Optimization of pressurized liquid extraction using a multivariate chemometric approach and comparison of solid phase extraction cleanup steps for the determination of polycyclic aromatic hydrocarbons in mosses

A factorial design was used to optimize the extraction of polycyclic aromatic hydrocarbons (PAHs) from mosses, plants used as biomonitors of air pollution. The analytical procedure consists of pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) cleanup, in association with analysis by high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). For method development, homogeneous samples were prepared with large quantities of the mosses Isothecium myosuroides Brid. and Hypnum cupressiforme Hedw., collected from a Spanish Nature Reserve. A factorial design was used to identify the optimal PLE operational conditions: 2 static cycles of 5 min at 80°C. The analytical procedure performed with PLE showed similar recoveries (∼70%) and total PAH concentrations (∼200 ng g-1) as found using Soxtec extraction, with the advantage of reducing solvent consumption by 3 (30 mL against 100 mL per sample), and taking a fifth of the time (24 samples extracted automatically in 8 h against 2 samples in 3.5 h). The performance of SPE normal phases (NH2, Florisil®, silica and activated aluminium) generally used for organic matrix cleanup was also compared. Florisil® appeared to be the most selective phase and ensured the highest PAH recoveries. The optimal analytical procedure was validated with a reference material and applied to moss samples from a remote Spanish site in order to determine spatial and inter-species variability

Biosurveillance des retombées atmosphériques à l'aide de bryophytes : suivi des hydrocarbures aromatiques polycycliques (HAP) à diverses échelles spatio-temporelles

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants (POP) émis par les activités humaines suite à la combustion incomplète de la matière organique (industrie, chauffage, trafic routier...). En raison de leur faible taux de dégradation, de leur toxicité, de leur capacité à être transportés sur de longues distances et de leur bioaccumulation dans la matière organique, les HAP font l'objet d'une surveillance atmosphérique (directive cadre européenne 96/62/CE du 27 septembre 1996). Les bryophytes, plus communément appelées mousses, sont utilisées depuis une trentaine d'années pour la biosurveillance de la qualité de l'air. Leurs caractéristiques biologiques font de ces plantes terrestres d'excellents bioaccumulateurs des dépôts de polluants atmosphériques. Cependant, elles ont été peu utilisées pour la biosurveillance des HAP et pour des échelles spatiales et temporelles très limitées. Dans ce contexte, la thèse a pour objectif de valider l'utilisation de bryophytes comme outils pour la biosurveillance des retombées atmosphériques de HAP à différentes échelles spatio-temporelles. La première phase de ce travail a consisté en un développement analytique afin de valider l'analyse des HAP par chromatographie liquide associée à une détection par fluorescence (CLHP-FLD), l'extraction liquide-liquide des HAP contenus dans les dépôts totaux et l'extraction par liquide pressurisé (PLE) des HAP contenus dans les mousses. Cette dernière a été optimisée, grâce à la réalisation d'un plan d'expériences, et a été complétée par des tests de purification par extraction sur phase solide (SPE). Des études de terrain ont ensuite été réalisées afin d'évaluer les tendances temporelles à l'échelle saisonnière, annuelle et historique, ainsi que les tendances spatiales à l'échelle locale et régionale, des teneurs en HAP dans les mousses. Une étude menée sur des mousses d'herbier prélevées dans le Parc Naturel de Bertiz (Navarre, Espagne) entre 1879 et 2007 a permis d'identifier les sources historiques d'émission des HAP. Les bryophytes apparaissent comme de bons « témoins » de la contamination atmosphérique du passé. Une étude annuelle (2010-2011) de biosurveillance active menée à Bertiz a permis ensuite d'observer des tendances saisonnières des teneurs en HAP dans les mousses Hylocomium splendens (Hedw.) Schimp., liées aux variations des émissions atmosphériques et des conditions météorologiques, en particulier des précipitations. De plus, l'étude annuelle a révélé un fractionnement lors de l'accumulation par les mousses des HAP contenus dans les dépôts totaux, qui résulte d'une affinité des HAP pour les dépôts liée à leur solubilité et d'une affinité des HAP pour le végétal liée à leur propriété lipophile (KOW). Ainsi, le suivi des flux atmosphériques de HAP dans les dépôts totaux ne permet pas de prévoir a priori les teneurs de HAP dans les écosystèmes. Enfin, une analyse multivariée de la distribution spatiale des teneurs en HAP dans l'espèce Hypnum cupressiforme Hedw. prélevée en Espagne (Navarre), France (Île-de-France) et Suisse (Plateau Suisse et région de Bâle) a permis de caractériser les sources de contamination atmosphériques des régions étudiées et l'influence de paramètres environnementaux tels que l'altitude et la pluviométrie. ABSTRACT : Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) emitted by human activities during incomplete combustion of organic matter (industry, domestic heating, road traffic…). Due to their low degradation rates, toxicity, long-range transport and bioaccumulation in organic matter, PAHs are monitored in the atmosphere (Council Directive 96/62/EC of 27 September 1996). Bryophytes, more commonly called mosses, have been used during the past three decades for biomonitoring air quality. Because of their biological characteristics, these terrestrial plants are excellent bioaccumulators of airborne pollutants. However, few studies concern PAH biomonitoring and they are usually restricted to studies limited in time and space. In this context, the thesis aims to validate bryophytes as tools for biomonitoring atmospheric deposition of PAHs on various space-time scales. The first step of this work consisted in developing the analytical procedures in order to validate PAH analysis by liquid chromatography associated with fluorescence detection (HPLC-FLD), liquid-liquid extraction of PAHs from bulk deposition and pressurized liquid extraction (PLE) of PAHs from mosses. The latter was optimized with a factorial design and was completed by purification tests with solid phase extraction (SPE). Field studies were then carried out to evaluate temporal trends of PAH levels in mosses at seasonal, annual and historical scales, as well as spatial trends, at local and regional scales. A study undertaken on herbarium mosses sampled in Bertiz Natural Park (Navarra, Spain) between 1879 and 2007 leaded to the identification of PAH historical emission sources. Bryophytes appear as excellent “witnesses” of the past atmospheric contamination. An annual biomonitoring study (2010-2011) at Bertiz showed seasonal trends of PAH content in mosses Hylocomium splendens (Hedw.) Schimp., which are related to variations of PAH emissions and weather conditions, particularly precipitations. Moreover, the annual study revealed a fractionation of PAHs while they are transferred from total deposition to the mosses, as the affinity of PAHs for deposition samples was related with their solubility and their affinity for the plant was related to their lipophilic property (KOW). Thus, monitoring PAH fluxes in total deposition does not give an a priori estimation of PAH contents in the ecosystems. Finally, multivariate analysis of the spatial distribution of PAHs in the species Hypnum cupressiforme Hedw. sampled in Spain (Navarra), France (Île-de-France) and Switzerland (Swiss Plateau and Basel area) leaded to the characterization of the atmospheric contamination sources in the different areas and the influence of environmental parameters such as elevation and pluviometry

