Exposure to crude oils has been shown to induce CYP1A enzymes and cause chronically toxic effects in aquatic organisms. Earlier studies indicated that polycyclic aromatic hydrocarbons (PAHs) present in crude oil are primarily responsible for the chronic toxicity. Crude oil contains a variety of PAHs; the majority of them are alkyl substituted. In this work, we have used an effects-driven fractionation and analysis approach (EDFA) to isolate and characterize PAHs present in Alaskan North Slope and Scotian Light crude oils that are toxic to fish. The crude oil components were first fractionated into four fractions using a low temperature vacuum distillation technique. Among them, the heavy gas oil fraction (boiling range 287°C – 461°C) of both oils caused highest toxicity to fish. To isolate the PAHs from waxes present in this fraction, a low temperature wax precipitation method was developed and optimized. CYP1A induction results showed that the extract contained a large number of CYP1A inducers while the residue contained none. Chemical analyses confirmed that most of the PAHs were partitioned into the extract fraction. Alkyl PAHs present in the extract were further fractionated into five fractions based on the number of aromatic rings using a normal phase HPLC method. Chemical analysis and the toxicity testing of these fractions indicated that alkyl PAHs belonging to classes such as phenanthrene, fluorene, naphthobenzothiophene, and chrysene are likely responsible for the observed toxic effects. To aid the EDFA scheme, a new HPLC-DAD method for the analysis of alkyl PAHs was developed. The alkyl PAHs were first fractionated based on the number of aromatic rings using a normal phase column followed by their analysis using reverse phase HPLC–DAD technique. The reverse phase analysis involved classifying the alkyl PAH peaks into different PAH classes based on their DAD spectra. Then, alkyl carbon numbers for each peak were assigned based on their retention time. To analyze co-eluting alkyl PAH isomers an offline multi-dimensional HPLC method was developed. Orthogonal separation was achieved by first fractionating the alkyl PAHs on a normal phase column followed by the RP-HPLC-DAD analysis. Using these data a 2D contour plot was developed and used for the detailed analysis of alkyl PAHs isomers. Analysis results showed good agreement with a gas-chromatography-mass-spectrometric (GC-MS) analysis method, and the new method was able to distinguish some PAH types which could not be identified by GC-MS. / Thesis (Ph.D, Chemistry) -- Queen's University, 2007-11-19 13:46:18.602

[pt] O objetivo desta pesquisa foi a substituição de técnicas convencionais de extração de amostras para análise hidrocarbonetos policíclicos aromáticos (HPAs) em amostras de tecido para um procedimento confiável, rápido e ambientalmente mais sustentáveis. O novo método foi desenvolvido usando um método de extração com solvente pressurizado, avaliando dois materiais de referência padrão (peixe e mexilhão) e amostras de sardinhas fortificadas (Sardinella sp.). Cinco procedimentos de extração diferentes foram avaliados e o melhor desempenho obtido foi pela extração de 1 g de tecido liofilizado em conjunto com 5 g de sílica desativada (5 por cento), mistura de diclorometano: metanol (4: 1 v / v), temperatura de 80 graus C, três ciclos, 10 min de tempo estático e 90 s de tempo de purga. Este método foi ainda validado pela análise de nove réplicas do material de referência número 2974a(Orgânicos em Tecido de Mexilhão Liofilizado) do National Institute of Standard and Technology (NIST), resultando em uma recuperação média de 85 mais ou menos 14 por cento. As médias e as incertezas obtidos para cada HPA foram equivalentes aos do material de referência, corroborando a confiabilidade do método desenvolvido. Um tempo de processamento mais curto, menos uso de solventes e reagentes e menor manipulação do extrato resultou em um método eficaz e alinhado às diretrizes da química verde. / [en] The aim of this research was the replacement of conventional sample extraction techniques for polycyclic aromatic hydrocarbons (PAH) in tissue samples for a reliable, fast and environmentally more sustainable. The methodwas developed using a pressurized solvent extraction method and assessing two different standard reference materials (fish and mussel) and freeze-dried and fortified sardine samples (Sardinella sp.). Five different extraction procedures were evaluated and the best performance comprised 1 g of lyophilized tissue, 5 g of deactivated (5 percent) silica, a dichloromethane:methanol (4:1 v/v) mixture, a temperature of 80 C degrees, three cycles, 10 min of static time and 90 s of purge time. The method selected following these tests was further validated through the analysis of nine replicates of the National Institute of Standard and Technology (NIST) reference material No. 2974a (Organics in Freeze-Dried Mussel Tissue), resulting in an effective recovery of 85 more or less 14 percent. The means and uncertainties attained for each PAH were equivalent to those of the reference material, corroborating the reliability of the developed method. A shorter processing time, less use of solvents and reagents and lower extract manipulation resulted in an effective method aligned with green-chemistry guidelines.