Later Life Consequences of Subteratogenic Exposure to a Complex PAH Mixture in the Atlantic Killifish (Fundulus heteroclitus)

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.

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.

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.

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.

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.

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.

Predicting toxic gas concentrations resulting from enclosure fires using local equivalence ratio concept linked to fire field models

A practical CFD method is presented in this study to predict the generation of toxic gases in enclosure fires. The model makes use of local combustion conditions to determine the yield of carbon monoxide, carbon dioxide, hydrocarbon, soot and oxygen. The local conditions used in the determination of these species are the local equivalence ratio (LER) and the local temperature. The heat released from combustion is calculated using the volumetric heat source model or the eddy dissipation model (EDM). The model is then used to simulate a range of reduced-scale and full-scale fire experiments. The model predictions for most of the predicted species are then shown to be in good agreement with the test results

Other uses of palladacycles in synthesis

Summary: This chapter contains sections titled: * Introduction * Chiral Palladacycles in Aldol and Related Transformations * Catalytic Allylic Rearrangements * Catalytic C-C Bond-Forming Reactions * Oxidations Involving Palladacycles * Conclusion * References

A regional chemostratigraphically-defined correlation framework for the late Triassic TAG-I Formation in Blocks 402 and 405a, Algeria

The Triassic Argilo-Gréseux Inférieur Formation (TAG-I) is one of the principal hydrocarbon reservoirs in the Berkine Basin of Algeria. Sedimentological studies have shown that it exhibits marked spatial and temporal facies variations on both a local field scale and a regional basinal scale. This variability, combined with a lack of diagnostic flora and fauna, makes regional correlation within the unit difficult. In turn, the lack of a consistent regional stratigraphic framework hampers the comparison of the various correlation schemes devised by operators in the basin. Contrasting the TAG-I in Blocks 402 and 405a exemplifies the problems encountered when attempting regionally to define a correlation framework for the interval. Between these two blocks, a distance of approximately 200 km, there are marked changes in the style of deposition from sand-dominated, proximal fluvial systems in the SW (Block 405a, MLN, MLC, KMD and MLNW fields) to a more distal, more clay-prone system in the NE (Block 402, ROD/BRSE/BSFN, SFNE and BSF fields). A chemostratigraphic study of the TAG-I in these two blocks has allowed a four-fold correlation framework to be defined, where each chemostratigraphic package has distinctive geochemical features. Chemostratigraphic Package 10, the oldest unit, lies above the Hercynian Unconformity, but beneath a geochemically identifiable hiatal surface. Chemostratigraphic Package 20 lies above the hiatal surface but is separated from the overlying packages by a mineralogical change identifiable in both claystone and sandstone geochemistry. Chemostratigraphic Packages 30 and 40 are chemically somewhat similar, but are separated by a regional event interpreted as a period of dolocrete and lacustrine development. By combining the geochemical differentiation of the units and recognition of their stratal boundaries, it is possible to define a correlation for the TAG-I between Blocks 402 and 405a. The proposed correlation between the two blocks suggests that the northern parts of Block 405a may have been occupied by a spur or subsidiary channel from the main SW–NE-trending fluvial system, resulting in one of the chemically defined packages being demonstrably absent in the MLNW, MLN, KMD and MLC fields when compared with the other areas of the study.

Effects Of Aromatic-Hydrocarbons On The Metabolism Of The Digestive Gland Of The Mussel Mytilus-Edulis-L

1. The results presented in this paper show that the exposure of mussels to a sublethal concentration of oil-derived aromatic hydrocarbons (30 μg 1−1) for a period of 4 months significantly decreases the protein level in the digestive gland of the animals (−17%). 2. The activity of the nuclear RNA polymerase I and II is also significantly decreased in the digestive gland of hydrocarbon-exposed mussels (−64% and −18%, respectively). 3. The RNAase(s) activity present in the nuclei from the digestive gland cells increases following the exposure of the mussels to aromatic hydrocarbons. This effect is particularly evident at high ionic strength [200 mM (NH4)2SO4]. 4. The analysis of some characteristics of the nuclear RNAase(s) (most of which is soluble and shows a maximum of activity at pH 4−5) could indicate that part of this hydrolytic enzyme may have a lysosomal origin. 5. This fact appears to be in agreement with the finding that in the mussels exposed for 4 months to aromatic hydrocarbons the lysosomal stability decreases drastically and the total content of lysosomal enzymes is significantly increased (+42.4%).

