Influence of deep water formation by open-sea convection on the transport of low hydrophobicity organic pollutants in the NW Mediterranean Sea.

The significance of the offshore vertical convection currents in the transport and sinking of water-soluble organic pollutants into marine deep basins has been evaluated. For this purpose, sediment cores were collected in the Gulf of Lion (GoL) at sites between 26 and 2330 m water depth. The top core layers were analyzed for aromatic and aliphatic hydrocarbons and organochlorine compounds. Organic compounds with logKAW (air water partition coefficient) between -2 and -4, e.g. lindane, PCB 28, PCB 52, phenanthrene, methylphenanthrenes, dimethylphenanthrenes, C14-C23n-alkanes, are found in higher concentrations or exhibit relative concentration increases in the sediments deposited in the continental rise as consequence of the open-sea convection processes associated with the formation of Western Mediterranean Deep Water (WMDW). In contrast, the organic pollutants with intermediate air-water distribution coefficients, logKAW between -2 and 0, and high octanol water distribution coefficients (logKow > 6), e.g. highly chlorinated PCBs, DDTs, DDEs, DDDs, C25-C35n-alkanes, and polycyclic aromatic hydrocarbons with molecular weight higher than 200, occur in association to sediment particles, which are mainly transported by the Northern current along the continental shelf forming the mud belt. The Rhône prodelta is therefore the area of the GoL showing the highest concentrations of this group of organic compounds, which are preferentially associated with water particles. Overall, the results show that vertical open-sea convection processes related with offshore formation of WMDW may have an important role in the transport and accumulation of water soluble pollutants to deep marine environments of the GoL (>2000 m water depth).

Removal of bias in C37 alkenone-based sea surface temperature measurements by high-performance liquid chromatography fractionation.

Sea surface temperature determinations based on marine sedimentary C37 alkenone distributions have provided a wealth of data for paleoclimatic studies, including those performed at high resolution. The success of this approach results from several characteristics of alkenone compounds, e.g. their geochemical properties (such as unequivocal synthesis by certain widespread haptophyte algae, plus chemical stability/preservation of the original alkenone distributions during sedimentation), and their analytical properties (such as fast clean up procedures using alkaline hydrolysis of sediment extracts, followed by robust instrumental methods allowing large scale sample processing). Here we show that, in sediments under the influence of continental inputs, coelution of these compounds with cholest-5-enyl 3ß-undecenyl ether and 24-methylcholesta-5,22-dienyl 3ß-undecenyl ether deviate the SST measurements despite alkaline hydrolysis. Here, we report a new high performance liquid chromatrography fractionation method which eliminates these interfering compounds and gathers all the alkenones into a single fraction. These fractions can then be analysed by gas chromatography as in the initial approach, providing large amounts of data as required in high resolution studies.

Assessment of analytical methods to determine pyrethroids content of bednets.

OBJECTIVE: To present and evaluate simple, cost-effective tests to determine the amount of insecticide on treated materials. METHODS: We developed and evaluated a competitive immunoassay on two different platforms: a label-free impedimetric biosensor (EIS biosensor) and a lateral flow. Both approaches were validated by gas chromatography (GC) and ELISA, gold standards for analytical methods and immunoassays, respectively. Finally, commercially available pyrethroid-treated ITN samples were analysed. Different extraction methods were evaluated. RESULTS: Insecticide extraction by direct infusion of the ITN samples with dichloromethane and dioxane showed recovery efficiencies around 100% for insecticide-coated bednets, and >70% for insecticide-incorporated bednets. These results were comparable to those obtained with standard sonication methods. The competitive immunoassay characterisation with ELISA presented a dynamic range between 12 nm and 1.5 µm (coefficient of variation (CV) below 5%), with an IC50 at 138 nm, and a limit of detection (LOD) of 3.2 nm. EIS biosensor had a linear range between 1.7 nm and 61 nm (CV around 14%), with an IC50 at 10.4 nm, and a LOD of 0.6 nm. Finally, the lateral flow approach showed a dynamic range between 150 nm and 1.5 µm, an IC50 at 505 nm and a LOD of 67 nm. CONCLUSIONS: ELISA can replace chromatography as an accurate laboratory technique to determine insecticide concentration in bednets. The lateral flow approach developed can be used to estimate ITN insecticide concentration in the field. This new technology, coupled to the new extraction methods, should provide reliable guidelines for ITN use and replacement in the field.

Broad spectrum analysis of polar and apolar organic compounds in submicron atmospheric particles.

