Zou, Huiling. / Thesis submitted in: November 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 159-176). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of contents --- p.viii / List of tables --- p.x / List of figures --- p.xiii / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Background --- p.1 / Chapter 1.1.1 --- Physicochemical properties of polycyclic aromatic hydrocarbons --- p.1 / Chapter 1.1.2 --- Sources of PAHs --- p.4 / Chapter 1.1.3 --- Toxicity of PAHs --- p.5 / Chapter 1.1.4 --- Fate of PAHs in environment --- p.6 / Chapter 1.1.5 --- Soil physicochemical and microbiological properties --- p.16 / Chapter 1.1.6 --- Geography and climate of Hong Kong --- p.17 / Chapter 1.1.7 --- Traffic status in Hong Kong --- p.17 / Chapter 1.1.8 --- Research status in Hong Kong --- p.18 / Chapter 1.2 --- "Significant, objectives and outline of this study" --- p.18 / Chapter 1.2.1 --- Research significance --- p.18 / Chapter 1.2.2 --- Research objectives and thesis outline --- p.19 / Chapter Chapter 2 --- PAH concentrations and their seasonal variations in roadside soils in Hong Kong / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.22 / Chapter 2.2.1 --- Soil sampling --- p.22 / Chapter 2.2.2 --- Soil physicochemical properties analysis --- p.24 / Chapter 2.2.3 --- Soil PAH analysis --- p.25 / Chapter 2.2.3.1 --- Extraction of PAHs --- p.25 / Chapter 2.2.3.2 --- Cleanup and concentration of the extract --- p.25 / Chapter 2.2.3.3 --- Determination of PAHs --- p.26 / Chapter 2.2.3.4 --- Calibration standards and recovery --- p.26 / Chapter 2.2.4 --- Statistical analysis --- p.28 / Chapter 2.3 --- Results and discussion --- p.29 / Chapter 2.3.1 --- Soil PAH contents and their relationships with soil physicochemical properties and AADT --- p.29 / Chapter 2.3.1.1 --- Soil PAHs --- p.29 / Chapter 2.3.1.2 --- Soil physicochemical properties --- p.38 / Chapter 2.3.1.3 --- Relationships of PAH contents with soil physicochemical properties and AADT --- p.39 / Chapter 2.3.2 --- Seasonal variations of PAH contents of roadside soils --- p.50 / Chapter 2.4 --- Conclusion --- p.56 / Chapter Chapter 3 --- "PAH concentrations in roadside vegetation, dusts and soils" / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and methods --- p.59 / Chapter 3.2.1 --- Sampling --- p.59 / Chapter 3.2.2 --- Soil physicochemical properties analysis --- p.60 / Chapter 3.2.3 --- PAHs analysis --- p.60 / Chapter 3.2.3.1 --- Extraction of PAHs --- p.60 / Chapter 3.2.3.2 --- Cleanup and concentration of the extract --- p.60 / Chapter 3.2.3.3 --- Determination of PAHs --- p.61 / Chapter 3.2.3.4 --- Calibration standards and recovery --- p.61 / Chapter 3.2.4 --- Statistical analysis --- p.61 / Chapter 3.3 --- Results and discussion --- p.62 / Chapter 3.3.1 --- Soil physicochemical properties --- p.62 / Chapter 3.3.2 --- PAH concentrations --- p.62 / Chapter 3.3.2.1 --- Soil PAHs --- p.62 / Chapter 3.3.2.2 --- Dust PAHs --- p.65 / Chapter 3.3.2.3 --- Vegetation PAHs --- p.71 / Chapter 3.3.3 --- PAH profile --- p.80 / Chapter 3.3.4 --- PAH sources --- p.83 / Chapter 3.3.5 --- PCA and HCA --- p.88 / Chapter 3.3.6 --- "Relationships of PAH contents between vegetation, dust and soil, and soil physicochemical properties and AADT" --- p.99 / Chapter 3.4 --- Conclusion --- p.124 / Chapter Chapter 4 --- Vertical and horizontal distribution of PAHs in roadside soil and their influences on soil microbial characteristics / Chapter 4.1 --- Introduction --- p.126 / Chapter 4.2 --- Materials and methods --- p.127 / Chapter 4.2.1 --- Sampling --- p.127 / Chapter 4.2.2 --- Soil physicochemical properties analysis --- p.128 / Chapter 4.2.3 --- Soil microbial biomass carbon analysis --- p.128 / Chapter 4.2.4 --- Soil microbial community analysis --- p.128 / Chapter 4.2.5 --- Soil enzyme activity analysis --- p.129 / Chapter 4.2.6 --- Soil PAH analysis --- p.130 / Chapter 4.2.7 --- Statistical analysis --- p.130 / Chapter 4.3 --- Results and discussion --- p.131 / Chapter 4.3.1 --- Vertical distribution --- p.131 / Chapter 4.3.2 --- Horizontal distribution --- p.137 / Chapter 4.3.3 --- Influences of roadside soil PAH on microbial characteristics --- p.142 / Chapter 4.4 --- Conclusion --- p.153 / Chapter Chapter 5 --- General conclusion / Chapter 5.1 --- Summary of findings --- p.155 / Chapter 5.2 --- Limitations of the study --- p.157 / Chapter 5.3 --- Implications for further studies --- p.158 / References --- p.159