Responses Of Mytilus-Edulis On Exposure To The Water-Accommodated Fraction Of North-Sea-Oil

Individuals of Mytilus edulis L., collected from the Erme estuary (S.W. England) in 1978, were exposed to low concentrations (7 to 68 μg l-1) of the water-accommodated fraction (WAF) of North Sea crude oil. The pattern of accumulation of petroleum hydrocarbons in the body tissues was affected by the presence of algal food cells, the period of exposure, the hydrocarbon concentration in seawater, the type of body tissue and the nature of the hydrocarbon. Many physiological responses (e.g. rates of oxygen consumption, feeding, excretion, and scope for growth), cellular responses (e.g. lysosomal latency and digestive cell size) and biochemical responses (e.g. specific activities of several enzymes) were significantly altered by short-term (4 wk) and/or long-term (5 mo) exposure to WAF. Stress indices such as scope for growth and lysosomal latency were negatively correlated with tissue aromatic hydrocarbons.

Cellular-Responses To Polycyclic Aromatic-Hydrocarbons And Phenobarbital In Mytilus-Edulis

Certain polycyclic aromatic hydrocarbons and phenobarbital induced an increase in the activity of microsomal NADPH neotetrazolium reductase (linked to mixed function oxygenase systems) in the blood cells of Mytilus edulis. Phenanthrene and methylated naphthalenes caused lysosomal destabilisation which is believed to be directly related to the mechanism of cytotoxicity in the digestive cells. The use of these cytochemical techniques as indices of aromatic hydrocarbon contamination is discussed.

The effect of pollution of intertidal sediments on the viability of calanoid copepod eggs

Calanoid copepod eggs have been extracted from intertidal sediments and hatched in the laboratory. Although most of the eggs which hatched did so within < 7 days, the hatching of some continued over a more prolonged period (> 20 days). This indicates that there were a significant number of diapausing or delayed hatching eggs. The species of calanoids present include some of which are known to produce diapausing eggs. Hatching of nauplii from incubated sediment samples was slower than from the extracted eggs indicating dormancy induced by the effects of burial in the sediment. Viability of the eggs has been related to chronic industrial or urban pollution as indicated by polycyclic aromatic hydrocarbon levels. These hatchings were conducted simultaneously with those for cleaner locations. The viability of eggs was significantly depressed in the more heavily polluted sites. An oil spill arising from the grounding of the "Sea Empress" at Milford Haven, UK, in February 1996 has provided a comparison of the impact of an acute situation with these chronic effects. An immediate drastic reduction in viability was followed by a recovery in the year following the spill. The data have provided no evidence for differences in the response to pollution between diapausing and subitaneous eggs.