A method for the quantitative analysis of organic compounds on submicron particulate matter (PM1) collected on quartz filters was developed. The compounds analyzed encompassed C22-C35 alkanes, polycyclic aromatic hydrocarbons (PAHs), quinones, levoglucosan, cis-pinonic acid and short chain dicarboxylic acids such as malonic, succinic, glutaric, adipic, suberic, azelaic, malic and phthalic acids. The method included extraction with a pressure liquid extraction system, sample filtration though glass fibre filter, fractionation by high performance liquid chromatography and subsequent analysis by gas chromatography coupled to mass spectrometry. The study of the extraction efficiency of different solvent mixtures showed that DCM:MeOH 1:1 was the one providing the highest recoveries for all compounds. Extraction temperatures of 100°C provided better results than 60°C or 80°C. This method provided comparable extraction efficiency and qualitative and quantitative data to those involving Soxhlet extraction. Method recoveries for alkanes, most PAH, quinones and polar compounds calculated from spiked real samples were 52-72%, 78-101%, 50-62% and 76-104%, respectively, reproducibilities were 2-28%, 7-29%, 10-27% and 5-28%, respectively, limits of quantification were 0.01-0.1ng/m(3), 0.01-0.27ng/m(3), 0.04ng/m(3) and 0.32-2.8ng/m(3), respectively, which affords the quantification of a broad number of primary and secondary organic constituents of submicron aerosols.

A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.

Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

Persistent organic pollutant accumulation in seasonal snow along an altitudinal gradient in the Tyrolean Alps.

The snow capacity for storage of a large number of pollutants such as polybromodiphenyl ethers (PBDE), including BDE-209, polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs), hexachlorocyclohexanes (HCHs; α- and γ-isomers), endosulfans (α- and ß-isomers and the sulphate residue) and hexachlorobenzene (HCB), in a steep altitudinal gradient (1,101-2,500 m above sea level (asl); maximum planar distance 16 km) in a typical European mountain system, the Tyrolean Alps (Austria), was studied here for the first time. Snow samples representing the whole snowpack accumulated at the end of the cold season were collected in all cases. The snow specific surface area (SSA) of these samples, 140-260 cm(2) g(-1), was characteristic of aged snow with low retention capacity. PAHs were the pollutant group in highest concentrations (500-8,400 pg L(-1)). PCBs and PBDEs were found in concentrations of 460-900 and 8.5-290 pg L(-1), respectively. From the fourteen investigated BDE congeners, only BDE-47, BDE-99, BDE-100 and BDE-209 were found above the detection limit, which is consistent with the results found in the only previous study in the Tatra Mountains (Slovakia) which also involved a steep gradient (1,683-2,634 m asl; maximum planar distance 5 km; Arellano et al. 2011) and confirm the capacity of these low-volatile compounds for long-range transport from distant sources. HCB was found in a concentration range of 34-55 pg L(-1). Snow deposition fluxes of PCB-118, PCB-153, γ-HCH, α-endosulfan and BDE-47 showed statistically significant correlations with altitude, involving higher values at higher elevation. This trend may reflect cold trapping effects in view of the snow particle contents and SSA values. However, these gradients were only significant for this limited number of compounds within each pollutant group which may be explained by differences in physical-chemical properties of the compounds and the limited capacity of the aged snow for organic pollutant retention. In some other cases, for example benzo[a]pyrene, the observed vertical gradients may reflect higher preservations at lower temperatures.

Changes in lipid content and fatty acid composition along the reproductive cycle of the freshwater mussel Dreissena polymorpha: its modulation by clofibrate exposure.

Total lipids and fatty acid profiles were determined along the reproductive cycle of the zebra mussel (Dreissena polymorpha). A total of 33 fatty acids with carbon atoms from 14 to 22 were identified: palmitic acid (16:0) was the most abundant fatty acid (13-24%) followed by docosahexaenoic acid (DHA; 22:6n-3), eicosapentaenoic acid (EPA; 20:5n-3) and palmitoleic acid (16:1n-7). Some individual fatty acids (16:0, 16:2n-4, 18:1n-7, 18:2n-6, 18:3n-4, 18:4n-3, 20:4n-3, 20:5n-3) were strongly related to reproductive events, while others having structural-type functions (18:0 and 22:6n-3) were rather stable during the study period. Multivariate analysis of the whole data set using the multivariate curve resolution alternating least squares method confirmed the strong relationship of fatty acid profiles with the reproductive cycle of zebra mussel. Additionally, the effects of the pharmaceutical clofibrate on lipid composition and fatty acid profiles were assessed following 7-day exposure of zebra mussels to a wide range of concentrations (20 ng/L to 2 mg/L). A significant reduction in total triglycerides (38%-48%) together with an increase in the amount of fatty acids per gram wet weight (1.5- to 2.2-fold) was observed in the exposed mussels. This work highlights the ability of clofibrate to induce changes on the lipidome of zebra mussels at concentrations as low as 200 ng/L.