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 102-117). Also available on the World Wide Web.

Since 2008, large numbers of Nile crocodile (Crocodylus niloticus) carcasses were found in the Kruger National Park (KNP), South Africa. Most of the crocodile carcasses were found in the Olifants Gorge, which is situated below the Letaba and Olifants river confluence, before the Mozambique border and Massingir Dam. The Massingir Dam is an important resource and it plays a significant role in the welfare of the local Mozambican population. Autopsies performed on the crocodiles indicated that the adipose tissue colour changed from normal white to yellow and this is usually a sign of pansteatitis. Pansteatitis is caused by lipid peroxidation in an organism and it is characterised by the lack of vitamin E. This disease is recognisable by the hardening of the fatty tissue and yellow discolouration, and is mostly associated with aquatic organisms from polluted ecosystems. There are speculations that the crocodile fatalities may be associated with the Massingir Dam that backed up into the Olifants Gorge after flooding. After the dam was reconstructed, it flooded the Olifants Gorge, causing it to act like a localised sediment trap as the water flow slowed down and as a result, caused pollutants to build-up. Sediment samples were collected from selected rivers and ponds within the KNP. These samples were analysed for selected elements, persistent organic pollutants (POPs), and polycyclic aromatic hydrocarbons (PAHs). The sediment samples were analysed in Norway for POPs and PAHs with the use of a high-resolution gas chromatography/mass spectrometry (GC/MS) and the heavy metals were analysed in South Africa with the use of inductively-coupled plasma mass spectrometry (ICP/MS). In order to identify which elements may have affected the health of the crocodiles, a series of sediment quality indices were used. These indices made it possible to determine which elements may have been involved. The order of probability of heavy metals causing harm was Se>As>Ni>Cr>Cu>I>V>Mn>Co>Fe>Cd>Hg>Zn>Pb>Ba>U. The data was compared to selected international guidelines. All the information was used to determine which of the sampled sites had the highest contamination. The sites sampled with the highest concentrations were in the Crocodile, Nkomati, Olifants, and Letaba Rivers. Concentrations of the elements, POPs, and PAHs were also quantifiable in the Olifants Gorge. The following elements (Fe, Co, Cu, Cr, Pb, V, As, and Ni) were quantified at elevated levels and may therefore have caused negative effects on the crocodiles in the Olifants Gorge. These elevated concentrations, in combination with the dramatic change in the physical environment due to the dam, could have added additional stress that may have contributed to the observed crocodile mortalities in the Olifants Gorge. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Since 2008, large numbers of Nile crocodile (Crocodylus niloticus) carcasses were found in the Kruger National Park (KNP), South Africa. Most of the crocodile carcasses were found in the Olifants Gorge, which is situated below the Letaba and Olifants river confluence, before the Mozambique border and Massingir Dam. The Massingir Dam is an important resource and it plays a significant role in the welfare of the local Mozambican population. Autopsies performed on the crocodiles indicated that the adipose tissue colour changed from normal white to yellow and this is usually a sign of pansteatitis. Pansteatitis is caused by lipid peroxidation in an organism and it is characterised by the lack of vitamin E. This disease is recognisable by the hardening of the fatty tissue and yellow discolouration, and is mostly associated with aquatic organisms from polluted ecosystems. There are speculations that the crocodile fatalities may be associated with the Massingir Dam that backed up into the Olifants Gorge after flooding. After the dam was reconstructed, it flooded the Olifants Gorge, causing it to act like a localised sediment trap as the water flow slowed down and as a result, caused pollutants to build-up. Sediment samples were collected from selected rivers and ponds within the KNP. These samples were analysed for selected elements, persistent organic pollutants (POPs), and polycyclic aromatic hydrocarbons (PAHs). The sediment samples were analysed in Norway for POPs and PAHs with the use of a high-resolution gas chromatography/mass spectrometry (GC/MS) and the heavy metals were analysed in South Africa with the use of inductively-coupled plasma mass spectrometry (ICP/MS). In order to identify which elements may have affected the health of the crocodiles, a series of sediment quality indices were used. These indices made it possible to determine which elements may have been involved. The order of probability of heavy metals causing harm was Se>As>Ni>Cr>Cu>I>V>Mn>Co>Fe>Cd>Hg>Zn>Pb>Ba>U. The data was compared to selected international guidelines. All the information was used to determine which of the sampled sites had the highest contamination. The sites sampled with the highest concentrations were in the Crocodile, Nkomati, Olifants, and Letaba Rivers. Concentrations of the elements, POPs, and PAHs were also quantifiable in the Olifants Gorge. The following elements (Fe, Co, Cu, Cr, Pb, V, As, and Ni) were quantified at elevated levels and may therefore have caused negative effects on the crocodiles in the Olifants Gorge. These elevated concentrations, in combination with the dramatic change in the physical environment due to the dam, could have added additional stress that may have contributed to the observed crocodile mortalities in the Olifants Gorge. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

