Abstracts, A - GMultifunctional Nanostructured Materials as Adsorbents
for Environmental Remediation Synthesis of nanostructured materials with hybrid properties from inorganic precursor and organic templates will produce materials with well-defined pore and ultra high surface area that are suitable as hosts for environmental remediation. Lead ions in soils and stormwater run-off from small arms firing range (SAFR) are a major concern. Various nanomaterials as adsorbents have been studied to remediate the problem of lead species releases to the environment. These nanomaterials as adsorbents demonstrated potential for SAFR applications with the greatest range of pH, and a consistent performance in the presence of competing ions and increasing temperatures. In conclusion, nanostructured materials with hybrid properties can be used to remediate lead (II) ions.
Best Management Practices for Managing Risk from
Nanomaterials in a University Research Laboratory Environment Limited published information exists on the potential
adverse health effects of occupational exposure to nanomaterials (NM),
however concerns suggested by laboratory animal studies (Poland et al.
2008; Takagi et al. 2008) are compelling Environment, Health, and Safety
(EHS) professionals to establish safe workplace practices following the
general principles of prudent practice.
Although the pace of NM research is accelerating , there are currently
no occupational exposure limits governing workplace exposure. Limited
information, inadequate sampling equipment, incomplete assessment guidance
and scant EHS budgets also contribute to the difficulties of creating
informed decisions for workplace safety. The research reported
here examined ways to provide informed guidance to those working with
NM on a laboratory-scale in a university environment, taking into account
the limited information and resources available. This research evaluated
three common laboratory practices for preparing NM: material transfer
(pouring) into a waste container outside of a chemical fumehood, sonication
of NM solution, and weighing dry NM in an unventilated enclosure. The
assessment procedure used to study laboratory use of NM was developed
in a NIOSH-funded project that combined two direct-reading, real-time
instruments to characterize concentrations in laboratory air (Methner,
2008). Combining available information with simplified assessment procedures
helped to make informed decisions when confronted with limited resources. Analytical
results, combined with standard chemical hygiene practices, were used
to develop workplace practices for these common laboratory procedures. Effect of Natural Organic Matter on the Behavior
of Quantum Dots in Aquatic Environment The anticipation of the increased production and commercialization of semiconductor quantum dots (QDs) has been paralleled with increasing concerns regarding the potential environmental impacts of these materials. Information on the mobility and persistence of QDs in water is key to evaluating potential ecological hazards posed by QDs in the environment. Therefore, a study was performed to examine the role of natural organic matter (NOM) in the partitioning behavior of CdSe QDs capped with trioctylphosphine-oxide (TOPO) from an organic solvent into water. Results show that humic and fulvic acids, which have been used as model NOM, facilitate the stabilization of organic-capped QDs in water in less than 24 h. Phase transfer of the QD to the aqueous phase was observed for particles of different sizes. Solution pH and ionic strength also influenced the rate of phase transfer, which was favored at lower pH and lower ionic strength (absence of Ca2+). Spectroscopic evidence suggest that HA and FA interact with the surface capping groups of QDs instead of metal coordination; these were revealed by dynamic light scattering, transmission electron microscopy and infrared spectroscopy studies. The results observed with the Suwannee River HAs and FAs translated to the natural surface water samples collected from local creeks. This study presents the first evidence of stabilization of QDs in water by humic substances in real environmental samples, illustrating that NOM will have a significant role in the fate and transport of QDs in the aquatic systems. Fate Modeling of Titanium Dioxide Nanoparticles in
the Water Compartment by Colloid Chemistry Modeling a New Separation Technology Using Microwave-Driven
Nanopores Zeolites are inorganic nanoporous materials that can be fabricated into
membranes used for a variety of applications including environmental
separations. The separation mechanism for volatile organic compounds
is often based on molecular sieving, which exploits size difference to
exclude molecules that are too big to fit into the particular nanopores.