Transformation of PBDE mixtures during sediment transport and resuspension in marine environments (Gulf of Lion, NW Mediterranean Sea).

Polybromodiphenyl ethers (PBDEs) in superficial sediments from the Gulf of Lion were studied. They were largely predominated by BDE 209 (98.7% of all PBDEs) indicating that the main source of these pollutants was the commercial mixture deca-BDE. This compound and the less brominated BDE exhibited a southwestward decreasing concentration gradient following the dominant marine currents and bottom relief, e.g. the Mud Belt, the submarine canyons and the Open Continental Slope. All PBDEs exhibited statistically significant correlations confirming the common origin. However, a progressive transformation of the dumped BDE 209 was identified showing a depletion paralleled by increases of the less brominated BDEs (from 8.6% to 22%). These less brominated compounds were accumulated at about 100-140 km away from the Rhone prodelta, e.g., at the end of the submarine canyons, evidencing that these transformation compounds can be accumulated at long distances from the dumping sites in the marine system.

Influences of natural emission sources (wildfires and Saharan dust) on the urban organic aerosol in Barcelona (Western Mediterranean Basis) during a PM event.

The urban air quality in Barcelona in the Western Mediterranean Basin is characterized by overall high particulate matter (PM) concentrations, due to intensive local anthropogenic emissions and specific meteorological conditions. Moreover, on several days, especially in summer, natural PM sources, such as long-range transported Saharan dust from Northern Africa or wildfires on the Iberian Peninsula and around the Mediterranean Basin, may influence the levels and composition of the organic aerosol. In the second half of July 2009, daily collected PM(10) filter samples in an urban background site in Barcelona were analyzed on organic tracer compounds representing several emission sources. During this period, an important PM peak event was observed. Individual organic compound concentrations increased two to five times during this event. Although highest increase was observed for the organic tracer of biomass burning, the contribution to the organic aerosol was estimated to be around 6 %. Organic tracers that could be related to Saharan dust showed no correlation with the PM and OC levels, while this was the case for those related to fossil fuel combustion from traffic emissions. Moreover, a change in the meteorological conditions gave way to an overall increase of the urban background contamination. Long-range atmospheric transport of organic compounds from primary emissions sources (i.e., wildfires and Saharan dust) has a relatively moderate impact on the organic aerosol in an urban area where the local emissions are dominating.

Role of dense shelf water cascading in the transfer of organochlorine compounds to open marine waters.

Settling particles were collected by an array of sediment trap moorings deployed along the Cap de Creus (CCC) and Lacaze-Duthiers (LDC) submarine canyons and on the adjacent southern open slope (SOS) between October 2005 and October 2006. This array collected particles during common settling processes and particles transferred to deep waters by dense shelf water cascading (DSWC). Polychlorobiphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs), chlorobenzenes (CBzs)--pentachlorobenzene and hexachlorobenzene--and hexachlorocyclohexanes were analyzed in all samples. The results show much higher settling fluxes of these compounds during DSWC than during common sedimentation processes. The area of highest deposition was located between 1000 and 1500 m depth and extended along the canyons and outside them showing their channelling effects but also overflows of dense shelf water from these canyons. Higher fluxes were observed near the bottom (30 m above bottom; mab) than at intermediate waters (500 mab) which is consistent with the formation and sinking of dense water close to the continental shelf and main displacement through the slope by the bottom. DSWC involved the highest settling fluxes of these compounds ever described in marine continental slopes and pelagic areas, e.g., peak values of PCBs (960 ng · m(-2) · d(-1)), DDTs (2900 ng · m(-2) · d(-1)), CBzs (340 ng · m(-2) · d(-1)) and lindane (180 ng · m(-2) · d(-1)).

Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the most common airborne fungal pathogen for humans. In this mold, iron starvation induces production of the siderophore triacetylfusarinine C (TAFC). Here we demonstrate a link between TAFC and ergosterol biosynthetic pathways, which are both critical for virulence and treatment of fungal infections. Consistent with mevalonate being a limiting prerequisite for TAFC biosynthesis, we observed increased expression of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron starvation, reduced TAFC biosynthesis following lovastatin-mediated Hmg1 inhibition, and increased TAFC biosynthesis following Hmg1 overexpression. We identified enzymes, the acyl-CoA ligase SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of mevalonate and TAFC, deficiency of which under iron starvation impaired TAFC biosynthesis, growth, oxidative stress resistance, and murine virulence. Moreover, inactivation of these enzymes alleviated TAFC-derived biosynthetic demand for mevalonate, as evidenced by increased resistance to lovastatin. Concordant with bilateral demand for mevalonate, iron starvation decreased the ergosterol content and composition, a phenotype that is mitigated in TAFC-lacking mutants.

