Aquatic Mesocosm Strategies for the Environmental Fate and Risk Assessment of Engineered Nanomaterials.

In the past decade, mesocosms have emerged as a useful tool for the environmental study of engineered nanomaterials (ENMs) as they can mimic the relevant exposure scenario of contamination. Herein, we analyzed the scientific outcomes of aquatic mesocosm experiments, with regard to their designs, the ENMs tested, and the end points investigated. Several mesocosm designs were consistently applied in the past decade to virtually mimic various contamination scenarios with regard to ecosystem setting as well as ENMs class, dose, and dosing. Statistical analyses were carried out with the literature data to identify the main parameters driving ENM distribution in the mesocosms and the potential risk posed to benthic and planktonic communities as well as global ecosystem responses. These analyses showed that at the end of the exposure, mesocosm size (water volume), experiment duration, and location indoor/outdoor had major roles in defining the ENMs/metal partitioning. Moreover, a higher exposure of the benthic communities is often observed but did not necessarily translate to a higher risk due to the lower hazard posed by transformed ENMs in the sediments (e.g., aggregated, sulfidized). However, planktonic organisms were generally exposed to lower concentrations of potentially more reactive and toxic ENM species. Hence, mesocosms can be complementary tools to existing standard operational procedures for regulatory purposes and environmental fate and risk assessment of ENMs. To date, the research was markedly unbalanced toward the investigation of metal-based ENMs compared to metalloid- and carbon-based ENMs but also nanoenabled products. Future studies are expected to fill this gap, with special regard to high production volume and potentially hazardous ENMs. Finally, to take full advantage of mesocosms, future studies must be carefully planned to incorporate interdisciplinary approaches and ensure that the large data sets produced are fully exploited.

Comparison of Nanomaterials for Delivery of Double-Stranded RNA in Caenorhabditis elegans.

RNA interference is a promising crop protection technology that has seen rapid development in the past several years. Here, we investigated polyamino acid biopolymers, inorganic nanomaterials, and hybrid organic-inorganic nanomaterials for delivery of dsRNA and efficacy of gene knockdown using the model nematode Caenorhabditis elegans. Using an oral route of delivery, we are able to approximate how nanomaterials will be delivered in the environment. Of the materials investigated, only Mg-Al layered double-hydroxide nanoparticles were effective at gene knockdown in C. elegans, reducing marker gene expression to 66.8% of that of the control at the lowest tested concentration. In addition, we identified previously unreported injuries to the mouthparts of C. elegans associated with the use of a common cell-penetrating peptide, poly-l-arginine. Our results will allow the pursuit of further research into promising materials for dsRNA delivery and also allow for the exclusion of those with little efficacy or deleterious effects.

Aqueous aging of a silica coated TiO2 UV filter used in sunscreens: investigations at the molecular scale with dynamic nuclear polarization NMR.

Short-term, aqueous aging of a commercial nanocomposite TiO2 UV filter with a protective SiO2 shell was examined in abiotic simulated fresh- and seawater. Under these conditions, the SiO2 layer was quantitatively removed (∼88-98%) within 96 hours, as determined using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). While these bulk ICP-AES analyses suggested almost identical SiO2 shell degradation after aging in fresh- and seawater, surface sensitive 29Si dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance (SSNMR), with signal enhancements of 5-10× compared to standard SSNMR, was able to distinguish differences in the aged nanocomposites at the molecular level. DNP-SSNMR revealed that the attachment of the silica layer to the underlying TiO2 core rested on substantial Si-O-Ti bond formation, bonds which were preserved after freshwater aging, yet barely present after aging in seawater. The removal of the protective SiO2 layer is due to ionic strength accelerated dissolution, which could present significant consequences to aqueous environments when the photoactive TiO2 core becomes exposed. This work demonstrates the importance of characterizing aged nanocomposites not only on the bulk scale, but also on the molecular level by employing surface sensitive techniques, such as DNP-NMR. Molecular level details on surface transformation and elemental speciation will be crucial for improving the environmental safety of nanocomposites.