Often one is faced with the need to separate molecules of roughly similar
size, relying instead on differences in, e.g., molecular polarities. We
have performed simulations to explore whether sorption selectivities
can be tuned by applying microwave fields to zeolite-guest systems. Such
an approach would provide a new method for separating volatile organics. Conflicting
reports on microwave effects have established a certain controversy over
whether such effects are real or imagined. To address this issue at a
fundamental level, we have developed and applied simulation methods to
study microwave-driven sorption in zeolites. We provide a brief overview
of these methods, followed by a description of the main results. In general,
we find that microwave heating can produce novel energy distributions,
which can lead to different molecules in a mixture seeming to diffuse
and sorb at different but simultaneous temperatures. Implications for
these findings in modern chemical processing is discussed. Nanoparticle-Polymer/Nanoparticle-Protein
Supramolecular Complexes as Biosensors Cellular signatures present on the surfaces of bacteria or mammalian cells provide a means for rapid and efficient identification. With bacteria, cell surface functionality can potentially be used to differentiate between harmless and pathogenic species and strains. In the case of mammalian cells, rapid screening of cell surfaces provides a potential means of screening for cancer and other disease states. Rapid and effective determination of cell surface signatures is challenging due to the wide array of proteins, sugars, and lipids at the cell surface. To address this complexity, we have developed an array-based “chemical nose” approach to recognize cellular signatures that relies on the selective interactions between multiple reporter elements and the target analytes. These arrays are able to present chemical diversity to respond differentially to a variety of analytes. Using this approach, we have developed sensors capable of distinguishing between species, and even individual strains of a single species. We have achieved similar success in mammalian cells, demonstrating effective differentiation between normal, cancerous and metastatic mammalian cell types as well as different isogenic cell lines. The presentation addresses the application of array-based sensing of microbes and cells in both environmental and biomedical contexts.\ Biological Oxidative Stress to Understand and Characterize
Exposures to Nanomaterials Background: Rapid developments and commercialization of nanotechnologies has resulted in a fast pace synthesis of novel nanomaterials (NMs). A need exists for fast screening of these NMs at the development stage to enable a proactive approach to potential health risks and to develop greener technologies. Biological oxidative stress (BOS) has been recognized as a key mechanism of toxicity of particulate matter and holds potential as a novel global metric for NMs exposures and for rapid toxicity screening. A ‘Ferric Reducing Ability of Serum (FRAS)’ assay was recently optimized by our group as a screening tool to quantitate the degree of biological oxidative damage in human blood serum and this approach was used to screen several NMs for their ability to cause BOS. This research had two main objectives: (i) to investigate the relationship between several physico-chemical parameters of NMs and FRAS-measured BOS in an effort to explain the observed results and to evaluate their potential utility as exposure metrics for NMs; and (ii) to investigate the utility of these findings for greening nanomanufacturing processes and NMs exposure assessment. Methods: A diverse set of 19 commercially important NMs, including a series of carbon blacks, fullerenes, several CNTs, carbon nanohorns, TiO2, aluminum oxide, and nano silver were tested for BOS with the FRAS assay. In-depth physico-chemical characterization was performed for each material, including measurements of their specific surface area, surface charge, soluble and total transition metals, crystallinity, organic carbon, and PAHs. Relationships between the FRAS BOS and physico-chemical properties of materials were explored in multivariate regression analyses. Results: BOS was strongly correlated with the specific surface area (SSA) and select transition metals (Fe, Cr, Co, Zn), which when combined explained 80-98 percent of the observed BOS. Neither metric was a sufficient predictor of BOS on its own. Peculiar behaviors were seen within the same series of materials, especially TiO2 and carbon nanotubes. Other measures were of lesser significance. Interestingly, a threshold phenomena was observed for BOS for selected parameters. Conclusion: Biological oxidative stress potential of NMs as measured by FRAS in human serum appears to be a valid approach for screening purposes and online, real-time measurement of the oxidative damage potential of particulate matter seems a credible proposition. Ongoing research to evaluate relationships between