Size determination and quantification of engineered cerium oxide nanoparticles by flow field-flow fractionation coupled to inductively coupled plasma mass spectrometry.

Facing the lack of studies on characterization and quantification of cerium oxide nanoparticles (CeO2 NPs), whose consumption and release is greatly increasing, this work proposes a method for their sizing and quantification by Flow Field-flow Fractionation (FFFF) coupled to Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Two modalities of FFFF (Asymmetric Flow- and Hollow Fiber-Flow Field Flow Fractionation, AF4 and HF5, respectively) are compared, and their advantages and limitations discussed. Experimental conditions (carrier composition, pH, ionic strength, crossflow and carrier flow rates) are studied in detail in terms of NP separation, recovery, and repeatability. Size characterization of CeO2 NPs was addressed by different approaches. In the absence of feasible size standards of CeO2 NPs, suspensions of Ag, Au, and SiO2 NPs of known size were investigated. Ag and Au NPs failed to show a comparable behavior to that of the CeO2 NPs, whereas the use of SiO2 NPs provided size estimations in agreement to those predicted by the theory. The latter approach was thus used for characterizing the size of CeO2 NPs in a commercial suspension. Results were in adequate concordance with those achieved by transmission electron microscopy, X-ray diffraction and dynamic light scattering. The quantification of CeO2 NPs in the commercial suspension by AF4-ICP-MS required the use of a CeO2 NPs standards, since the use of ionic cerium resulted in low recoveries (99 ± 9% vs. 73 ± 7%, respectively). A limit of detection of 0.9 µg L(-1) CeO2 corresponding to a number concentration of 1.8 × 1012 L(-1) for NPs of 5 nm was achieved for an injection volume of 100 µL.

Thallium occurrence and partitioning in soils and sediments affected by mining activities in Madrid province (Spain).

Thallium (Tl) and its compounds are toxic to biota even at low concentrations but little is known about Tl concentration and speciation in soils. An understanding of the source, mobility, and dispersion of Tl is necessary to evaluate the environmental impact of Tl pollution cases. In this paper, we examine the Tl source and dispersion in two areas affected by abandoned mine facilities whose residues remain dumped on-site affecting to soils and sediments of natural water courses near Madrid city (Spain). Total Tl contents and partitioning in soil solid phases as determined by means of a sequential extraction procedure were also examined in soils along the riverbeds of an ephemeral and a permanent streams collecting water runoff and drainage from the mines wastes. Lastly, electronic microscopy and cathodoluminescence probe are used as a suitable technique for Tl elemental detection on thallium-bearing phases. Tl was found mainly bound to quartz and alumino-phyllosilicates in both rocks and examined soils. Besides, Tl was also frequently found associated to organic particles and diatom frustules in all samples from both mine scenarios. These biogenic silicates may regulate the transfer of Tl into the soil-water system.

Spread and partitioning of arsenic in soils from a mine waste site in Madrid province (Spain).

The formation of scorodite is an important mechanism for the natural attenuation of arsenic in a wide range of environments. It is dumped on site by metallurgical industries to minimize arsenic release. However, the long-term stability of these deposits is unclear. Sequential As extractions and synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy were used to determine both As and Fe speciation in a small catchment area affected by a scorodite-rich waste pile at an abandoned smelting factory. Our results indicate that this deposit behaves as an acute point source of As and metal pollution and confirms the strong association of As(V) with Fe(III) oxide phases, highlighting the important role of ferrihydrite as an As scavenger in natural systems. In this seasonally variable system, other trapping forms such as jarosite-like minerals also play a role in the attenuation of As. Overall, our results demonstrate that scorodite should not be considered an environmental stable repository for As attenuation when dumped outside because natural rainfall and the resulting runoff drive As dispersion in the environment and indicate the need to monitor and reclamate As-rich mine deposits.

Detection and characterization of silver nanoparticles and dissolved species of silver in culture medium and cells by AsFlFFF-UV-Vis-ICPMS: application to nanotoxicity tests.