Monitoring the Environmental Aging of Nanomaterials: An Opportunity for Mesocosm Testing?

Traditional aging protocols typically examine only the effects of a limited number of stresses, and relatively harsh conditions may trigger degradation mechanisms that are not observed in actual situations. Environmental aging is, in essence, the complex interaction of multiple mechanical, physicochemical and biological stresses. As yet, there is no (pre)standardized procedure that addresses this issue in a satisfactory manner. Mesocosm experiments can be designed to specifically cover the aging of nanomaterials while characterizing the associated exposure and hazard. The scenario of exposure and the life time of the nanomaterial appear as the predominant factors in the design of the experiment, and appropriate precautions need to be taken. This should the subject of guidance that may be divided into product/application categories.

Non-linear release dynamics for a CeO2 nanomaterial embedded in a protective wood stain, due to matrix photo-degradation.

The release of CeO2-bearing residues during the weathering of an acrylic stain enriched with CeO2 nanomaterial designed for wood protection (Nanobyk brand additive) was studied under two different scenarios: (i) a standard 12-weeks weathering protocol in climate chamber, that combined condensation, water spraying and UV-visible irradiation and (ii) an alternative accelerated 2-weeks leaching batch assay relying on the same weathering factors (water and UV), but with a higher intensity of radiation and immersion phases. Similar Ce released amounts were evidenced for both scenarios following two phases: one related to the removal of loosely bound material with a relatively limited release, and the other resulting from the degradation of the stain, where major release occurred. A non-linear evolution of the release with the UV dose was evidenced for the second phase. No stabilization of Ce emissions was reached at the end of the experiments. The two weathering tests led to different estimates of long-term Ce releases, and different degradations of the stain. Finally, the photo-degradations of the nanocomposite, the pure acrylic stains and the Nanobyk additive were compared. The incorporation of Nanobyk into the acrylic matrix significantly modified the response of the acrylic stain to weathering.

Stealth Biocompatible Si-Based Nanoparticles for Biomedical Applications.

A challenge regarding the design of nanocarriers for drug delivery is to prevent their recognition by the immune system. To improve the blood residence time and prevent their capture by organs, nanoparticles can be designed with stealth properties using polymeric coating. In this study, we focused on the influence of surface modification with polyethylene glycol and/or mannose on the stealth behavior of porous silicon nanoparticles (pSiNP, ~200 nm). In vivo biodistribution of pSiNPs formulations were evaluated in mice 5 h after intravenous injection. Results indicated that the distribution in the organs was surface functionalization-dependent. Pristine pSiNPs and PEGylated pSiNPs were distributed mainly in the liver and spleen, while mannose-functionalized pSiNPs escaped capture by the spleen, and had higher blood retention. The most efficient stealth behavior was observed with PEGylated pSiNPs anchored with mannose that were the most excreted in urine at 5 h. The biodegradation kinetics evaluated in vitro were in agreement with these in vivo observations. The biocompatibility of the pristine and functionalized pSiNPs was confirmed in vitro on human cell lines and in vivo by cytotoxic and systemic inflammation investigations, respectively. With their biocompatibility, biodegradability, and stealth properties, the pSiNPs functionalized with mannose and PEG show promising potential for biomedical applications.

Nanoparticle Uptake in Plants: Gold Nanomaterial Localized in Roots of Arabidopsis thaliana by X-ray Computed Nanotomography and Hyperspectral Imaging.