FRAS BOS with chemical and cellular electron spin resonance and cellular toxicity assays will further elucidate the utility of FRAS as a screening assay for NM toxicity. Advances in nZVI Coupled with Enhanced Bioremediation Recent results from a very large pilot-scale nZVI injection illustrate the transition of abiotic CAH degradation to enhanced bioremediation of CAHs. This transition appears to be driven by aquifer redox conditioning in response to nZVI reactivity, coupled with the addition of a complex source of soluble carbon electron donor (soy protein), which is mechanistically used to suspend and disperse the nano-particles for increased radii of influence from injection wells. This transition and continued degradation of CAHs continued for over one year and achieved a groundwater concentration target of 5 ppb for trichloroethene (TCE) without build-up of intermediate degradation products. Microbial analysis (PFLA) of pre, during and post pilot conditions indicates a substantial biomass increase, a shift from more simple community structures to more complex structures and some associated physiological stress that decreased over time. Additionally, as compared to 14 other pilots performed by Golder, results further show the most significant transport distance we have yet experienced (60 to 100 feet), visually observed in down gradient monitoring wells as a black water and as determined by total iron analysis. This suggests that a combined remedy of nZVI injection and long-term enhanced bioremediation may be a very strong candidate technology for a number of CAH impacted sites, especially in fractured bedrock sites that often contain residual NAPL materials not amenable to extraction. Aqueous Toxicity and Food Chain Transfer of Quantum
Dots in Freshwater Algae and Ceriodaphnia dubia Innovative research and diagnostic techniques for biological
testing have advanced during recent years due to the development of semiconductor
nanocrystals. Although these commercially available fluorescent
nanocrystals have a protective organic coating, the inner core contains
cadmium and selenium. Because these metals have the potential for
detrimental environmental effects, concerns have been raised from the
lack of understanding of the environmental fate of these products. U.S.
Environmental Protection Agency test protocol and fluorescence microscopy
was used to determine the fate and effect of quantum dots (QD® 545 ITK™
Carboxyl QDs) using standard aquatic test organisms. Although no
lethality was measured following 48-h exposure of Ceriodaphnia dubia
to suspensions as high as 110 ppb, 96-h median lethal concentration (LC50)
to Pseudokirchneriella subcapitata was measured at 37.1 ppb QDs. Transfer
of QDs from dosed algae to C. dubia was verified with fluorescence microscopy.
These results indicate that coatings present on nanocrystals provide
protection from metal toxicity during laboratory exposures but the transfer
of core metals from intact nanocrystals may occur at levels well above
toxic threshold values indicating potential exposure of higher trophic
levels. Studies on the fate and effects of nanoparticles can be
incorporated into models for predictive toxicology of these emerging
contaminants. Removal of Metallic Ions from Water: Adsorption onto
Plant Materials and Microwave Radiation for the Fabrication of Nanometals In the current paper we describe a combined procedure
composed of two known technologies namely, adsorption of metallic ions
on plant materials and converting the adsorbed heavy ions into metallic
nanoparticles. The combined processes offer a promising Approach for
removal of heavy metals from wastewater and recycling of the adsorbed
metals into marketable product of metallic nanoparticles. In the second stage of the research, plant cuticular materials were used as sorbents for the metallic ions (Ag+). Plant cuticular materials have been recently described as highly efficient natural sorbents for organic pollutants; however, their role as adsorption agent for metal ions and metallic particles is unknown. The objective was to study the adsorption and fabrication of silver ions and nanoparticles by the Agave americana cuticle and its structural components. The two sides of the A. americana cuticle exhibited different behavior with respect to the adsorption of silver nanoparticles produced by microwave-assisted polyol reduction. Only the outside of the cuticle was found to be coated with silver nanoparticles. A mechanistic investigation showed that the first step in this reaction is the formation of nanoparticles in solution. Then the metallic nanoparticles were adsorbed by the cuticle. Silver ions were not adsorbed by the cuticle and therefore the interaction mechanism with Agave americana cuticle was different from the one observed with the aquatic plant Azolla filiculoides. Assessing the Fate and Effects of Nano Aluminum Oxide