"Better people make better players": exploring the role of character in rugby union talent identification

Character and rugby union are historically linked with governing bodies, stakeholders and media all citing its importance for rugby union performance. Recent years have seen an increase in the prevalence of talent identification programmes in professional sports, rugby union included. However, little is known about the importance of psychological characteristics, less so character in rugby union talent identification. A literature review of psychological components of talent identification was conducted, with a focus on the application to Rugby Union. A more concerted effort needs to be made by researchers to define and differentiate the psychological components clearly to allow for more diverse research methodologies to take place and further the rugby union talent identification literature. To investigate the role character in the talent identification process in rugby union, a qualitative design was used. Interviews were conducted with 9 rugby union talent identification experts which focused on the components of character they valued most in potential professional players. A multidimensional understanding of character was used to analyse the aspects most valuable. No unified understanding of character exists in rugby union. However, it was deemed as especially important by stakeholders especially performance character virtues such as industriousness, resilience, and perseverance. Moral character was second most valued by participants including virtues, self-regulation, self-awareness, and honesty. These findings were then discussed in relation to current practices and further research directions were outlined

Reconstructing historical trends of polycyclic aromatic hydrocarbon deposition in a remote area of Spain using herbarium moss material

Herbarium mosses from 1879-1881, 1973-1975 and 2006-2007 were used to investigate the historical changes of atmospheric deposition of polycyclic aromatic hydrocarbons (PAHs) at a remote site located in Northern Spain. Natural abundance of nitrogen and carbon isotopes was also measured in order to assess the evolution of emissions from anthropogenic sources. Nitrogen concentrations, 13C, 13N and PAH levels were significantly higher in 19th century samples with respect to the present century samples. Moreover, PAH distribution varied over the centuries, following a tendency of light PAH enrichment. The carbon, nitrogen and PAH levels measured in the mosses tally with the historic evolution of anthropogenic emissions in the area, mainly influenced by changes of economic activities, domestic heating and road traffic density. Mosses provided by herbaria seem to offer the possibility to study long-term temporal evolution of atmospheric PAH deposition

History of the New Caledonia Barrier Reef over the last 1.2Myrs: links with regional palaeoceanography and palaeoclimate

