Benjamin Espinasse

More than the Ions: The Effects of Silver Nanoparticles on Lolium multiflorum

Silver nanoparticles (AgNPs) are increasingly used as antimicrobial additives in consumer products and may have adverse impacts on organisms when they inadvertently enter ecosystems. This study investigated the uptake and toxicity of AgNPs to the common grass, Lolium multiflorum. We found that root and shoot Ag content increased with increasing AgNP exposures. AgNPs inhibited seedling growth. While exposed to 40 mg L(-1) GA-coated AgNPs, seedlings failed to develop root hairs, had highly vacuolated and collapsed cortical cells and broken epidermis and rootcap. In contrast, seedlings exposed to identical concentrations of AgNO(3) or supernatants of ultracentrifuged AgNP solutions showed no such abnormalities. AgNP toxicity was influenced by total NP surface area with smaller AgNPs (6 nm) more strongly affecting growth than did similar concentrations of larger (25 nm) NPs for a given mass. Cysteine (which binds Ag(+)) mitigated the effects of AgNO(3) but did not reduce the toxicity of AgNP treatments. X-ray spectro-microscopy documented silver speciation within exposed roots and suggested that silver is oxidized within plant tissues. Collectively, this study suggests that growth inhibition and cell damage can be directly attributed either to the nanoparticles themselves or to the ability of AgNPs to deliver dissolved Ag to critical biotic receptors.

Long-term transformation and fate of manufactured ag nanoparticles in a simulated large scale freshwater emergent wetland.

Transformations and long-term fate of engineered nanomaterials must be measured in realistic complex natural systems to accurately assess the risks that they may pose. Here, we determine the long-term behavior of poly(vinylpyrrolidone)-coated silver nanoparticles (AgNPs) in freshwater mesocosms simulating an emergent wetland environment. AgNPs were either applied to the water column or to the terrestrial soils. The distribution of silver among water, solids, and biota, and Ag speciation in soils and sediment was determined 18 months after dosing. Most (70 wt %) of the added Ag resided in the soils and sediments, and largely remained in the compartment in which they were dosed. However, some movement between soil and sediment was observed. Movement of AgNPs from terrestrial soils to sediments was more facile than from sediments to soils, suggesting that erosion and runoff is a potential pathway for AgNPs to enter waterways. The AgNPs in terrestrial soils were transformed to Ag(2)S (~52%), whereas AgNPs in the subaquatic sediment were present as Ag(2)S (55%) and Ag-sulfhydryl compounds (27%). Despite significant sulfidation of the AgNPs, a fraction of the added Ag resided in the terrestrial plant biomass (~3 wt % for the terrestrially dosed mesocosm), and relatively high body burdens of Ag (0.5-3.3 μg Ag/g wet weight) were found in mosquito fish and chironomids in both mesocosms. Thus, Ag from the NPs remained bioavailable even after partial sulfidation and when water column total Ag concentrations are low (<0.002 mg/L).

Emerging Contaminant or an Old Toxin in Disguise? Silver Nanoparticle Impacts on Ecosystems

The use of antimicrobial silver nanoparticles (AgNPs) in consumer-products is rising. Much of these AgNPs are expected to enter the wastewater stream, with up to 10% of that eventually released as effluent into aquatic ecosystems with unknown ecological consequences. We examined AgNP impacts on aquatic ecosystems by comparing the effects of two AgNP sizes (12 and 49 nm) to ionic silver (Ag(+); added as AgNO3), a historically problematic contaminant with known impacts. Using 19 wetland mesocosms, we added Ag to the 360 L aquatic compartment to reach 2.5 mg Ag L(-1). Silver treatments and two coating controls were done in triplicate, and compared to four replicate controls. All three silver treatments were toxic to aquatic plants, leading to a significant release of dissolved organic carbon and chloride following exposure. Simultaneously, dissolved methane concentrations increased forty-fold relative to controls in all three Ag treatments. Despite dramatic toxicity differences observed in lab studies for these three forms of Ag, our results show surprising convergence in the direction, magnitude, and duration of ecosystem-scale impacts for all Ag treatments. Our results suggest that all forms of Ag changed solute chemistry driving transformations of Ag which then altered Ag impacts.