DIESE Final Report

The DIESE program (Determination of relevant Indicators for Environmental monitoring: A Strategy for Europe) brought together seven French and British research teams, a private company and the agencies responsible for the management of water bodies of the two countries (ONEMA and the Environmental Agency) in a joint effort to document the ecotoxicological effects related to the presence of chemicals in the environment. To contribute to a better understanding and management of the environment, the program has expanded its efforts to (1) use existing knowledge, or new information acquired during the research program, to identify important biological problems affecting wildlife, (2) increase our understanding of toxicological mechanisms involved and thus be able to identify the causes of the identified dysfunctions and (3) to hone our expertise and vigilance systems in order to better monitor changes in the environment and make appropriate diagnoses. The first part of the program identified clear biological effects, and using biological tests representative of the mechanisms of action of compounds, identified the responsible compounds present in the environment. In connection with the feminization observed in many fish species in European streams, a search for estrogenic and anti-androgenic compounds was conducted. A new test identifying estrogenic compounds has been developed in roach and the ER-Calux test for anti-androgenic effects has been implemented. The results showed that, in addition to biocides such as triclosan and chlorophène, many aromatic hydrocarbon compounds are likely to disturb the physiology of living organisms by interacting with the androgen receptor. Six of these were identified in sediment extracts: benzanthrone, fluoranthene, 1,2- benzodiphenylene sulfide, benzo[a]pyrene, benz[a] anthracene, and 9-phenylcarbazole. The second part of the program aimed at documenting and understanding the mechanisms of action of chemicals leading to physiological changes. This work represents a particular challenge when dealing with molluscs, as knowledge about their physiology and endocrinology is still fragmentary. Thus, new technologies including metabolomic and transcriptomic analyses have been implemented in order to obtain a comprehensive picture of the effects on molluscs. Metabolomic research demonstrated that estrogenic compounds are able to alter the metabolism of eicosanoids and amines, while transcriptomic strategies identified genes whose expression is altered in intersex clams. Because these genes mainly appear as “male” genes, the results suggest that these profound physiological changes result from demasculinisation of male clams. Proteomic studies have also been carried out to elucidate the mechanisms of action of pollutants on fish physiology. These studies generally included a set of molecular marker measurements in an integrative and ecological perspective. The results showed that not only male fish physiology is altered but also female reproductive status is impaired. Moreover, it appeared that other alterations of the fish endocrine system, such as androgenic effects, are at work and that the immune system is also subject to chemical pressure including effects from environmental estrogens. Notably, the immune system, like the endocrine system, seems to show periods of particular sensitivity during development. Measurements on growth and on the general metabolism emphasize the importance of environmental conditions in the physiology of aquatic organisms and in particular the inter-site variability due to temperature,hypoxic conditions, and fish development strategies. They thus provide a unique perspective that allow us to better understand the context and consequences of natural conditions on the population. In a third part of the program, the research conducted had the objective of developing and testing a biomarker strategy to support the environmental management methodologies. Two lanes of specific studies have been followed. The first was to implement, over all or part of the study area, robust biomarkers to establish maps that highlight the water bodies at risk and provide information on sources of compounds and associated disturbances. The second part of the work aimed at exploring methodologies to take advantage of biomarker measurements and to integrate them in a very simple and clear index. Partial or comprehensive maps of the Channel area were produced to report the presence of mutagenic or anti-androgenic compounds in the sediments, intersex fish and clams, and imposex. These maps may remain to be completed and work will be necessary to confront this information in order to learn relevant lessons for management of the environment, a goal that the DIESE program has contributed to by providing some necessary and original information.

Polychaete burrows harbour distinct microbial communities in oil-contaminated coastal sediments

Previous studies have shown that the bioturbating polychaete Hediste (Nereis) diversicolor can affect the composition of bacterial communities in oil-contaminated sediments, but have not considered diversity specifically within bioturbator burrows or the impact on microbial eukaryotes. We tested the hypothesis that H. diversicolor burrows harbour different eukaryotic and bacterial communities compared with un-bioturbated sediment, and that bioturbation stimulates oil degradation. Oil-contaminated sediment was incubated with or without H. diversicolor for 30 days, after which sediment un-affected by H. diversicolor and burrow DNA/RNA samples were analysed using quantitative reverse transcription PCR (Q-RT-PCR) and high-throughput sequencing. Fungi dominated both burrow and un-bioturbated sediment sequence libraries; however, there was significant enrichment of bacterivorous protists and nematodes in the burrows. There were also significant differences between the bacterial communities in burrows compared with un-bioturbated sediment. Increased activity and relative abundance of aerobic hydrocarbon-degrading bacteria in the burrows coincided with the significant reduction in hydrocarbon concentration in the bioturbated sediment. This study represents the first detailed assessment of the effect of bioturbation on total microbial communities in oil-contaminated sediments. In addition, it further shows that bioturbation is a significant factor in determining microbial diversity within polluted sediments and plays an important role in stimulating bioremediation.