The timing of glacial-interglacial cycles shows a clear dependence on the periodic variations in the Earth’s orbital parameters. However, the Earth’s climate is an extremely complex, non-linear system, with many internal feedback mechanisms and there are still features of the climate record for which a definitive explanation remains elusive. Understanding reef history is important due to significant predicted feedbacks between changes in global climate and carbonate production via the carbon cycle; phases of rapid reef growth in shallow water areas being associated with increased release of carbon dioxide to the atmosphere. Previous work on Pleistocene reef history, investigated via reef boreholes, shows a large global expansion of reefs between 800-400ka; approximately concurrent with one of the major unexplained alterations in the climate system, the Mid-Pleistocene Transition (MPT). Quaternary reef history is usually investigated via reef boreholes, which provide limited spatial information and are subject to dating uncertainties of the order of ±100kyrs. This means that any inferences made about the relationship between reef expansion and specific changes in the climate system are not well constrained. This thesis instead, presents a novel approach to reconstructing reef growth history, using a trial site near the island of New Caledonia, in the south west Pacific. The initiation of carbonate production on shallow shelves is known to produce a signal in the surrounding deeper basins, via sediment shedding. Therefore, this research set out to independently verify the proposed expansion of the New Caledonia Barrier Reef at ~ 400ka (Marine Isotope Stage [MIS] 11) by examining the composition of turbidites deposited in the New Caledonia Trough. Deep sea sediment core MD06-3019, was collected south west of the New Caledonia Barrier Reef (22oS, 165oE; 3,500m water depth). It is predominantly composed of pelagic carbonate ooze, into which 79 sandy turbidite layers have been deposited. These layers interrupt, but do not disturb, the background sedimentation and source material from the shallow shelf, which is carried to the deep sea via submarine canyons. A core age model based principally on orbital tuning, yields a core bottom age of 1,260ka, ~MIS38. This chronology has allowed the timing of deposition of the turbidite layers within the core to be assigned to within ±10kyr. Turbidite layers vary in width (1-35cm), grain size (φ=4 to -2) and composition, containing among other shelf derived material, well preserved coral fragments from 1.26Ma through to the present day. Patterns in turbidite timing and frequency, grain size and composition (investigated via point counting, carbonate coulometry and aragonite content) have been analysed, to assess whether there are any temporal changes which may reflect variation in shallow shelf reef extent. This included the development of XRF scanning measurements for [Sr], as a new proxy for the aragonite content of samples. A calibration line with the equation: Aragonite %=0.0011* Sr count +2.64 (R2 =0.6105, p-value < 0.001) was obtained for turbidite samples from sediment core MD06-3019. The method shows significant promise as a new proxy for quickly establishing the aragonite content of sediment samples. Corroborating the work of previous investigators, turbidites deposited since MIS11 show an increase in average bulk carbonate and aragonite content, a greater dominance of shallow water bioclasts and a higher occurrence of coral fragments. Additionally, both coarse and fine grained turbidites are present, whereas directly before this period only fine grained turbidites occur. However, there is another significant shift in depositional style further back in the record. Prior to MIS23 both coarse and fine grained turbidites are present, the average carbonate content of turbidite layers is higher and there is a greater dominance of shallow water biota. Coral abundance for turbidites at the base of the core can equal values for turbidites at the top of the core. These results challenge the assumption that the only significant evolution on the western New Caledonia margin over the last 1.2Myrs was the expansion of the barrier reef at MIS11. This suggests that the history of the western New Caledonia margin may be more complicated than initially anticipated. These temporal variations in turbidite deposition are interpreted as reflecting changes in the level of carbonate production on the shallow shelf over the course of the 1.26Myr record. Shallow water carbonate production having decreased substantially during the period MIS23-MIS11. There are many possible controls on the shallow water carbonate production; such as: sea surface temperature and salinity, sea-level and nutrient availability. However, it is hypothesised that the principal control is glacial-interglacial sea-level change. It is proposed that prior to MIS23 sea-level was high enough during certain interglacial periods for significant carbonate production to occur on the shelf. However, from MIS23 onwards the climate proceeded into a period of ‘lukewarm’ interglacials which were both cooler and had lower sea-levels. It is hypothesised that during this period sea-level did not rise enough during highstands to flood the shelf sufficiently to allow for significant shallow water carbonate production. The high sea-levels of the long, warm MIS11then allowed for the expansion of the barrier reef (perhaps on substrates provided by former siliciclastic coast lines, deposited between MIS23-11) and its continuation during subsequent interglacial periods until the current day. This pattern of shallow shelf carbonate production is similar to those proposed for the Belize margin and the Gulf of Papua over the last 1.2Myrs. This thesis provides one of the first detailed investigations of gravity deposits in the New Caledonia Trough, providing information on their composition and timing over an unprecedented 1.26Myr time period. This study demonstrates that deep sea turbidites, sourced from shallow shelf areas, can be used to help reconstruct tropical reef growth histories. The results corroborate the work of previous researchers in the area and provide new insights into the history of reefs along the western New Caledonian margin. The main advantage of this method, compared to traditional borehole techniques, is the 10-fold reduction in the age uncertainty of events, to ±10 kyr. In addition, because turbidite material is sourced from a wide area along the coast, the method is able to provide information on reef history over a larger spatial area than single reef boreholes. This method can now be extended globally to help improve knowledge of the timing and history of tropical reef growth during the Quaternary. This will enable a better understanding of how reefs have impacted on, and been affected by, changes in climate, linked by feedbacks mechanisms via the global carbon cycle