in the Terrestrial Invertebrate, Eisenia fetida Nano aluminum, through transition to its oxidized form (Al2O3), offers great promise in commercial and military technology applications. The unique size and surface area of nano- Al2O3 may play a role in the material’s ability to strengthen coatings, increase propellant burning rates, and enhance explosive impacts. Due to the potential for wide dispersal in soil systems, we chose to investigate the fate and effects of nano-Al2O3 in a terrestrial organism. The toxicity and bioaccumulation potential of bulk (50-200µm) and nano (11 nm) Al2O3 was assessed through acute and chronic exposures using the terrestrial invertebrate, Eisenia fetida. Acute (48 hour) filter paper exposures were conducted to investigate the potential for dermal uptake of nano and bulk-Al2O3. Chronic (28 day) studies exposed E. fetida to nano and bulk-Al2O3 spiked reference soils to assess long term exposure effects. In addition to toxicity and bioaccumulation studies, an acute (48 hour) behavioral bioassay was conducted. The behavioral study utilized soil avoidance wheels in which E. fetida were given a choice of habitat between control versus nano and bulk-Al2O3 amended soils. In chronic soil exposures, reproductive decline and higher aluminum body burdens were observed in nano-Al2O3 treatments relative to control and bulk-Al2O3 treatments of equal concentration. In soil avoidance bioassays, E. fetida showed a preference for control soil relative to bulk and nano-Al2O3 at the highest concentrations (>5,000 mg/kg). Our results indicate that nano-Al2O3 may impact the life cycle and behavior of E. fetida, although at levels unlikely to be found in the environment. Due to increasing use of nano-Al2O3 materials in both commercial and military applications, additional studies are needed to determine the fate and transport of this material in terrestrial systems. Life Cycle Assessment of Developmental Carbon Nanotube
Products As nanotechnology advances from development to commercialization
at a rapid rate, it is necessary to gain a more comprehensive understanding
of the environmental impacts associated with various nanomanufacturing
processes. The use of carbon nanotubes (CNT) in applications such
as structural polymers, energy conversion, batteries, sensors, and shielding
represents particularly promising opportunities. Though CNTs possess
unique mechanical, electrical and thermal properties, great uncertainty
remains regarding their environmental impacts, health effects, and economic
viability. To address these concerns while these processes are
still in the developmental phase, models have been developed to assess
the environmental attributes of manufacturing processes using CNTs. Influence of Surface Chemistry on the Colloidal Properties
of Carbon Nanotubes Surface chemistry is expected to play a pivotal role in determining the behavior of nanomaterials, because the majority of the atoms are located at or near the surface. For carbon nanotubes, one of the most important changes in surface chemistry occurs as a result of oxidation. This can occur either deliberately as a surface functionalization strategy or unintentionally as a result of CNT exposure to powerful oxidants present in the environment, such as ozone. During these oxidative treatments, oxygen containing functional groups form at the CNT’s exposed surfaces, preferentially at open ends and defect sites. In this presentation we discuss the influence that both the oxygen concentration and oxygen functional-group distribution have on the colloidal stability of multi-walled carbon nanotubes, prepared by exposing pristine MWCNTs to common oxidizing agents and reaction conditions. As a metric of colloidal stability, we have studied the influence that surface chemistry exerts on the aggregation kinetics of O-MWCNTs as a function of pH and electrolyte composition. The key finding from these studies is that even comparatively small changes in the concentration and distribution of surface oxides exert a profound influence on the colloidal stability of multi-walled carbon nanotubes and furthermore that mathematical relationships exist that can be used to correlate the concentration of surface oxygen, the surface charge and the colloidal stability of oxidized MWNCTs (O-MWCNTs) under environmentally relevant pH conditions. In contrast, electrophoretic mobility measurements did not provide a reliable measure of the colloidal stability of O-MWCNTs. In addition to the oxygen concentration, the distribution of oxygen-containing functional groups also influences the colloidal stability of O-MWCNTs, with carboxylic acid groups playing the dominant role. The overarching conclusion of this study is that changes in a nanoparticle’s surface composition lead to predictable changes in phenomenological properties that have a direct bearing on the health and safety effects of nanomaterials, such as particle stability. Furthermore, in a broader context this study suggests that structure-function relationships can be established to help understand and predict the environmental health and safety risks posed by surface modified nanomaterials.
