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.

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.

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.

Purification and molecular properties of an alpha-galactosidase synthesized and secreted by Aspergillus nidulans.

alpha-Galactosidases from mycelial extract and culture filtrate of Aspergillus nidulans have been purified to homogeneity and utilised to obtain polyclonal antibodies anti-alpha-galactosidase. The enzymatic characteristics and the cross reactivity of the antibodies suggest that alpha-galactosidases isolated from the two sources were the same enzyme. Thus, A. nidulans synthesized and secreted only one enzymatic form of alpha-galactosidase which is a multimeric enzyme of 370 kDa composed of four monomers of 87 kDa and a pI of 6.3. The optimum temperature of activity was 50 degrees C and the optimum pH 4-5. The enzyme was stable over a wide range of pH but quite unstable to temperature. alpha-Galactosidase of A. nidulans is a very specific enzyme, it is active only on p-nitrophenyl-alpha-D-galactoside (PNPG), melibiose and raffinose. When PNPG was utilised as substrate melibiose, raffinose, galactose and glucose were competitive inhibitors of the activity.

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.

Rapid separation of soybean globulins by reversed-phase high-performance liquid chromatography.

A rapid separation of the main soybean proteins (7S and 11S globulins) was carried out by reversed-phase high-performance liquid chromatography. For this purpose, a linear binary gradient acetonitrile--water--0.1% trifluoroacetic acid, at a flow-rate of 1 ml/min and 50 degrees C temperature was designed. Under the experimental conditions of this work, it was possible to separate five peaks corresponding to the globulins from a soybean protein isolate in 9 min. The characterization of soybean proteins was accomplished by analyzing the 7S and 11S purified fractions obtained from a soybean protein isolate. The method was applied to the separation of soybean proteins from commercial foodstuffs: soybean flour, textured soybean and soybean milks.

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.

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.

[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.

[PSA and PSAD study in patients with renal dysfunction]. / Estudio de PSA y PSAD en pacientes con disfunción renal.

This study analyzes the changes in serum and urinary PSA values in 28 subjects; 13 with creatinine clearance under 75 ml/mn and 15 with creatinine clearance over 75 ml/mn. Both groups were compared for prostate size, measured by transrectal ultrasound, prostate weight, serum PSA (SPSA), 24h urine PSA (PSAO), PSA clearance (PSACl), serum creatinine (SCr), creatinine clearance (CrCl), PSA density (PSAD), PSA/creatinine ratio (PSA/Cr) and PSACl/CrCl ratio. Mean values of SPSA and PSAO were 4.5 +/- 0.8 and 222 +/- 29.7 ng/ml respectively, values for SCr, CrCl and PSACl averaging 1.62 +/- 0.2 mgr/dl, 71.6 +/- 6.5 ml/mn and 150.5 +/) 32.9 ml/mn. Median prostate size was 32.6 +/- 3.9 cc, with weights of 40.3 +/- 4.9 g and mean PSA density (PSAD) 0.13 +/- 0.02. The results of the homogeneity study showed that there are no significant differences between both groups with regard to the variables considered in the study. SPSA values were higher in patients with CrCl < 75; 3.4 vs 5.7, but not significantly. There are no significant differences between PSAO and PSACl values for both groups, even though PSAO levels were higher in patients with CrCl < 75 ml/min (p = 0.1). PSAD values for patients with CrCl > 75 ml/mn were lower than those for patients with CrCl < 75 ml/mn; 0.09 vs 0.17 (p = 0.08). In the entire sample, PSAD levels showed correlation with SPSA and PSA/Cr values; R = 0.63 (P = 0.0003) and r = 0.5 (p = 0.009) respectively. Also, they were significantly but inversely correlated with PSACl levels; r = - 0.5 (p = 0.006) and PSACl/CrCl; r = - 0.048 (p = 0.01). No correlation was seen between PSAD values and the following parameters; PSAO (p = 0.7), SCr (p = 0.5) and CrCl (p = 0.27). When the group of patients with CrCl < 75 ml/mn is considered, PSAD values are correlated exclusively with PSACl values; r = - 0.69 (p = 0.008) and PSACl/CrCl; r = 0.68 (p = 0.009). Our data appear to indicate that there is a certain relationship between PSAD and the renal function although the physiopathological mechanism responsible for that is unknown. Nevertheless, considering the sample size, more comprehensive studies will be necessary to obtain more convincing results.

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.

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).

Ultrastructural changes in Cladosporium cucumerinum during pathogenesis.

Three ultrastructural patterns, each one associated with a different stage of pathogenesis, were observed in Cladosporium cucumerinum infecting cucumber cytoledons. (i) Fungal prepenetration structures (conidia and germ tubes) contained numberous lipid bodies. Microbodies were abundant and often associated with lipid bodies. Mitochondria were also abundant. (ii) During the invasion stage, no lipid bodies and relatively few microbodies were observed. Mitochondria were abundant, but differed markedly morphologically from those observed in the prepenetration structures. (iii) In the colonizing hyphae, microbodies were the most abundant organelle, lipid bodies were seldom found, and mitochondria were as observed during invasion. These changes in numbers of organelles may be associated with differences in the metabolic activities of the pathogen at various stages of pathogenesis.

[Degradation of oil derivatives by Acinetobacter calcoaceticus MM5]. / Degradación de derivados del petróleo por Acinetobacter calcoaceticus MM5.

This paper describes the isolation of microorganisms from polluted heating oil. The growth of one of them has been studied (Acinetobacter calcoaceticus MM5) in several linear and branched hydrocarbons as well as the effect of its growth on commercial diesel oil. Acinetobacter calcoaceticus MM5 is not capable of using glucose as its only source of carbon, and it needs the presence of nitrogen and phosphorus sources to degrade any petroleum by-product.

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.

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.