Ergosterol biosynthesis pathway in Aspergillus fumigatus.

The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus.

Altitudinal gradients of PBDEs and PCBs in fish from European high mountain lakes.

A first case of temperature-dependent distribution of polybromodiphenyl ethers (PBDEs) in remote areas is shown. Analysis of these compounds in fish from Pyrenean lakes distributed along an altitudinal transect shows higher concentrations at lower temperatures, as predicted in the global distillation model. Conversely, no temperature-dependent distribution is observed in a similar transect in the Tatra mountains (Central Europe) nor in fish from high mountain lakes distributed throughout Europe. The fish concentrations of polychlorobiphenyls (PCBs) examined for comparison showed significant temperature correlations in all these studied lakes. Cold trapping of both PCBs and PBDEs concerned the less volatile congeners. In the Pyrenean lake transect the concentrations of PCBs and PBDEs in fish were correlated despite the distinct use of these compounds and their 40 year time lag of emissions to the environment. Thus, temperature effects have overcome these anthropogenic differences constituting at present the main process determining their distributions. These cases of distinct PBDEs and PCBs behavior in high mountains likely reflect early stages in the environmental distribution of the former since they have been under secondary redistribution processes over much shorter time than the latter.

Reassessment of the structural composition of the alkenone distributions in natural environments using an improved method for double bond location based on GC-MS analysis of cyclopropylimines.

The usefulness of n-propyl-, iso-propyl-, and cyclopropylamines for the location of double bonds positions in C37-C40 alkenones after formation of imino derivatives has been evaluated. Cyclopropylamine is the best reagent for its high reaction yields, GC retention time difference between derivatives and precursor compounds, and absence of generation of byproducts. The use of this C3 amine involves higher sensitivity and ease of application than previously reported C5 amines. Examination of a large group of alkenones from cultures of Emiliania huxleyi, water particles, and recent and ancient sediments with cyclopropylamine derivatization shows that, in all cases, the double bonds were located at the same carbon atom distance from the carbonyl group, and spaced in intervals of five methylene groups either from the carbonyl or between them, e.g., at sites 7, 14, 21, and 28. This result represents a correction from previous assumptions in which double-bond positions were situated by reference to the methyl end. 4,4-Dimethyloxazoline derivatization of hexatriacontenoates showed that these compounds have also their unsaturations with seven carbon atom spacing and counting by reference to the carboxyl group. The concurrence of both series of isomers in compounds of different oxygen functionalities indicates that the precursor haptophycean algal species have a major biosynthetic pathway leading to the formation of these lipids. The data presented in this work unify the structures of the known alkenones in the present and the recent past under a common metabolic pathway.

Aspergillus fumigatus C-5 sterol desaturases Erg3A and Erg3B: role in sterol biosynthesis and antifungal drug susceptibility.

Two erg3 genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatus were characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3A and erg3B genes and a double gene knockout, erg3A- erg3B-, showed that they are not essential for A. fumigatus viability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatus ergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3A and erg3B were knocked out alone and in combination.

Phenyl- and cyclopentylimino derivatization for double bond location in unsaturated C(37)-C(40) alkenones by GC-MS.

A method for the identification of double bond locations in polyunsaturated long chain alkenones adapted to nanogram amounts as currently analyzed by gas chromatography coupled to mass spectrometry (GC-MS) has been developed. The method is based on interpretation of the electron impact mass spectra of the imino derivatives of the carbonyl groups using either cyclopentyl or phenyl substitutents. Other complementary derivatization methods such as elaidization and hydrogenation have also been used for structural characterization of these compounds. This application has led to the identification of a novel homologous series of di-, tri-, and tetraunsaturated ketones with carbon number chain lengths between 37 and 40 in coastal hypersaline sediments. The novel series identified shows a distribution in which the double bond position between different homologs is established by reference to the distance from the carbonyl group whereas the previously known alkenones were constituted by unsaturated homologs with double bonds located at defined distances of the terminal methyl. This difference points out to a dissimilar, but still unknown, biogenic precursor of these novel alkenones.