Sediment contaminant surveillance in Milford Haven Waterway

Sediment contaminants were monitored in Milford Haven Waterway (MHW) since 1978 (hydrocarbons) and 1982 (metals), with the aim of providing surveillance of environmental quality in one of the UK’s busiest oil and gas ports. This aim is particularly important during and after large-scale investment in liquefied natural gas (LNG) facilities. However, methods inevitably have changed over the years, compounding the difficulties of coordinating sampling and analytical programmes. After a review by the MHW Environmental Surveillance Group (MHWESG), sediment hydrocarbon chemistry was investigated in detail in 2010. Natural Resources Wales (NRW) contributed their MHW data for 2007 and 2012, collected to assess the condition of the Special Area of Conservation (SAC) designated under the European Union Habitats Directive. Datasets during 2007-2012 have thus been more comparable. The results showed conclusively that a MHW-wide peak in concentrations of sediment polycyclic aromatic hydrocarbons (PAHs), metals and other contaminants occurred in late 2007. This was corroborated by independent annual monitoring at one centrally-located station with peaks in early 2008 and 2011. The spatial and temporal patterns of recovery from the 2007 peak, shown by MHW-wide surveys in 2010 and 2012, indicate several probable causes of contaminant trends, as follows: atmospheric deposition, catchment runoff, sediment resuspension from dredging, and construction of two LNG terminals and a power station. Adverse biological effects predictable in 2007 using international sediment quality guidelines, were independently tested by data from monitoring schemes of more than a decade duration in MHW (starfish, limpets), and in the wider SAC (grey seals). Although not proving cause and effect, many of these potential biological receptors showed a simultaneous negative response to the elevated 2007 contamination following intense dredging activity in 2006. Wetland bird counts were typically at a peak in the winter of 2005-2006 previous to peak dredging. In the following winter 2006-2007, shelduck in Pembroke River showed their lowest winter count, and spring 2007 was the largest ever drop in numbers of broods across MHW between successive breeding seasons. Wigeon counts in Pembroke River were again low in late 2012 after further dredging nearby. These results are strongly supported by PAH data reported previously from invertebrate bioaccumulation studies in MHW 2007-2010, themselves closely reflecting sediment

Polycyclic aromatic hydrocarbons (PAHS) (I): Toxicity, population exposure and involved foods

Food is one of the main exogenous sources of genotoxic compounds. In heated food products, polycyclic aromatic hydrocarbons (PAHs) represent a priority group of genotoxic, mutagenic and/or carcinogenic chemical pollutants with adverse long-term health effects. People can be exposed to these compounds through different environments and via various routes: inhalation, ingestion of foods and water and even percutaneously. The presence of these compounds in food may be due to environmental contamination, to industrial handling and processing of foods and to oil processing and refining. The highest levels of these compounds are found in smoked foods, in seafood which is found in polluted waters, in grilled meats and, to a lesser extent, in vegetable fats and oils. Lower levels of PAHs are found in vegetables and in cereals and its products.

Polycyclic aromatic hydrocarbons (PAHS) (I): Measures of prevention and control

Measures of prevention and control against polycyclic aromatic hydrocarbons (PAHs) focus on an official food control, a code of best practice to reduce PAHs levels by controlling industry and in the development of a chemopreventive strategy. Regulation (EU) 835/2011 establishes maximum levels of PAHs for each food group. In addition, Regulations (EU) 333/2007 and 836/2011 set up the methods of sampling and analysis for its official control. Scientific studies prove that the chemopreventive strategy is effective against these genotoxic compounds effects. Most chemopreventive compounds studied with proven protective effects against PAHs are found in fruit and vegetables.

The Effects of Several Paper Characteristics and Application Methods on the Sublimation Rate of Cyclododecane

Cyclododecane (CDD) is a waxy, solid cyclic hydrocarbon (C12H24) that sublimes at room temperature and possesses strong hydrophobicity. In paper conservation CDD is used principally as a temporary fixative of water-soluble media during aqueous treatments. Hydrophobicity, ease of reversibility, low toxicity, and absence of residues are reasons often cited for its use over alternative materials although the latter two claims continue to be debated in the literature. The sublimation rate has important implications for treatment planning as well as health and safety considerations given the dearth of reliable information on its toxicity and exposure limits. This study examined how the rate of sublimation is affected by fiber type, sizing, and surface finish as well as delivery in the molten phase and as a saturated solution in low boiling petroleum ether. The effect of warming the paper prior to application was also evaluated. Sublimation was monitored using gravimetric analysis after which samples were tested for residues with gas chromatography-flame ionization detection (GC-FID) to confirm complete sublimation. Water absorbency tests were conducted to determine whether this property is fully reestablished. Results suggested that the sublimation rate of CDD is affected minimally by all of the paper characteristics and application methods examined in this study. The main factors influencing the rate appear to be the thickness and mass of the CDD over a given surface area as well as temperature and ventilation. The GC-FID results showed that most of the CDD sublimed within several days of its disappearance from the paper surface regardless of the application method. Minimal changes occurred in the water absorbency of the samples following complete sublimation.