<p>Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.</p><p>A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses. </p><p>The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.</p><p>Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.</p><p>Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.</p><p>My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.</p> / Dissertation

Within the last few decades, epidemiological evidence has linked exposure to air pollution, both its particles and its organic components, with cardiovascular disease (CVD) progression. CVD is a life long disease with the disruption of the endothelium being the inaugural event in this inflammatory process. The vascular endothelium is extremely susceptible to environmental insults given its tremendous surface area and that it is in constant contact with blood and components circulating within the blood, including xenobiotics. The endothelium is important as a barrier from blood constituents however, dysfunction of this barrier leads to the influx of lymphocytes and granulocytes that lead to the fatty build‐up characteristic of atherosclerosis. The studies presented in this dissertation tested the hypothesis that two unique environmental contaminants, alumina nanoparticles and benzo[a]pyrene (B[a]P), lead to increased endothelial cell dysfunction, characterized by increased adhesion molecule expression. Alumina nanoparticles induced vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and E‐selectin (ELAM‐1), as well as increased monocyte adhesion to activated endothelium. Polystyrene nanoparticles did not elicit this response. B[a]P induced ICAM‐1 expression, but only after toxification by aryl hydrocarbon receptor (AhR) controlled enzymes. Silencing of either AhR or the membrane microdomains called caveolae attenuated the B[a]P‐induced ICAM‐1 response. It was also shown that the induction of ICAM‐1 occurred by signaling through MEK, p‐38 MAPK, and activator protein‐1 (AP‐1). These data provide a novel mechanism by which air pollutants like B[a]P may cause increased atherosclerosis and describe a new toxicant, alumina nanoparticles, as a possible threat for the development of inflammatory diseases, such as atherosclerosis. Little is known about dietary interventions capable of alleviating xenobiotic‐induced toxicity. Nutrition is an obtainable and inexpensive means of possible preventative therapy. With this in mind, it was also hypothesized that plant polyphenols, such as flavonoids, can down‐regulate B[a]P‐induced ICAM‐1. Selective flavonoids, containing both a 4’ B‐ring hydroxyl substitution and a 2‐3 C‐ring double bond, protected against B[a]P‐induced ICAM‐1 activation, however this protection did not correlate with the flavonoid’s antioxidant capacity.

ABSTRACT To compare the degree of biodegradation of petroleum hydrocarbons in sediments from Ikarama and Okwori in the Niger delta, Nigeria, concentrations of n-alkanes and polycyclic aromatic hydrocarbons in the sediments were measured. Analysis was conducted with gas chromatography using mass spectrometry detector. While the decrease in concentrations of n-alkanes and polycyclic aromatic hydrocarbons confirmed the process of biodegradation in the sediments it was not solely fit to substantiate the degree of biodegradation in the sediments. Hence the percentage proportion of n-alkanes and polycyclic aromatic hydrocarbons was used. The degree of biodegradation of n-alkanes in both Okwori and Ikarama was almost similar. However, it was observed that polycyclic aromatic hydrocarbons were biodegraded in Okwori sediments than Ikarama sediments and this indicates the degree of biodegradation of petroleum hydrocarbons impacted sediments in Okwori is greater than that of Ikarama.