The increasing epidemiological and experimental studies indicate that the cardiovascular diseases induced by nanoparticle exposure have been hypothesized to be the crucial factors in nanotoxicity relative fields. The post-exposure transportation pathway and the subsequent reacts on endothelial system were considered to be one of the most essential mechanisms for nanoparticle induced cardiovascular disorders. Iron oxide nanoparticle-ferric oxide (Fe2O3) and ferroso-ferric oxide (Fe3O4), as the potential widely application materials are required to evaluate their transportation and the post-exposure potential toxicities. In the study, the respiratory exposure of 22nm-sized, radioactive 59Fe2O3 particles to male Sprague Dawley (SD) rats was used to monitor the transportation and the coagulation system responses after exposure. The direct effects of Fe2O3 and Fe3O4 nanoparticles on human aortic endothelial cells (HAECs) and the indirect effects of them which mediated by monocytes (U937) phagocytosis were investigated to approach the potential risks of the particles on endothelial system. Our results showed that the intratracheally instilled
22nm-sized 59Fe2O3 passed through the alveolar-capillary
barrier into systemic circulation within 10 min, which was consistent
with the one-compartment model kinetics. The elimination half-life in
blood was calculated as 22.8 day, indicating the systemic accumulation
had occurred and thus probably enhanced the interaction between nanoparticle
and endothelial system. The prolonged coagulation parameters of PT and
APTT presented somewhat degree of coagulatory system disorders. The in
vitro study demonstrated that Fe2O3 and Fe3O4
nanoparticles could translocate into human aortic endothelial cells (HAECs)
and localize in vesicles. Both two types iron oxide nanoparticles could
induce inflammatory responses of ICAM-1 expression and IL-8 secretion
in cells. The adhesion of human monocyte (U937) to HAECs increased as
well, which might result in downstream inflammation responses and cardiovascular
toxicities. The disturbance of NO synthesis and secretion and the decreased
viability in HAECs were also observed. Our results demonstrate that the respiratory exposed iron oxide nanoparticle can transfer across the alveolar-capillary barrier at an extremely fast speed into systemic circulation. The metabolism of nano-Fe2O3 in blood was consistent with one-compartment model kinetics as typically small molecules. The extrapulmonary transferred iron oxide nanoparticles in vessels may induce human aortic endothelial cells cytotoxicities and coagulation system disorders. Iron oxide nanopoarticles could provoke inflammation responses and dysfunction on human aortic endothelial cells and decreased the cell viability. Moreover, they also prolonged the coagulatory process as a sub-acute response of post-exposure. MultiCriteria Mapping of Stakeholder
Preferences in Regulating Nanotechnology A number of recent publications on governance of nanomaterials have pointed to stakeholder deliberation as a key element for nanotechnology to reach its full potential and to secure democratic and transparent decision-making process. In order to promote and/or facilitate such discussions on whether or not and how to regulate nanotechnology, we used the open-source program termed MultiCriteria Mapping (MCM) to structure and facilitate 26 interviews with various stakeholders in the U.S including academics, NGOs, public interest groups, regulators and global, medium and small nanomaterial producing companies. MCM offers a systematic part quantitative, part qualitative approach to clarify why some regulatory options were deemed to be acceptable/unacceptable by various stakeholders and which criteria are used to evaluate the different regulatory options. In order to set the stage equal for all the interviews a number of policy options on how to regulate/not regulate nanotechnology had been pre-defined ranging from a ban for nanotechnology and materials to relying on voluntary measures and no additional regulation over moratorium on R&D and/or commercialization After considering the policy options interviewees where asked to list their “criteria” for assessing the pros and cons of the different policy options after which they performed a quantitative evaluation of the relative performance of the options under each of the criteria. Finally, interviewees were asked to assign values concerning the relative importance of the different criteria and a final ranking of the various policy options was generated and discussed. Of the predefined options adopting an incremental approach and implementing
