SILAC-based quantitative proteomics identifies size-dependent molecular mechanisms involved in silver nanoparticles-induced toxicity.

Silver nanoparticles are currently one of the most widely used metallic nanoparticles. Due to their antibacterial properties, they are applied in textiles, house-holds items, and medical devices, among many other products. Understanding the potential toxicity associated with silver nanoparticles and the differential effect that nanoparticles of different size might induce is crucial, due to the increasing human and environmental exposure to this type of nanoparticles. In this work, we explored the different biomolecular mechanisms underlying the toxicity of silver nanoparticles in a size-dependent manner. Quantitative proteomic analysis of hepatic cells exposed to 10 and 60 nm silver nanoparticles demonstrated the alteration of a different set of proteins depending on the particle size. We demonstrated that while 10 nm silver nanoparticles induce nucleolar stress and ribosome biogenesis halt, both types of nanoparticles induce DNA damage and oxidative stress but through different pathways. In addition, both types of nanoparticles also affected cell proliferation, disrupted the cell cycle and ultimately, induced apoptosis. The alteration of different cellular mechanisms in a size-dependent manner, have relevant implications not only from a toxicity point of view, but also for the potential applications of silver nanoparticles.

In vivo bioconcentration of a metal mixture by Danio rerio eleutheroembryos.

Exposure to heavy metals has represented one of the most serious health risks of environmental pollution over the last 50 years. Most of the bioconcentration studies that have been carried out to date explored only individual contaminants, unlike the real situations that occur in the environment. In this work, zebrafish eleutheroembryos were exposed to a mixture of CH3Hg(II), iAs(III), Ag(I) and Cd(II), and new BCFs were calculated and compared with those calculated from single metal exposures. In both cases, experimental conditions meet the OECD Test 305 conditions established for aquatic systems. In addition, spatial imaging obtained by laser ablation coupled to inductively plasma mass spectrometry (LA-ICP/MS), has been directly performed in these samples providing complementary information. The new BCF's have revealed some differences compared to single metal exposures when eleutheroembryos were exposed to the metal mixture, especially for iAs(III) and Cd(II). LA-ICP/MS images are in good agreement with the BFC's found, representing an interesting approach to get spatial distribution of metals that reinforces the toxicokinetic information.

Development of a thiourea derivative polymer combined to a Direct Mercury Analyser for screening and pre-concentration of mercury species.

A new selective polymer has been developed (Patent: P201500704) with high mercury sorption capacity. The combination of this material with a Direct Mercury Analyser (DMA80) has overcome important limitations of this equipment for mercury determination of complex matrix samples, like high saline content, due to the ability of this material to selective and efficient mercury retention. Furthermore, application for fast screening of mercury species in food samples, like fish, is also presented. The polymer material has been synthesized by the precipitation technique, using Azobisisobutyronitrile (AIBN) as initiator, 2-(Methacryloylamino) ethyl 2-Methyl Acrylate (NOBE) as cross-linker, and 1-phenyl-3-(-3-vinyl phenyl) thiourea as monomer. It has been characterized by SEM, TGA, FTIR, and binding isotherms have been established through Langmuir and Freundlich models. Selectivity has been proven by carrying out cross-reactivity experiments.

Bioconcentration of ionic cadmium and cadmium selenide quantum dots in zebrafish larvae.

The concern related to the use of nanomaterials is growing nowadays, especially the risk associated with their emission or exposure. One type of nanomaterials that has attracted much attention is quantum dots (QDs). QDs incorporation in consumer goods increases the probability of their entering in the environment and then into living organisms and human. In order to evaluate their potential to be bioconcentrated, zebrafish larvae have been exposed to SeCd/ZnS QDs, after performing an exhaustive characterization of these nanoparticles under the assay conditions. These data were compared with those obtained when zebrafish larvae were exposed to ionic cadmium. Finally, distribution of ionic Cd and QDs in exposed zebrafish larvae have been evaluated by Laser Ablation ICP-MS.

A new specific polymeric material for mercury speciation: Application to environmental and food samples.

A new polymeric material (Patent: P201400535) highly specific for mercury is presented. Its great capability to pre-concentrate and selectively elute inorganic mercury and methylmercury are the main figures of merit. The polymer can be reused several times. To our knowledge, this is the only polymer proposed in the literature for direct inorganic mercury and methylmercury speciation without need of chromatography or quantification by difference. The polymer formation is based on the reaction of a vinyl derivative of 8-hydroxiquinoline as monomer, and 2-(Methacryloylamino) ethyl 2-Methyl Acrylate (NOBE) as co-monomer. Random radical polymerization by the precipitation method was carried out using Azobisisobutyronitrile (AIBN) as initiator. The polymer was characterized by SEM and FTIR. Adsorption binding isotherms were evaluated using Langmuir and Freundlich models, showing high adsorption capacity for both inorganic and organic mercury species. The polymer was employed to sequentially determine inorganic mercury and methylmercury, using a solid phase extraction (SPE) scheme. Cross reactivity of several ions, as well as matrix effects from a high saline matrix like seawater was irrelevant as the retained fractions mostly eluted during the washing step. The procedure was first validated by analyzing a certified reference material (BCR 464) and finally applied to commercial fish samples. The speciation proposed procedure is cheap, fast, and easy to use and minimizes reagents waste.