Terrestrial plants can internalize and translocate nanoparticles (NPs). However, direct evidence for the processes driving the NP uptake and distribution in plants is scarce at the cellular level. Here, NP-root interactions were investigated after 10 days of exposure of Arabidopsis thaliana to 10 mg·L-1 of negatively or positively charged gold NPs (∼12 nm) in gels. Two complementary imaging tools were used: X-ray computed nanotomography (nano-CT) and enhanced dark-field microscopy combined with hyperspectral imaging (DF-HSI). The use of these emerging techniques improved our ability to detect and visualize NP in plant tissue: by spectral confirmation via DF-HSI, and in three dimensions via nano-CT. The resulting imaging provides direct evidence that detaching border-like cells (i.e., sheets of border cells detaching from the root) and associated mucilage can accumulate and trap NPs irrespective of particle charge. On the contrary, border cells on the root cap behaved in a charge-specific fashion: positively charged NPs induced a higher mucilage production and adsorbed to it, which prevented translocation into the root tissue. Negatively charged NPs did not adsorb to the mucilage and were able to translocate into the apoplast. These observations provide direct mechanistic insight into NP-plant interactions, and reveal the important function of border cells and mucilage in interactions of plants with charged NPs.

Dynamic Nuclear Polarization NMR as a new tool to investigate the nature of organic compounds occluded in plant silica particles.

The determination of the chemical nature of the organic matter associated with phytoliths remains a challenge. This difficulty mainly stems from amounts of organic carbon (C) that are often well below the detection limit of traditional spectroscopic tools. Conventional solid-state 13C Nuclear Magnetic Resonance (NMR) is widely used to examine the nature and structure of organic molecules, but its inherent low sensitivity prohibits the observation of diluted samples. The recent advent of commercial microwave source in the terahertz range triggered a renewed interest in the Dynamic Nuclear Polarization (DNP) technique to improve the signal to noise ratio of solid-state NMR experiments. With this technique, the 13C spectrum of a phytolith sample containing 0.1% w/w C was obtained overnight with sufficient quality to permit a semi-quantitative analysis of the organic matter, showing the presence of peptides and carbohydrates as predominant compounds. Considering the natural abundance of the 13C isotope, this experiment demonstrates that DNP NMR is sufficiently sensitive to observe spin systems present in amounts as low as a few tens of ppm.

Remote Biodegradation of Ge-Imogolite Nanotubes Controlled by the Iron Homeostasis of Pseudomonas brassicacearum.

The toxicity of high-aspect-ratio nanomaterials (HARNs) is often associated with oxidative stress. The essential nutrient Fe may also be responsible of oxidative stress through the production of reactive oxygen species. In the present study, it has been examined to what extent adding Fenton reaction promoting Fe impacted the toxicity of an alumino-germanate model HARN. Structural addition of only 0.95% wt Fe to Ge-imogolite not only alleviated the toxicity observed in the case of Fe-free nanotubes but also stimulated bacterial growth. This was attributed to the metabolization of siderophore-mobilized Fe from the nanotube structure. This was evidenced by the regulation of the homeostasis-monitoring intracellular Fe levels. This was accompanied by a biodegradation of the nanotubes approaching 40%, whereas the Fe-free nanomaterial remained nearly untouched.

Involvement of nitrogen functional groups in high-affinity copper binding in tomato and wheat root apoplasts: spectroscopic and thermodynamic evidence.

Carboxylic groups located in plant cell walls (CW) are generally considered to be the main copper binding sites in plant roots, despite the presence of other functional groups. The aim of this study was to investigate sites responsible for copper binding in root apoplasts, i.e. CW and outer surface of the plasma membrane (PM) continuum. Binding sites in root apoplasts were investigated by comparing isolated CW of a monocotyledon (Triticum aestivum L.) and dicotyledon (Solanum lycopersicum L.) crop with their respective whole roots. Copper speciation was examined by X-ray absorption (XAS) and (13)C-nuclear magnetic resonance spectroscopies while the affinity of ligands involved in copper binding was investigated by modeling copper sorption isotherms. Homogeneous speciation and binding of copper was found in wheat and tomato root apoplasts. Only Cu-N and Cu-O bonds were detected in wheat and tomato root apoplasts. Nitrogen/oxygen ligands were identified in slightly higher proportions (40-70%) than single oxygen ligands. Furthermore, low- and high-affinity binding sites contributed in an almost equivalent proportion to copper binding in root apoplasts. The high-affinity N functional groups embedded in root apoplasts participated in copper binding in the same magnitude than the low-affinity carboxylic groups.