A methodology based on Asymmetric Flow Field-Flow Fractionation (AsFlFFF) coupled with UV-Vis absorption spectrometry and ICP mass spectrometry (ICPMS) has been developed and applied to the study of silver nanoparticles (AgNPs) and dissolved species of silver in culture media and cells used in cytotoxicity tests. The effect of a nano-silver based product (protein stabilized silver nanoparticles ca. 15 nm average diameter) on human hepatoma (HepG2) cell viability has been studied. UV-Vis absorption spectrometry provided information about the nature (organic vs. nanoparticle) of the eluted species, whereas the silver was monitored by ICPMS. A shift towards larger hydrodynamic diameters was observed in the AgNPs after a 24 hour incubation period in the culture medium, which suggests a "protein corona" effect. Silver(I) associated with proteins present in the culture medium has also been detected, as a consequence of the oxidation process experimented by the AgNPs. However, the Ag(I) released into the culture medium did not justify the toxicity levels observed. AgNPs associated with the cultured HepG2 cells were also identified by AsFlFFF, after applying a solubilisation process based on the use of tetramethylammonium hydroxide (TMAH) and Triton X-100. These results have been confirmed by transmission electronic microscopy (TEM) analysis of the fractions collected from the AsFlFFF. The effect of AgNPs on HepG2 cells has been compared to that caused by silver(I) as AgNO3 under the same conditions. The determination of the total content of silver in the cells confirms that a much larger mass of silver as AgNPs with respect to AgNO3 (16 to 1) is needed to observe a similar toxicity.

Size characterization and quantification of silver nanoparticles by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry.

A method for determining the size of silver nanoparticles and their quantification by asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (ICP-MS) is proposed and was tested in consumer products. Experimental conditions were studied in detail to avoid aggregation processes or alteration of the original size distributions. Additionally, losses from sorption processes onto the channel membrane were minimized for correct quantification of the nanoparticles. Mobile phase composition, injection/focusing, and fractionation conditions were evaluated in terms of their influence on both separation resolution and recovery. The ionic strength, pH, and the presence of ionic and nonionic surfactants had a strong influence on both separation and recovery of the nanoparticles. In general, better results were obtained under those conditions that favored charge repulsions with the membrane. Recovery values of 83 ± 8% and 93 ± 4% with respect to the content of silver nanoparticles were achieved for the consumer products studied. Silver nanoparticle standards were used for size calibration of the channel. The results were compared with those obtained by photon correlation spectroscopy and images taken by transmission electron microscopy. The quantification of silver nanoparticles was performed by direct injection of ionic silver standard solutions into the ICP-MS system, integration of the corresponding peaks, and interpolation of the fractogram area. A limit of detection of 5.6 µg L(-1) silver, which corresponds to a number concentration of 1×10(12) L(-1) for nanoparticles of 10 nm, was achieved for an injection volume of 20 µL.

Study of the size-based environmental availability of metals associated to natural organic matter by stable isotope exchange and quadrupole inductively coupled plasma mass spectrometry coupled to asymmetrical flow field flow fractionation.

The determination of the isotopically exchangeable fraction of metals in environmental solid samples (soils, composts, sediments, sludges, etc.) is used to know the amount of metal potentially available (E-value). Stable isotopes can be used for determination of E-values through the analysis of the aqueous phases from spiked suspensions. However, the presence of isotopically non-exchangeable metal forms in the aqueous phase led to overestimation of the E-values. In this paper, a method for monitoring the degree of isotopic exchange in function of the molecular mass and/or size of the metal form has been developed based on the direct coupling of asymmetrical flow field flow fractionation (AsFlFFF) with inductively coupled plasma mass spectrometry (ICP-MS) for on-line isotope ratio measurements. ICP-MS data acquisition parameters were stressed to avoid degradation of isotope ratio precision. Two sets of fractionation conditions were selected: a colloids separation, which allowed the separation of substances up to 1 µm, and a macromolecules separation, designed to resolve small size substances up to 50 kDa. The methodology was applied to study the environmental availability of copper and lead in compost samples, where metals are mainly associated to different forms of organic matter. No significant differences on isotopic exchange were observed over the size range studied, validating the E-values determined by direct analysis of the aqueous phases.

Metal associations to microparticles, nanocolloids and macromolecules in compost leachates: size characterization by asymmetrical flow field-flow fractionation coupled to ICP-MS.