Aryl hydrocarbon hydroxylase activity was studied in cultured human lymphocytes using 3-methylcholanthrene, 1,2- benzanthracene, and 4'-bromoflavone as inducers. The substrates used to run the 60 minute assay were benzo(α)pyrene and diphenyloxazole. At the optimum bromoflavone concentration for induction of aryl hydrocarbon hydroxylase, the induced enzymatic activity compared favorably with that of aryl hydrocarbon hydroxylase induced by 3MC in a 96 hour lymphocyte culture using BP as the assay substrate. The whole cell human lymphocyte system was found to have as much or more activity in 20 ml vials using Joklik's-Modified Minimum Essential Medium at a pH optimum of 7.5 with no co-factor added as did the Roswell Park assay system. The whole cell assay showed that levels of aryl hydrocarbonhydroxylase inducibility in lumphocytes from smokers and non-smokers varied without regard to the subjects' smoking habits. The assay system also indicated that intact lymphocytes generate a similar group of benzo(α)pyrene metabolites as that produced by a hepatic microsomal preparation from C57B1/6J mice.

In the search for the conformation of extant or extinct life in an extraterrestrial setting the detection of organic molecular species which may be considered diagnostic of life is a key objective. These molecular targets comprise a range of distinct chemical species, with recognisable spectroscopic features. This project aims to use these features to develop an in-situ molecular specific Raman spectroscopic methodology which can provide structural information about the organic–inorganic interface. The development of this methodology identified a surface enhanced Raman spectroscopic technique, that required minimal sample preparation, allowed for the detection of selected organic species immobilised on an inorganic matrix and was effective for quantities below those which conventional dispersive Raman spectroscopy would detect. For the first time spectral information was gained which allowed analysis of the organic–inorganic interface to be carried out, this gave an insight into the orientation with which molecules arrange on the surfaces of the matrices. Additionally a method for the detection of organic residues intercalated into the interlamellar space of smectite type clays was developed. An evaluation of the effectiveness of uni and multivariate methods for the analysis of large datasets containing a small number of organic features was also carried out, with a view to develop an unsupervised methodology capable of performing with minimal user interaction. It has been shown that a novel use of the Hotellings T2 test when applied to the principal component analysis of the datasets combined with SERS allows identification of a small number of organic features in an otherwise inorganic dominated dataset. Both the SERS and PCA methods hold relevance for the detection of organic residues within interplanetary exploration but may also be applied to terrestrial environmental chemistry.

Doctor of Philosophy / Department of Agronomy / Ganga M. Hettiarachchi / Urban soils may contain harmful levels of potentially toxic contaminants. These contaminants transfer to humans via two exposure pathways: direct transfer (soil-humans by soil ingestion, dermal exposure and inhalation) and food chain transfer (soil-plant-humans). Soil amendments alter the speciation of the contaminants in soils and thereby modify their bioavailability. The objectives of this research were to access the plant availability of lead (Pb), arsenic (As), and polycyclic aromatic hydrocarbons (PAHs); bioaccessibility and speciation of soil Pb, and As; and dermal absorption of soil PAHs in contaminated urban soils; and effectiveness of soil organic amendments on reducing contaminant bioavailability. Two field experiments were conducted in Kansas City, MO and Indianapolis, IN. Both sites had elevated concentrations of Pb in soils (Kansas City site: 30-380 mg kg⁻¹ and Indianapolis site: 200-700 mg kg⁻¹) . Indianapolis site’s soils also had elevated concentrations of As (40-100 mg kg⁻¹) and PAHs (benzo[a]pyrene: 1-10 mg kg⁻¹) . A control treatment (no-compost) and compost-types (leaf compost and/or composted biosolids, non-composted biosolids, mushroom compost) were used as treatments. A leafy vegetable, a fruiting vegetable and a root crop were grown for two growing seasons. The treatments were arranged in split-plot design (main plot factor: compost; sub-plot factor plant-type). An in vitro steady fluid experiment was conducted using human skins to examine the dermal transfer of soil PAHs. The concentrations of Pb, As, and PAHs in the vegetables were low, except Pb in root crops. Compost reduced the bioaccessibility of Pb, but did not change the bioaccessibility of As. Selected soil samples were analyzed for speciation of Pb using extended x-ray absorption fine structure spectroscopy. The predominant Pb species were Pb sorbed to Fe oxy(hydr)oxide and to organic C. Stable Pb phosphates (pyromorphite) was formed during the in vitro extraction. Dermal transfer experiments showed PAHs in the contaminated soils did not transfer through the skin. Stratum conium of the skin acted as a barrier for dermal transfer of soil PAHs. In general, the risk of food chain transfer of soil Pb, As, and PAHs were low in the studied sites and can be further reduced by compost addition. Bioaccessibility of Pb and As in urban soils were low. Dermal absorption of soil PAHs was insignificant.