Zebrafish (Danio rerio) eleutheroembryo-based procedure for assessing bioaccumulation.

This paper reports on the development and preliminary evaluation of a new bioaccumulation test based on the use of zebrafish (Danio renio) eleutheroembryos (72 h after hatching, corresponding to 144 h post fertilization, hpf) as an alternative to adult fish-based procedures for regulatory purposes regarding REACH application. The proposed test accomplished the OECD 305 guideline and consists of a 48 h uptake period followed by a 24 h depuration step. Bioaccumulation experiments were performed for a selected of hyper hydrophobic chemicals (log Kow> 7.6), that is, PCB 136 and PBDE 154 at two concentration levels corresponding roughly to 1% and 0.1% the chemicals LC50(nominal concentrations of 4.0 and 12.0 µg/L for PCB 136, and 1.0 and 5.0 µg/L PBDE 154, respectively). Toxicokinetic models were used to calculate the bioconcentration factors (BCFs) based on of the chemical concentrations found in the contaminated eleutheroembryos and their surrounding media. The experimentally determined accumulation profiles show bioaccumulation by zebrafish eleutheroembryos of both chemicals, and that the process is more complex than simple water-lipid partition. Calculated log BCFs using a first-order accumulation model(3.97 and 3.73 for PCB 136, and 3.95 and 4.29 for PBDE 154) were in the range of those previously reported in the literature. The suitability of this new nonprotected life stage bioaccumulation protocol for BCF estimation was evaluated by application to widely divergent micropollutants with different accumulation mechanisms. The results were compared with those in the MITE-NITE database for adult rice fish (Oryzias latipes).

Comparison of bioconcentration of ionic silver and silver nanoparticles in zebrafish eleutheroembryos.

The production of silver nanoparticles has reached nowadays high levels. Bioconcentration studies, information on persistence and toxicity are fundamental to assess their global risk and thus necessary to establish legislations regarding their use. Previous studies on silver nanoparticle toxicity have determined a clear correlation between their chemical stability and toxicity. In this work, experimental conditions able to assure silver nanoparticles stability have been optimized. Then, zebrafish (Danio rerio) eleutheroembryos were exposed to ionic silver and to Ag NPs for comparison purposes. A protocol alternative to the OECD 305 technical guideline was used. To determine silver concentration in both the eleutheroembryos and the exposure media, an analytical method consisting in ultrasound assisted extraction, followed by inductively coupled plasma mass spectrometry and graphite furnace atomic absorption spectrometry, was developed. Then, bioconcentration factors were calculated. The results revealed that ionic silver was more accumulative for zebrafish eleutheroembryos than nanoparticles at the levels tested.

Zebrafish larvae as a model for the evaluation of inorganic arsenic and tributyltin bioconcentration.

The European REACH legislation establishes the need to study the toxicity, persistence and bioaccumulation of those chemicals with an exceeding production of 100tons and/or chemicals considered PBTs substances (Persistence, Bioaccumulation and Toxicity). Currently, the OECD technical guideline 305 is the most used protocol to determine bioconcentration factors of contaminants in aquatic environments. However, this procedure implies high cost and amount of adult fishes. Zebrafish (Danio Rerio) has been selected since this animal model has several advantageous features over other vertebrates, mainly fast embryonic development and easy growth. The analytical methodology here developed has been applied to calculate the bioconcentration factors (BCFs) of two contaminants: inorganic arsenic and tributyltin (measured as arsenic and tin). The method is based on the use of an ultrasonic probe assisted extraction for accelerating the sample treatment followed by detection using graphite furnace atomic absorption spectrometry with Zeeman correction (ZGFAAS). Results obtained for the BCFs values are in good agreement with previously reported data on freshwater aquatic organisms. In the case of arsenic, after exposing larvae to concentrations of 5 and 50µgL(-1), very low BCFs were observed (between 2.2 and 9.5); while for tributyltin, the BCFs observed were within the range 840-1280 after exposure to concentrations of 0.2 and 2.0µgL(-1), respectively. This study shows the use of zebrafish larvae together with the proposed analytical approach as a promising alternative to the OECD 305 test to evaluate the BCFs of classical and emergent contaminants.

Arsenic speciation in rice, straw, soil, hair and nails samples from the arsenic-affected areas of Middle and Lower Ganga plain.