A methodological approach based on the size characterization of environmental microparticles (size larger than 1 microm), nanocolloids (1 microm to 15 nm) and macromolecules (lower than 1000 kDa) by asymmetrical flow field-flow fractionation (AsFlFFF), taking advantage of both normal and steric elution modes, is presented. The procedure was optimized to minimize the potential alteration of the size distribution and metal associations of the species characterized. Prior to separation by AsFlFFF, samples are subjected to gravitational settling of the solid suspension, followed by a centrifugation of the settled sample. The comparison between the fractograms of the settled and the centrifuged samples allows the characterization of the microparticles, which are eluted in steric mode in the AsFlFFF system. The characterization of nanocolloids and macromolecules is carried out on the centrifuged sample by applying different operational conditions under normal mode in the AsFlFFF system. A comparison with the conventional frontal filtration through 0.45 microm pore size membranes have shown that filtration removes particles below their nominal pore size, modifying the size distribution of the samples respect to the centrifugation. The methodology proposed has been applied to the size characterization of compost leachates. The contribution of these three differentiated fractions to the mobilization of metals has been determined by coupling the AsFlFFF system to an inductively coupled plasma mass spectrometer (ICP-MS).

Multielement characterization of metal-humic substances complexation by size exclusion chromatography, asymmetrical flow field-flow fractionation, ultrafiltration and inductively coupled plasma-mass spectrometry detection: a comparative approach.

The use of three different separation techniques, ultrafiltration (UF), high performance size exclusion chromatography (HPSEC) and asymmetrical flow field-flow fractionation (AsFlFFF), for the characterization of a compost leachate is described. The possible interaction of about 30 elements with different size fractions of humic substances (HS) has been investigated coupling these separation techniques with UV-vis absorption spectrophotometry and inductively coupled plasma-mass spectrometry (ICP-MS) as detection techniques. The organic matter is constituted by a polydisperse mixture of humic substances ranging from low molecular weights (around 1kDa) to significantly larger entities. Elements can be classified into three main groups with regard to their interaction with HS. The first group is constituted by primarily the monovalent alkaline metal ions and anionic species like B, W, Mo, As existing as oxyanions all being not significantly associated to HS. The second group consists of elements that are at least partly associated to a smaller HS size fraction (such as Ni, Cu, Cr and Co). A third group of mainly tri- and tetravalent metal ions like Al, Fe, the lanthanides, Sn and Th are rather associated to larger-sized HS fractions. The three separation techniques provide a fairly consistent size classification for most of the metal ions, even though slight disagreements were observed. The number-average molecular weight (Mn), the weight-average molecular weight (Mw) and the polydispersity (rho) parameters have been calculated both from AsFlFFF and HPSEC experiments and compared for HS and some metal-HS species. Differences in values can be partly explained by an overloading effect observed in the AsFlFFF experiments induced by electrostatic repulsion effects in the low ionic strength, high pH carrier solution. Size information obtained from ultrafiltration is not as resolved as for the other methods. Molecular weight cut-offs (MWCO) of the individual filter membranes refer to globular proteins and molecular weight information may therefore, deviate from that given by the other methods after calibration with polystyrene sulfonate (PSS) standards.

Slurry sampling electrothermal atomic absorption spectrometry as screening method for chromium in compost.

Ultrasonic slurry sample introduction was applied to the determination of total chromium in composted materials by electrothermal atomic absorption spectrometry (ETAAS). The effect of grinding on the heterogeneity of the test samples and on the attainable precision was studied. The repeatability was influenced by the heterogeneity of the test samples at the mug-level, the R.S.D. of the measurements being 15%. The reproducibility depended on the heterogeneity of the test sample at the mg level, and it could be improved from 11 to 7% by increasing the grinding time. The characteristic mass was 2.6pg and the detection limit for the optimised procedure at the 0.04% (w/v) slurry concentration, 370ngg(-1). Good agreement with a certified reference material and with the conventional microwave assisted digestion method was found by using external calibration with aqueous standards. The performance of the method for screening purposes was evaluated.

Reagent injection FIA system for lead determination by hydride generation - quartz-tube atomic absorption spectrometry.