Spatial distribution of PAH concentrations and stable isotope signatures (δ13C, δ15N) in mosses from three European areas – Characterization by multivariate analysis

Polycyclic aromatic hydrocarbon (PAH) concentrations and N, C stable isotope signatures were determined in mosses Hypnum cupressiforme Hedw. from 61 sites of 3 European regions: Île-de-France (France); Navarra (Spain); the Swiss Plateau and Basel area (Switzerland). Total PAH concentrations of 100-700 ng g-1, as well as δ13C values of -32 to -29‰ and δ15N values of -11 to -3‰ were measured. Pearson correlation tests revealed opposite trends between high molecular weight PAH (4-6 aromatic rings) content and δ13C values. Partial Least Square regressions explained the very significant correlations (r > 0.91, p < 0.001) between high molecular weight PAH concentrations by local urban land use (<10 km) and environmental factors such as elevation and pluviometry. Finally, specific correlations between heavy metal and PAH concentrations were attributed to industrial emissions in Switzerland and road traffic emissions in Spain

Mosses as an integrating tool for monitoring PAH atmospheric deposition: Comparison with total deposition and evaluation of bioconcentration factors. A year-long case-study

Polycyclic aromatic hydrocarbon (PAH) atmospheric deposition was evaluated at a remote site in Northern Spain using moss biomonitoring with Hylocomium splendens (Hedw.) Schimp., and by measuring the total deposition fluxes of PAHs. The year-long study allowed seasonal variations of PAH content in mosses to be observed, and these followed a similar trend to those of PAH fluxes in total deposition. Generally, atmospheric deposition of PAHs is greater in winter than in summer, due to more PAH emissions from domestic heating, less photoreactivity of the compounds, and intense leaching of the atmosphere by wet deposition. However, fractionation of these molecules between the environmental compartments occurs: PAH fluxes in total deposition and PAH concentrations in mosses are correlated with their solubility (r=0.852, p<0.01) and lipophilic properties (KOW, r=0.768, p<0.01), respectively. This annual study therefore showed that atmospheric PAH fluxes can be estimated with moss biomonitoring data if the bioconcentration or ‘enriching’ factors are known

A novel transcript of oil palm (Elaeis guineensis Jacq.), Eg707, is specifically upregulated in tissues related to totipotency

In this study, we report the molecular characterization of clone Eg707 isolated from cell suspension culture of the oil palm. The deduced polypeptide of clone Eg707 is highly similar to an unknown protein from Arabidopsis thaliana. The presence of an Ald-Xan-dh-C2 superfamily domain in the deduced protein sequence suggested that Eg707 protein might be involved in abscisic acid biosynthesis. Eg707 might be present as a single copy gene in the oil palm genome. This gene is highly expressed in tissue cultured materials compared to vegetative and reproductive tissues, suggesting a role of this gene during oil palm somatic embryogenesis or at the early stages of embryo development. Expression analysis of Eg707 by RNA in situ hybridization showed that Eg707 transcripts were present throughout somatic embryo development starting from proembryo formation at the embryogenic callus stages till the maturing embryo stages. Since proembryo formation within the embryogenic callus is one of the first key factors in oil palm somatic embryo development, it is suggested that Eg707 could be used as a reliable molecular marker for detecting early stage of oil palm somatic embryogenesis

Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L.

BackgroundCapsicum is a genus of important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava via a biolistic particle delivery system. In addition, in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established.ResultsIn this biolistic system study, explant target distance, bombardment helium (He) pressure and the size of microcarrier were the key parameters to be investigated. The optimized parameters based on screening of GFP expression were determined to be 6 cm target distance, 1350 psi of helium pressure and 1.6 ?m of gold particle (microcarrier) size. The greatest number of shoots were obtained from hypocotyl as explant using Murashige and Skoog medium supplemented with 5.0 mg/L BAP and 0.1 mg/L NAA. An average of 5 shoots per explant were formed. Out of which, one shoot managed to form root and developed into whole plant.ConclusionsWe have obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of the chili plants