An adaptable mesocosm platform for performing integrated assessments of nanomaterial risk in complex environmental systems.

Physical-chemists, (micro)biologists, and ecologists need to conduct meaningful experiments to study the environmental risk of engineered nanomaterials with access to relevant mechanistic data across several spatial and temporal scales. Indoor aquatic mesocosms (60L) that can be tailored to virtually mimic any ecosystem appear as a particularly well-suited device. Here, this concept is illustrated by a pilot study aimed at assessing the distribution of a CeO2-based nanomaterial within our system at low concentration (1.5 mg/L). Physico-chemical as well as microbiological parameters took two weeks to equilibrate. These parameters were found to be reproducible across the 9-mesocosm setup over a 45-day period of time. Recovery mass balances of 115 ± 18% and 60 ± 30% of the Ce were obtained for the pulse dosing and the chronic dosing, respectively. This demonstrated the relevance of our experimental approach that allows for adequately monitoring the fate and impact of a given nanomaterial.

Transformation of pristine and citrate-functionalized CeO2 nanoparticles in a laboratory-scale activated sludge reactor.

Engineered nanomaterials (ENMs) are used to enhance the properties of many manufactured products and technologies. Increased use of ENMs will inevitably lead to their release into the environment. An important route of exposure is through the waste stream, where ENMs will enter wastewater treatment plants (WWTPs), undergo transformations, and be discharged with treated effluent or biosolids. To better understand the fate of a common ENM in WWTPs, experiments with laboratory-scale activated sludge reactors and pristine and citrate-functionalized CeO2 nanoparticles (NPs) were conducted. Greater than 90% of the CeO2 introduced was observed to associate with biosolids. This association was accompanied by reduction of the Ce(IV) NPs to Ce(III). After 5 weeks in the reactor, 44 ± 4% reduction was observed for the pristine NPs and 31 ± 3% for the citrate-functionalized NPs, illustrating surface functionality dependence. Thermodynamic arguments suggest that the likely Ce(III) phase generated would be Ce2S3. This study indicates that the majority of CeO2 NPs (>90% by mass) entering WWTPs will be associated with the solid phase, and a significant portion will be present as Ce(III). At maximum, 10% of the CeO2 will remain in the effluent and be discharged as a Ce(IV) phase, governed by cerianite (CeO2).

Long-term aging of a CeO(2) based nanocomposite used for wood protection.

A multi-scale methodology was used to characterize the long-term behavior and chemical stability of a CeO2-based nanocomposite used as UV filter in wood stains. ATR-FTIR and (13)C NMR demonstrated that the citrate coated chelates with Ce(IV) through its central carboxyl- and its α-hydroxyl- groups at the surface of the unaged nanocomposite. After 42 days under artificial daylight, the citrate completely disappeared and small amount of degradation products remained attached to the surface even after 112 days. Moreover, the release/desorption of the citrate layer led to a surface reorganization of the nano-sized CeO2 core observed by XANES (Ce L3-edge). Such a surface and structural transformation of the commercialized nanocomposite could have implications in term of fate, transport, and potential impacts towards the environment.

Inhibition of sulfate reducing bacteria in aquifer sediment by iron nanoparticles.

Batch microcosms were setup to determine the impact of different sized zero valent iron (Fe(0)) particles on microbial sulfate reduction during the in situ bio-precipitation of metals. The microcosms were constructed with aquifer sediment and groundwater from a low pH (3.1), heavy-metal contaminated aquifer. Nano (nFe(0)), micro (mFe(0)) and granular (gFe(0)) sized Fe(0) particles were added to separate microcosms. Additionally, selected microcosms were also amended with glycerol as a C-source for sulfate-reducing bacteria. In addition to metal removal, Fe(0) in microcosms also raised the pH from 3.1 to 6.5, and decreased the oxidation redox potential from initial values of 249 to -226 mV, providing more favorable conditions for microbial sulfate reduction. mFe(0) and gFe(0) in combination with glycerol were found to enhance microbial sulfate reduction. However, no sulfate reduction occurred in the controls without Fe(0) or in the microcosm amended with nFe(0). A separate dose test confirmed the inhibition for sulfate reduction in presence of nFe(0). Hydrogen produced by Fe(0) was not capable of supporting microbial sulfate reduction as a lone electron donor in this study. Microbial analysis revealed that the addition of Fe(0) and glycerol shifted the microbial community towards Desulfosporosinus sp. from a population initially dominated by low pH and metal-resisting Acidithiobacillus ferrooxidans.