A method is proposed for the determination of lead by generation of its hydride and detection by quartz-tube AAS using a reagent injection FIA system based on the injection of sodium tetrahydroborate. Lead hydride generation was carried out using a combination of 0.5 M nitric acid, 10% m/ v hydrogen peroxide and 10% m/ v sodium tetrahydroborate. The characteristic concentration obtained was 3.1 ng mL(-1) and the detection limit was 2.6 ng mL(-1) for an injected volume of 0.125 mL of tetrahydroborate.

[Synchronous primary urologic triple neoplasia. Report of a new case and review of the literature]. / Triple neoplasia urológica primaria sincrónica. Aportación de un nuevo caso y revisión de la literatura.

Herein we present the case of a patient who went to the emergency department complaining of haematuria. With a conservative management, the radiologic and endoscopic studies show a bladder tumour and an incidental left renal mass. After several radical procedures, the last a cystoprostatectomy, it was shown that the patient had a new tumour inside his prostate gland. We comment the surgical procedures, the clinical evolution and the five years follow-up. We review the few cases described in the literature.

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.

The Aspergillus nidulans carnitine carrier encoded by the acuH gene is exclusively located in the mitochondria.

The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACUH with a His-tag at its N-terminus was over-expressed in Escherichia coli and purified by Ni(2+) affinity chromatography. The purified protein was utilised to raise polyclonal antibodies which were characterised by Western blotting. For localisation studies A. nidulans T1 strain, that contains the acuH gene under control of the strong promoter alcA(p), was derived. Results obtained demonstrate the exclusively mitochondrial localisation of ACUH and therefore exclude the targeting of the acuH gene product to the peroxisomal membrane.

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes.

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

Processes of liquefaction/solubilization of Spanish coals by microorganisms

Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization.

The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine/acylcarnitine carrier.

The Aspergillus nidulans acuH gene, required for growth on acetate and long-chain fatty acids, was cloned by complementation of the acuH13 mutation. Northern blotting analysis showed that transcription of the acuH gene occurs in acetate-grown mycelium and at higher levels in oleate-grown mycelium, but not during growth on glucose minimal medium. The acuH gene encodes a protein of 326 amino acids that belongs to the mitochondrial carrier family. The ACUH protein contains three related segments of approximately 100 amino acids in length, each segment comprising two hydrophobic domains that are probably folded into two transmembrane alpha-helices linked by an extensive polar region. Sequence comparisons suggest that the acuH gene of A. nidulans encodes the homologue of the carnitine/acylcarnitine carrier of rat and man. The uncharacterised proteins YOR100C of Saccharomyces cerevisiae, COLT of Drosophila melanogaster, and DIF-1 of Caenorhabditis elegans also seem to be homologues of ACUH. In addition to the motifs present in all members of the mitochondrial carrier family, we propose the highly conserved motif R(A,S)(V,F)PANAA(T,C)F within the sixth hydrophobic domain of these proteins as the characteristic feature of the carnitine carrier subfamily. The proposed function of the ACUH protein is the transport of acetylcarnitine molecules from the cytosol to the mitochondrial matrix, a process required during growth on acetate or on long-chain fatty acids.

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy.

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide.

Determination of polychlorinated biphenyls in soybean infant formulas by gas chromatography.

A method previously developed for the analysis of organohalogenated compounds in dairy products is now validated for polychlorinated biphenyls (PCBs) determination in soybean infant formulas. The results of this study are consistent with those found for PCBs in powdered full-fat milk. The methodology is based on a solid-liquid extraction step enabling a semi-selective extraction of the apolar lipids of the matrix without affecting the efficiency for the recovery of PCBs. Mean recoveries for the spiked coplanar congeners studied were in the 88-114% range, with relative standard deviations (R.S.D.s) lower than 9.8%. The R.S.D.s related to the determination of endogenous PCBs were in the 1.5-10.0% range. The validated methodology was applied to the PCB analysis in different trademarks of soybean infant formulas commercialised in Spain. Toxic tetraequivalents of tetrachlorodibenzo-p-dioxin and daily intake corresponding to each one were calculated and compared with values previously published and with those found in literature for human breast milk in different countries.