to the results obtained from the investigation of the Randle Reef sediment sample, extracts of sediment trap samples displayed significantly higher responses with the TA98-type strains. The compound(s) responsible for this biological activity were contained in the more polar polycyclic aromatic compound (PAC) fractions and did not require oxidative metabolism to manifest their activity. Further separation of the most biologically active fraction from the sediment trap samples revealed that the biological activity was contained in a very narrow elution time range in the reversed-phase high performance liquid chromatography (RP-HPLC) chromatogram. This narrow band of activity was collected and analysed usign gas chromatography-mass spectrometry (GC-MS). While alkyl-benzocarbazole derivatives were identified in these fractions, they were not considered to be the agents responsible for the observed biological activity.
The use of ultrasonic extraction and the alumina/Sephadex LH2- clean-up and normal phase HPLC compound class fractionation procedures were found to be effective for the preparation of a number of complex environmental samples. The chemical and biological profiles of the harbour sediment and suspended sediments indicate that PAH from resuspended coal tar contaminated sediment is a principal contributor to the chemical profile of suspended sediments in the harbour and a significant contributor to the genotoxicity of extracts of these samples. However, the biological profiles of the suspended sediments indicate the presence of additional mutagens other than PAH that are not present in the Randle Reef sediment sample. A comparison of the biological profiles of extracts of suspended sediments and air particulate material indicate that there may be a common mutagenic contaminant in both of these sample matrices.
Marvin, Christopher H., "A multi-media bioassay-directed investigation of the hamilton harbour area of western lake ontario" (1994). Open Access Dissertations and Theses. Paper 2410.