Exploring the impact of silver-based nanomaterial feed additives on green algae through single-cell techniques.

Silver in its various forms, including dissolved silver ions (Ag+) and silver nanoparticles (AgNPs), is a promising alternative to traditional antibiotics, largely used in livestock as feed additives and could contribute to the decrease and avoidance of the development of antibiotic resistance. The present study aims to assess the potential ecotoxicity of a silver-based nanomaterial (Ag-kaolin), the feed supplemented with the nanomaterial and the faeces since the latter are the ones that finally reach the environment. To this end, green alga Raphidocellis subcapitata was exposed to the extracts of Ag-kaolin, supplemented feed, and pig faeces for 72 h, along with Ag+ and AgNPs as controls for comparison purposes. Given the complexity of the studied materials, single-cell techniques were used to follow the changes in the cell numbers and chlorophyll fluorescence by flow cytometry, and the accumulation of silver in the exposed cells by single cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Changes in cell morphology were observed by cell imaging multimode reader. The results revealed a decrease in chlorophyll fluorescence, even at low concentrations of Ag-kaolin (10 µg L-1) after 48 h of exposure. Additionally, complete growth inhibition was found with this material like the results obtained by exposure to Ag+. For the supplemented feed, a concentration of 50 µg L-1 was necessary to achieve complete growth inhibition. However, the behaviour differed for the leachate of faeces, which released Ag2S and AgCl alongside Ag+ and AgNPs. At 50 µg L-1, inhibition was minimal, primarily due to the predominance of less toxic Ag2S in the leachate. The uptake of silver by the cells was confirmed with all the samples through SC-ICP-MS analysis. These findings demonstrate that the use of Ag-kaolin as a feed supplement will lead to a low environmental impact.

Nanosilver-based materials as feed additives: Evaluation of their transformations along in vitro gastrointestinal digestion in pigs and chickens by using an ICP-MS based analytical platform.

The use of a new nanomaterial in the feed chain requires a risk assessment that involves in vitro gastrointestinal digestions to predict its degradation and oral exposure to nanoparticles. In this study, a nanosilver-based material was incorporated into pig and chicken feed as a growth-promoting additive and subjected to the corresponding in vitro gastrointestinal digestions. An inductively coupled plasma mass spectroscopy (ICP-MS) analytical platform was used to obtain information about the silver released in the different digestion phases. It included conventional ICP-MS for total silver determination, but also single particle ICP-MS and coupling to hydrodynamic chromatography for detection of dissolved and particulate silver. The bioaccessible fraction in the intestinal phase accounted for 8-13% of the total silver, mainly in the form of dissolved Ag(I) species, with less than 0.1% as silver-containing particles. Despite the additive behaving differently in pig and chicken digestions, the feed matrix played a relevant role in the fate of the silver.

Comparative study of extraction methods of silver species from faeces of animals fed with silver-based nanomaterials.

Extractions methods based on ultrapure water, tetramethylammonium hydroxide (TMAH), and tetrasodium pyrophosphate (TSPP) were applied to faeces collected from two in vivo experiments of pigs and chickens fed with a silver-based nanomaterial to study the fate and speciation of silver. For TMAH extraction, cysteine and CaCl2 were used to evaluate their stabilization effect on the silver forms. The analytical techniques single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS), hydrodynamic chromatography hyphenated to ICP-MS (HDC-ICP-MS) and asymmetric flow field flow fractionation coupled to ICP-MS (AF4-ICP-MS) were applied to the simultaneous detection of particulate and dissolved silver. Results have shown that water extraction was a suitable option to assess the environmental release of silver, with percentages of 3 and 9% for faeces of pigs and chickens, respectively. The use of TMAH extraction combined with SP-ICP-MS analysis was useful to characterize Ag-containing particles (less than 1%). Both stabilizers, cysteine and CaCl2, have a similar effect on silver nanoparticle preservation for chicken faeces, whereas cysteine-Triton was better for pig samples. In any case, silver extraction efficiency with TMAH was low (39-42%) for both types of faeces due to a matrix effect. TSPP followed by ICP-MS enabled the fractionation of the silver in the faeces, with silver sulphide (41%) and ionic silver (62%) being the most abundant fractions.

How to trust size distributions obtained by single particle inductively coupled plasma mass spectrometry analysis.

Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) is a technique widely used to obtain direct information about the number concentration and the size distribution of nanoparticles in liquid suspensions. However, its methods still lack clear quality control strategies to confirm the validity of the information derived from them. Only the detection of the complete size distribution of the nanoparticles in a sample over the size critical value ensures obtaining unbiased quantitative information, otherwise information should be restricted to report the presence of nanoparticles over a certain size and number concentration since their actual total number concentration is underestimated and the size overestimated. Under the latter conditions, data processing produces histograms showing the tails of the incomplete size distributions, although apparently, complete distributions can also be obtained when particle events are recorded as peaks, as reported here for the first time. The occurrence of these misleading situations must be critically evaluated for each SP-ICP-MS analysis. An approach, based on estimation of size critical values and successive dilutions, is proposed for the assessment of the validity of the quantitative information obtained, together with specific criteria for reconsidering the information that can be derived from those measurements. The approach was verified with different case studies and applied to the analysis of complex nanomaterials, confirming the validity of the reported information by comparison with other techniques. A calculation tool is also included to facilitate the estimation of size critical values under experimental conditions.

Analytical applications of single particle inductively coupled plasma mass spectrometry: a comprehensive and critical review.

Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) refers to the use of ICP-MS as a particle counting technique. When ICP-MS measurements are performed at very high data acquisition frequencies, information about (nano)particles containing specific elements and their dissolved forms can be obtained (element mass per particle, size and number and mass concentrations). As a result of its outstanding performance, SP-ICP-MS has become a relevant technique for the analysis of complex samples containing inorganic nanoparticles. This review discusses the maturity level achieved by the technique through the methods developed for the detection, characterisation and quantification of engineered and natural (nano)particles. The application of these methods in different analytical scenarios is comprehensively reviewed and critically discussed, with special attention to their current technical and metrological limitations. The emergent applications of SP-ICP-MS in the field of nanoparticle-tagged immunoassay and hybridization methods are also reviewed.

Evaluation of hydrodynamic chromatography coupled to inductively coupled plasma mass spectrometry for speciation of dissolved and nanoparticulate gold and silver.

In this study, hydrodynamic chromatography coupled to inductively coupled plasma mass spectrometry has been evaluated for the simultaneous determination of dissolved and nanoparticulate species of gold and silver. Optimization of mobile phase was carried out with special attention to the column recovery of the different species and the resolution between them. Addition of 0.05 mM penicillamine to the mobile phase allowed the quantitative recovery of ionic gold and gold nanoparticles up to 50 nm, whereas 1 mM penicillamine was necessary for quantitative recovery of ionic silver and silver nanoparticles up to 40 nm. The resolution achieved between ionic gold and 10-nm gold nanoparticles was 0.7, whereas it ranged between 0.31 and 0.93 for ionic silver and 10-nm silver nanoparticles, depending on the composition of mobile phase. Best-case mass concentration detection limits for gold and silver species were 0.05 and 0.75 µg L-1, respectively. The developed methods allowed the simultaneous detection of nanoparticulate and dissolved species of gold and silver in less than 10 min. Size determination and quantification of gold and silver species were carried out in different dietary supplements, showing good agreement with the results obtained by electron microscopy and total and ultrafiltrable contents, respectively. Due to the attainable resolution, the quality of the quantitative results is affected by the relative abundance of nanoparticulate and dissolved species of the element and the size of the nanoparticles if present.

Increased water use efficiency does not prevent growth decline of Pinus canariensis in a semi-arid treeline ecotone in Tenerife, Canary Islands (Spain).

KEY MESSAGE: Intrinsic water-use efficiency of Pinus canariensis (Sweet ex Spreng.) growing at a semi-arid treeline has increased during the past 37 years. Tree-ring width by contrast has declined, likely caused by reduced stomatal conductance due to increasing aridity. CONTEXT: Rising atmospheric CO2 concentration (Ca ) has been related to tree growth enhancement accompanied by increasing intrinsic water-use-efficiency (iWUE). Nevertheless, the extent of rising Ca on long-term changes in iWUE and growth has remained poorly understood to date in Mediterranean treeline ecosystems. AIMS: This study aimed to examine radial growth and physiological responses of P. canariensis in relation to rising Ca and increasing aridity at treeline in Tenerife, Canary Islands, Spain. METHODS: We evaluated temporal changes in secondary growth (tree-ring width; TRW) and tree ring stable C isotope signature for assessing iWUE from 1975 through 2011. RESULTS: Precipitation was the main factor controlling secondary growth. Over the last 36 years P. canariensis showed a decline in TRW at enhanced iWUE, likely caused by reduced stomatal conductance due to increasing aridity. CONCLUSION: Our results indicate that increasing aridity has overridden the potential CO2 fertilization on tree growth of P. canariensis at its upper distribution limit.

Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE).

Detection, characterization and quantification of inorganic engineered nanomaterials: A review of techniques and methodological approaches for the analysis of complex samples.

The increasing demand of analytical information related to inorganic engineered nanomaterials requires the adaptation of existing techniques and methods, or the development of new ones. The challenge for the analytical sciences has been to consider the nanoparticles as a new sort of analytes, involving both chemical (composition, mass and number concentration) and physical information (e.g. size, shape, aggregation). Moreover, information about the species derived from the nanoparticles themselves and their transformations must also be supplied. Whereas techniques commonly used for nanoparticle characterization, such as light scattering techniques, show serious limitations when applied to complex samples, other well-established techniques, like electron microscopy and atomic spectrometry, can provide useful information in most cases. Furthermore, separation techniques, including flow field flow fractionation, capillary electrophoresis and hydrodynamic chromatography, are moving to the nano domain, mostly hyphenated to inductively coupled plasma mass spectrometry as element specific detector. Emerging techniques based on the detection of single nanoparticles by using ICP-MS, but also coulometry, are in their way to gain a position. Chemical sensors selective to nanoparticles are in their early stages, but they are very promising considering their portability and simplicity. Although the field is in continuous evolution, at this moment it is moving from proofs-of-concept in simple matrices to methods dealing with matrices of higher complexity and relevant analyte concentrations. To achieve this goal, sample preparation methods are essential to manage such complex situations. Apart from size fractionation methods, matrix digestion, extraction and concentration methods capable of preserving the nature of the nanoparticles are being developed. This review presents and discusses the state-of-the-art analytical techniques and sample preparation methods suitable for dealing with complex samples. Single- and multi-method approaches applied to solve the nanometrological challenges posed by a variety of stakeholders are also presented.

Assessment of metals bound to marine plankton proteins and to dissolved proteins in seawater.

Studies based on laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) have been performed to assess metal bound to dissolved proteins and proteins from marine plankton after two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Dissolved proteins were pre-concentrated from surface seawater (60 L) by tangential ultrafiltration with 10 kDa molecular weight cut-off (MWCO) membranes and further centrifugal ultrafiltration (10 kDa) before proteins isolation by methanol/chloroform/water precipitation. Proteins isolation from plankton was assessed after different trichloroacetic acid (TCA)/acetone and methanol washing stages, and further proteins extraction with a phenol solution. LA-ICP-MS analysis of the electrophoretic profiles obtained for dissolved proteins shows the presence of Cd, Cr, Cu, and Zn in five spots analyzed. These proteins exhibit quite similar molecular weights (within the 10-14 kDa range) and pIs (from 5.8 to 7.3). Cd, Cr, Cu, and Zn have also been found to be associated to proteins isolated from plankton samples. In this case, Cd has been found to be bound to proteins of quite different molecular weight (9, 13 and 22 kDa) and pIs (4.5, 5.2, 5.5, and 10). However, trace elements such as Cr, Cu and Zn appear to be mainly bound to plankton proteins of low molecular weight and variable pI.

Evaluation of gel electrophoresis techniques and laser ablation-inductively coupled plasma-mass spectrometry for screening analysis of Zn and Cu-binding proteins in plankton.

The determination of metal-binding proteins in plankton is important because of their involvement in photosynthesis, which is fundamental to the biogeochemical cycle of the oceans and other ecosystems. We have elaborated a new strategy for screening of Cu and Zn-containing proteins in plankton on the basis of separation of proteins by use of Blue-Native PAGE (BN-PAGE), which entails use of a non-denaturing Tris-tricine system and detection of metals in the proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). For comparison, denaturing PAGE based on Tris-glycine and Tris-tricine systems and Anodic-Native PAGE have also been investigated. A large number of protein bands with MW between 20 and 75 kDa were obtained by use of Tris-glycine PAGE but detection of metals by LA-ICP-MS was unsuccessful because of loss of metals from the proteins during the separation process. Different protein extraction, purification, and preconcentration methods were evaluated, focussing on both issues-achieving the best extraction and characterization of the proteins while maintaining the integrity of metal-protein binding in the plankton sample. Use of 25 mmol L(-1) Tris-HCl and a protease inhibitor as extraction buffer with subsequent ultrafiltration and acetone precipitation was the most efficient means of sample preparation. Two Cu and Zn proteins were detected, a protein band corresponding to a MW of 60 kDa and another poorly resolved band with a MW between 15 and 35 kDa.

Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders.

Characterization of metal-humic acid complexes by polyacrylamide gel electrophoresis-laser ablation-inductively coupled plasma mass spectrometry.

A new speciation methodology based on the use of 1D-PAGE (one dimensional-polyacrylamide gel electrophoresis), IEF (isoelectric focusing electrophoresis) and 2D-PAGE (two dimensional-polyacrylamide gel electrophoresis) as separation techniques and LA-ICP-MS (laser ablation-inductively coupled plasma mass spectrometry) as detection system have been applied to study the distribution of metal-humic acids (metal-HA) complexes in environmental samples. The use of 1D-Tris borate method using low current (10 mA) and Tris borate as trailing ion previous to LA-ICP-MS measurements, allowed to obtain the distribution of metals-HA complexes from a compost sample maintaining metals-HA complexes stability. It was observed that metals were associated with the smallest HA size fraction (around 3 kDa), validating results obtained by high performance size-exclusion chromatography (HP-SEC), cross-flow field flow fractionation (FIFFF) and ultrafiltration. IEF-LA-ICP-MS profiles indicated that there are two main regions: (A) with 35. The biggest metals-HA signals are obtained in A region (low pIs) corresponding to low molecular weight (MW) compounds which are predominant in our compost sample indicating a low stabilization of organic matter is low. These results are coincident with 1D-Tris borate PAGE-LA-ICP-MS results. The use of 2D-PAGE method followed by LA-ICP-MS confirmed the results obtained by 1D and IEF as PAGE methods and added information about humic acid molecular size distribution.

Metal-protein binding losses in proteomic studies by PAGE-LA-ICP-MS.

Some experiments to study the influence of electrophoresis conditions and subsequent LA-ICP-MS (laser ablation-inductively coupled plasma mass spectrometry) determination of two metal-binding proteins with different metal-protein affinities (superoxide dismutase, containing Cu and Zn, and alcohol dehydrogenase, containing Zn) are performed. In metal-binding proteins with weak metal-protein affinities, metal losses can happen during electrophoretic separation. It has been demonstrated that the detection of these metals bound to the proteins depends, not only on the nature of the electrophoretic process (naturing or non-denaturing) and post-separation gel treatment, but also on the trailing ion chosen and current applied in the electrophoretic method used. Non-denaturing methods are preferred to denaturing ones in the case of alcohol dehydrogenase being BN-PAGE (Blue Native-Polyacrylamide Gel Electrophoresis) with the use of Tricine as trailing ion the most recommended method. The concentration obtained for Zn in ADH applying BN-PAGE-LA-ICP-MS was 2.6+/-0.30 mg g(-1) very close to the one obtained for ADH solution by ICP-MS (3+/-0.23 mg g(1)). For superoxide dismutase either denaturing or non-denaturing electrophoresis conditions can be used, but a denaturing method based on the use of Tricine as trailing ion is recommended to preserve metals-protein binding when the use of non-denaturing conditions must be avoided. The found concentration for Cu and Zn in SOD after applying SDS-Tris-Tricine-PAGE-LA-ICP-MS was 2.5+/-0.33 and 2.4+/-0.37 mg g(-1) respectively, more or less close (especially for Cu) to the one obtained in SOD solution by ICP-MS (3+/-0.21 and 3.7+/-0.32 mg g(-1) for Cu and Zn). We observe that as higher current is applied the possibility of metal-protein binding losses is higher. In all cases staining of the gel prior to LA-ICP-MS is not recommended.

Some pitfalls in PAGE-LA-ICP-MS for quantitative elemental speciation of dissolved organic matter and metalomics.

An experimental approach to evaluate the capabilities and limitations of polyacrylamide gel electrophoresis (PAGE) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for quantitative elemental speciation is presented. Two metalloproteins (superoxide dismutase, containing Cu and Zn, and thyroglobulin, containing I) with high binding affinity for metals, and metal-dissolved organic matter (DOM) complexes (from a compost leachate sample) which show different types of metal binding are studied. Iodine can be quantitatively detected in thyroglobulin after PAGE-LA-ICP-MS using either sodium dodecyl sulfate (SDS) PAGE or native PAGE. However, detection of Cu and Zn in superoxide dismutase after PAGE-LA-ICP-MS depends on the conditions of the PAGE method because possible metal losses can occur (either with SDS-PAGE or with native PAGE). The use of PAGE-LA-ICP-MS to study the contribution of DOM to the mobilization of metals from environmental samples is possible, but it depends also on the PAGE separation conditions owing to disequilibrium effects of metal-DOM complexes.