In the present study, pressurised liquid extraction and ultrasound probe sonication, for the latter in combination with a mixed enzymatic treatment in case of rice and straw samples, were applied as sample preparation prior to arsenic speciation analysis by high pressure liquid chromatography coupled to inductively coupled plasma mass spectrometric detection (HPLC-ICP-MS). A significant number of samples as different as rice, straw, soil, nail and hair, all coming from the heavily arsenic-contaminated Middle and Lower Ganga plain area, could be investigated with validated methods, supported by high speed extraction methods. For rice and paddy samples, inorganic arsenic counted up to 70-98% of the total arsenic content, being the major species As (III). The levels of arsenic obtained from straw and soil samples are significantly higher than the background levels, being the major species As (V), thus increasing human exposure to arsenic via the soil-plant-animal-human pathway. Concentrations found in hair and nails were significantly higher than their background levels: 39- and 20-fold for hair and nails, respectively. These samples contained mainly inorganic arsenic in its tri- and pentavalent forms. Results indicate that, under the local frame conditions, arsenic mainly enters into the food chain via its more problematic inorganic forms. Arsenic speciation analysis proves to be a powerful tool for a complete analytical assessment in epidemiological studies covering the endemic areas.

Evaluation of a focused sonication probe for arsenic speciation in environmental and biological samples.

Arsenic speciation analysis suffers in general from high sample handling time required by sample preparation. In a previous work, ultrasonic probe has been proved to reduce sample treatment time for arsenic extraction in rice to only a few minutes. Base upon the obtained results, here several extraction media for chicken, fish and soil samples (SEAS G6RD-CT2001-00473) have been studied and evaluated employing the same technique. Chicken sample needed an enzymatic treatment in order to liberate the species linked to the protein matrix. Extraction of the major species in fish, AsB, was quantitatively achieved in water in 1 min. Also 1 min was enough to leach about 85% of species present in soils and sediments, mainly the inorganic ones, using H(3)PO(4). In all cases, no inter-conversion among As species was observed. The five species found in those samples were separated using an improved HPLC-ICP-MS method in only 11 min, with detection limits at the ng l(-1) level. The proposed methods were validated by analysing several Certified Reference Materials: SRM 1,568 a rice flour, CRM-627 tuna fish tissue, SOIL-7 soil and MURST-ISS-A1 Antarctic sediment.

Coupling pervaporation to AAS for inorganic and organic mercury determination. A new approach to speciation of Hg in environmental samples.

The design and development of a new approach for Hg speciation in environmental samples is described in detail. This method, consisting of the coupling of pervaporation and atomic absorption spectrometry, is based on a membrane phenomenon that combines the evaporation of volatile analytes and their diffusion through a polymeric membrane. It is proposed here as an alternative to gas chromatography for speciation of inorganic and organic Hg compounds, as the latter compounds are volatile and can be separated by applying the principles mentioned above. The interest of this method lies in its easy handling, low cost, and rapidity for the analysis of liquid and solid samples. This method has been applied to Hg speciation in a compost sample provided by a waste water treatment plant.

Determination of Cd in sonicate slurries and leachates of biological and environmental materials by FI-CV-AAS.

An analytical method for Cd analysis in solid samples which combines the ultrasonic slurry formation with cold vapour generation and atomic absorption spectrometry is described. The samples are suspended in HCl and sonicated until homogeneous and reduced particle size slurry formation. Several aspects were studied: acidity of the medium, sonication time, and slurry formation in different matrices. The procedure described permits the use of direct calibration, with KCN addition as masking agent of interfering ions (Cu, Pb, Ni and Zn) present in the environmental matrices. Supernatant analysis of these last samples experimentally shown that preparation of the suspension with 6 mol l(-1) HCl concentration led to quantitative extraction of Cd. Biological materials analysis needed the use of the standard addition calibration method due to the high matrix effect observed. Supernatant analysis in biological samples does not give a total Cd recovery for all of them. The detection limits observed for Cd were 0.05 and 0.2 mug l(-1) for supernatant and slurry analysis respectively in environmental samples, while in biological samples were 0.2 and 0.6 mug l(-1) for supernatant and slurry analysis, respectively. In all case the better precision was obtained for supernatant analysis (3-6%) than slurry analysis (6-12%). The results obtained by analysing different reference materials (sewage sludge, city waste incineration, Antarctic krill and human hair) showed good agreement with the certified value confirming the validity of such a method for Cd determination instead to wet digestion procedures.

Microwave assisted reduction of Se(VI) to Se(IV) and determination by HG/FI-ICP/MS for inorganic selenium speciation.

Speciation of inorganic selenium using hydride generation method is a widespread analytical method nowadays. However, a reduction step of Se(VI) to Se(IV) is necessary as the hydride-forming species is HSeO(3)(-) (oxydation state+IV). This paper describes the development of a batch assisted microwave system allowing a rapid (<5 min) conversion of Se(VI) to Se(IV). Hydride generation is performed by a flow injection system and detection by ICP/MS. Detection limits of 6 and 8 pg for Se(IV) and for Se(VI) (by using a sample loop of 200 mul) respectively have been achieved. This method has been validated by participating in a European certification exercise for inorganic Se speciation in aqueous solutions.