Influence of the length of imogolite-like nanotubes on their cytotoxicity and genotoxicity toward human dermal cells.

Physical-chemical parameters such as purity, structure, chemistry, length, and aspect ratio of nanoparticles (NPs) are linked to their toxicity. Here, synthetic imogolite-like nanotubes with a set chemical composition but various sizes and shapes were used as models to investigate the influence of these physical parameters on the cyto- and genotoxicity and cellular uptake of NPs. The NPs were characterized using X-ray diffraction (XRD), small angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Imogolite precursors (PR, ca. 5 nm curved platelets), as well as short tubes (ST, ca. 6 nm) and long tubes (LT, ca. 50 nm), remained stable in the cell culture medium. Internalization into human fibroblasts was observed only for the small particles PR and ST. None of the tested particles induced a significant cytotoxicity up to a concentration of 10(-1) mg·mL(-1). However, small sized NPs (PR and ST) were found to be genotoxic at very low concentration 10(-6) mg·mL(-1), while LT particles exhibited a weak genotoxicity. Our results indicate that small size NPs (PR, ST) were able to induce primary lesions of DNA at very low concentrations and that this DNA damage was exclusively induced by oxidative stress. The higher aspect ratio LT particles exhibited a weaker genotoxicity, where oxidative stress is a minor factor, and the likely involvement of other mechanisms. Moreover, a relationship among cell uptake, particle aspect ratio, and DNA damage of NPs was observed.

Is there a Trojan-horse effect during magnetic nanoparticles and metalloid cocontamination of human dermal fibroblasts?

This study investigates the issue of nanoparticles/pollutants cocontamination. By combining viability assays, physicochemical and structural analysis (to probe the As speciation and valence), we assessed how γFe(2)O(3) nanoparticles can affect the cytotoxicity, the intra- and extracellular speciation of As(III). Human dermal fibroblasts were contaminated with γFe(2)O(3) nanoparticles and As(III) considering two scenarios: (i) a simultaneous coinjection of the nanoparticles and As, and (ii) an injection of the nanoparticles after 24 h of As adsorption in water. In both scenarios, we did not notice significant changes on the nanoparticles surface charge (zeta potential ∼ -10 mV) nor hydrodynamic diameters (∼950 nm) after 24 h. We demonstrated that the coinjection of γFe(2)O(3) nanoparticles and As in the cellular media strongly affects the complexation of the intracellular As with thiol groups. This significantly increases at low doses the cytotoxicity of the As nonadsorbed at the surface of the nanoparticles. However, once As is adsorbed at the surface the desorption is very weak in the culture medium. This fraction of As strongly adsorbed at the surface is significantly less cytotoxic than As itself. On the basis of our data and the thermodynamics, we demonstrated that any disturbance of the biotransformation mechanisms by the nanoparticles (i.e., surface complexation of thiol groups with the iron atoms) is likely to be responsible for the increase of the As adverse effects at low doses.

Analysis of engineered nanomaterials in complex matrices (environment and biota): general considerations and conceptual case studies.