Multi-element analysis of compost by laser ablation-inductively coupled plasma mass spectrometry.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been applied to multi-element determination in compost samples. Since compost is a heterogeneous mixture of organic and inorganic materials, the influence of sample heterogeneity on the accuracy and precision of analysis was investigated. Several parameters related to the following were studied: laser (energy, laser-beam diameter, preablation. rastering speed, carrier-gas flow rate), sample preparation (use of compacted pellets, grinding time, particle size, sample amount, length of hydraulic press treatment, position of line scan), and the ICP-MS system (quantitative versus semiquantitative analysis, matrix-matched standards and liquid standards calibration). The main causes of imprecision in sample preparation were determined to be particle size and grinding time. The effect of sample heterogeneity on precision was also evaluated by using different test samples (pellets). For Ni, Zn and Pb, the greatest contribution to the total relative standard deviation (R.S.D.) was related to analyte determination. For Mn and Cu, sample heterogeneity and analyte determination contributed equally to the total R.S.D., whereas for Cr, Co, Cd and Hg sample heterogeneity accounted for most of the total R.S.D. A comparison of semiquantitative and quantitative analysis modes showed that better precision and very good agreement with certified reference material was obtained with the latter, but semiquantitative analysis could be a practical alternative. Although accuracy of results was improved with matrix-matched standards calibration the use of standard addition calibration with aqueous standards could be another possibility.

The measurement of neurovegetative activity during anesthesia and surgery in swine: an evaluation of different techniques.

In this study we evaluated, in 10 sevoflurane-anesthetized pigs undergoing abdominal surgery, different techniques for measuring autonomic nervous system (ANS) activity: ANSiscope index, spectral analysis of heart-rate variability, hemodynamic variables, and plasma catecholamines and cortisol levels. Animals underwent a 120-min anesthesia during which unilateral ovariectomy was performed. Cardiovascular and respiratory responses were monitored. ANSiscope indices (ANSindex sympathetic, ANSindex parasympathetic and balANSindex) were used to monitor ANS activity. Spectral analysis was performed using an autoregressive model with a parametric method. The low frequency (LF) and high frequency (HF) components were used to interpret the power spectral density of short-term electrocardiograms (ECGs). The relationship LF/(LF+HF) reflects sympathetic activity, HF/(LF+HF) indicates parasympathetic activity, and the LF/HF ratio gives the predominance of the system. Plasma concentrations of adrenaline, noradrenaline, and cortisol were determined at different times. Correlation (P < 0.01) was found between the balANSindex and adrenaline levels and between LF/HF ratio and plasma adrenaline concentrations. Moreover, a significant (P < 0.01) correlation was found between the balANSindex and LF/HF ratio. However, no correlation was seen between the registered ANSiscope indices and hemodynamic variables. The correlation seen in this study suggests that the balANSindex could be a useful tool to monitor ANS activity during anesthesia and surgery.

Bispectral index, spectral edge frequency 95% and median frequency recorded at varying desflurane concentrations in pigs.

The objective of this study is to evaluate the usefulness of bispectral index (BIS), spectral edge frequency 95% (SEF) and median frequency (MED) in relation to a simple descriptive scale (SDS) as indicators of anaesthetic depth at different desflurane concentrations in swine. Sixteen pigs were randomly allocated to four groups. Electroencephalograms (EEG) were recorded during desflurane anaesthesia, and BIS, SEF and MED were calculated from the EEG. The agent was administered in pure oxygen at 1, 1.25, 1.5 and 1.7 MAC in randomized order. Anaesthetic depth was evaluated on a SDS. BIS decreased significantly (P<0.001) at the different anaesthetic dosages used. SEF decreased significantly (P<0.001) from basal to 1 MAC of desflurane. MED decreased significantly (P<0.001) from basal to 1 MAC and from 1 to 1.75 MAC. Good correlation was seen between SDS scores and BIS values and between SDS scores and MED values. BIS appeared to be useful to predict changes in anaesthetic depth at clinically used dosages of inhalant anaesthesia.