Nanomaterials as possible contaminants: the fullerene example

An assessment of the potential risks posed by nanomaterials will require case-by-case evaluations of the processes controlling exposure and hazards such as toxicity. Factors that control fullerene transport and transformation in aqueous environments and their relationship to toxicity are discussed. Natural organic matter is observed to either increase or decrease nanoparticle stability while trends in reactive oxygen generation run counter to proposed mechanisms of possible fullerene toxicity.

Heparin modifies the immunogenicity of positively-charged proteins

The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.

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.

Fate of single walled carbon nanotubes in wetland ecosystems

We report here the first studies addressing fate and transport of single walled carbon nanotubes (SWNTs) in aquatic mesocosms. The experimental design was structured to study the impact of nanomaterials within a tightly controlled and highly instrumented wetland ecosystems (aka mesocosm) and to address questions including fate and transport, effect on community structure, effects on biogeochemical function, and effects on productivity of the ecosystem. We added well-dispersed CoMoCat SWNTs (cSWNT,0 = 2.5 mg L−1) to the water column of a wetland mesocosm and examined the resulting phase distribution over time. Rapid settling of SWNTs from the water column was observed within a period of 2 days (Cw,t/Cw,0 < 0.01) after spiking. Samples from all mesocosm compartments (e.g. aquatic/semi aquatic plants, biofilm, mosquitofish and sediment) were analyzed to evaluate the transport and fate of SWNTs in the ecosystem. SWNTs were quantified in organism and sediment extracts using near-infrared fluorescence spectroscopy (NIRF). This technique can be used to quantitatively detect SWNTs in sediment and biotic matrices at environmentally relevant concentrations (MDLwater 5 μg L−1 MDLsediment 0.5 μg g−1 MDLbiota 5 μg g−1 wet weight) and qualitatively characterize SWNT samples before and after the studies. Results indicated that rapid aggregation and settling of SWNT resulted in accumulation of SWNT in surficial sediment. Sediment concentrations were spatially variable across the mesocosm, and thus estimates of SWNT mass balance within the mesocosm ranged from 7–48%. No bioaccumulation of SWNT in aquatic plants or vertebrates was observed over the 10 month incubation. However, NIRF imaging analysis suggested that mosquitofish ingested SWNT-laden particles but that burdens of SWNTs were confined to gut contents and may have been rapidly eliminated.

Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH)

Recent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin, ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and unfractionated heparin (UFH) are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralisation and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.

Comparative Persistence of Engineered Nanoparticles in a Complex Aquatic Ecosystem

During nanoparticle environmental exposure, presence in the water column is expected to dominate long distance transport as well as initial aquatic organism exposure. Much work has been done to understand potential ecological and toxicological effects of these particles. However, little has been done to date to understand the comparative persistence of engineered particles in realistic environmental systems. Presented here is a study of the water column lifetimes of 3 different classes of nanoparticles prepared with a combination of surface chemistries in wetland mesocosms. We find that, when introduced as a single pulse, all tested nanoparticles persist in the water column for periods ranging from 36 h to 10 days. Specifically, we found a range of nanoparticle residence times in the order Ag > TiO2 > SWCNT > CeO2. We further explored the hypothesis that heteroaggregation was the primary driving factor for nanoparticle removal from the water column in all but one case, and that values of surface affinity (α) measured in the laboratory appear to predict relative removal rates when heteroaggregation dominates. Though persistence in the water column was relatively short in all cases, differences in persistence may play a role in determining nanoparticle fate and impacts and were poorly predicted by currently prevailing benchmarks such as particle surface preparation.