Advances in the study of the environmental fate, transport, and ecotoxicological effects of engineered nanomaterials (ENMs) have been hampered by a lack of adequate techniques for the detection and quantification of ENMs at environmentally relevant concentrations in complex media. Analysis of ENMs differs from traditional chemical analysis because both chemical and physical forms must be considered. Because ENMs are present as colloidal systems, their physicochemical properties are dependent on their surroundings. Therefore, the simple act of trying to isolate, observe, and quantify ENMs may change their physicochemical properties, making analysis extremely susceptible to artifacts. Many analytical techniques applied in materials science and other chemical/biological/physical disciplines may be applied to ENM analysis as well; however, environmental and biological studies may require that methods be adapted to work at low concentrations in complex matrices. The most pressing research needs are the development of techniques for extraction, cleanup, separation, and sample storage that introduce minimal artifacts to increase the speed, sensitivity, and specificity of analytical techniques, as well as the development of techniques that can differentiate between abundant, naturally occurring particles, and manufactured nanoparticles.

Polyaluminum chloride with high Al30 content as removal agent for arsenic-contaminated well water.

Polyaluminum chloride (PACl) is a well-established coagulant in water treatment with high removal efficiency for arsenic. A high content of Al(30) nanoclusters in PACl improves the removal efficiency over broader dosage and pH range. In this study we tested PACl with 75% Al(30) nanoclusters (PACl(Al30)) for the treatment of arsenic-contaminated well water by laboratory batch experiments and field application in the geothermal area of Chalkidiki, Greece, and in the Pannonian Basin, Romania. The treatment efficiency was studied as a function of dosage and the nanoclusters' protonation degree. Acid-base titration revealed increasing deprotonation of PACl(Al30) from pH 4.7 to the point of zero charge at pH 6.7. The most efficient removal of As(III) and As(V) coincided with optimal aggregation of the Al nanoclusters at pH 7-8, a common pH range for groundwater. The application of PACl(Al30) with an Al(tot) concentration of 1-5mM in laboratory batch experiments successfully lowered dissolved As(V) concentrations from 20 to 230 µg/L to less than 5 µg/L. Field tests confirmed laboratory results, and showed that the WHO threshold value of 10 µg/L was only slightly exceeded (10.8 µg/L) at initial concentrations as high as 2300 µg/L As(V). However, As(III) removal was less efficient (<40%), therefore oxidation will be crucial before coagulation with PACl(Al30). The presence of silica in the well water improved As(III) removal by typically 10%. This study revealed that the Al(30) nanoclusters are most efficient for the removal of As(V) from water resources at near-neutral pH.

TiO2-based nanoparticles released in water from commercialized sunscreens in a life-cycle perspective: structures and quantities.

This work investigates the physical-chemical evolution during artificial aging in water of four commercialized sunscreens containing TiO2-based nanocomposites. Sunscreens were analyzed in terms of mineralogy and TiO2 concentration. The residues formed after aging were characterized in size, shape, chemistry and surface properties. The results showed that a significant fraction of nano-TiO2 residues was released from all sunscreens, despite their heterogeneous behaviors. A stable dispersion of submicronic aggregates of nanoparticles was generated, representing up to 38 w/w% of the amount of sunscreen, and containing up to 30% of the total nano-TiO2 initially present in the creams. The stability of the dispersion was tested as a function of salt concentration, revealing that in seawater conditions, a major part of these nano-TiO2 residues will aggregate and sediment. These results were put in perspective with consumption and life cycle of sunscreens to estimate the amount of nano-TiO2 potentially released into AQUATIC environment.

Growth kinetic of single and double-walled aluminogermanate imogolite-like nanotubes: an experimental and modeling approach.

Atomic Force Microscopy (AFM) and in situ Small Angle X-ray Scattering (SAXS) were used to investigate the evolution of the aluminogermanate imogolite-like nanotubes concentration and morphology during their synthesis. In particular, in situ SAXS allowed quantifying the transformation of protoimogolite into nanotubes. The size distribution of the final nanotubes was also assessed after growth by AFM. A particular attention was focused on the determination of the single and double walled nanotube length distributions. We observed that the two nanotube types do not grow with the same kinetic and that their final length distribution was different. A model of protoimogolites oriented aggregation was constructed to account for the experimental growth kinetic and the length distribution differences.