a new regulatory framework scored the highest whereas a complete ban
and no additional regulation of nanotechnology scored the lowest. Criteria
applied by various stakeholders differ substantially and included environmental,
health, safety, social, ethics, and regulatory issues. Reading across
the ranking of each of the individual stakeholders, opinions of how to
move forward in regard to regulation of nanotechnology seems to be far
less polarized than expected. Colloidal behavior of the metal oxide nanoparticles (NP) needs to be studied with great care because of its increasing production and possible exposure to natural aquatic ecosystems. The colloidal behavior of structurally different humic acids (HA) coated Al2O3 NP was examined. Early stage aggregation kinetics experiments were conducted with dynamic light scattering (DLS) measurements. Al2O3 NP was coated with three structurally different HAs extracted sequentially from Amherst peat soil. The 1st, 3rd, and 7th fractions of HAs (HA1, HA3, and HA7) were used for the coating. Molecular size and hydrophobicity of HA fractions increased with increasing number of extraction. The structural characteristics of the adsorbed HAs layer on the Al2O3 NP surface govern their colloidal behavior in aqueous solution. The rate of colloidal aggregation was much higher in HA1, and HA3 coated Al2O3 NP, even at very low Ca2+ concentration at pH 5. However, HA7 coated nanoparticles did not show faster aggregation rate even at very high Ca2+ concentrations. In case of HA1, and HA3 coated nanoparticles cation bridging and collapsing of double layer in the presence of Ca2+ may facilitated the colloidal aggregation. On the contrary, weaker ionization and steric stabilization may hinder the rate of aggregation for HA7 coated Al2O3 NP. Therefore, physico-chemical nature of the HAs adsorbed on the Al2O3 NP surface significantly influenced their colloidal behavior. Synthesis, Characterization and Transport in Porous Media of Surface-Modified Nanoiron Particles Nano scale zero-valent iron particles (nZVI) have been shown to be very efficient in the transformation of chlorinated hydrocarbons, chromium and nitrate to innocuous end products. Therefore, nZVI are considered to be an excellent reactive agent for in situ remediation of contaminated sites if they can be injected into the subsurface and transported with the groundwater. However, the use of nZVIs for in situ remediation of contaminated sites has been hindered by their poor transport in groundwater due to their tendency to be retained by porous media during transport due to their tendency to deposit on soil grains and to agglomerate. Surface modifications of nZVI that minimize this aggregation behaviour are likely to make nZVI effective for in situ remediation. Reaction conditions were altered to manipulate the size ( 5.5 nm – 75 nm) and structure of CMC-nZVI. Different types of surface modifiers such as carboximethyl cellulose (CMC), poly styrene sulfonate (PSS), sodium butanesulfonae (BS), valeric acid (VA) are used to enhance colloidal stability of nZVI . Transmission electron microscopy (TEM) image analysis showed that the average particle sizes of bare and surface modified particle ranges between 55nm to 130nm. Sedimentation tests show that the CMC modified nZVI has the best colloidal stability. Transport experiment of CMC-nZVI through a sand packed column was studied to evaluate the effect of surface property after modification by carboxymethyl cellulose (CMC). Monitoring of the nanoiron concentrations in the column effluent shows that bare nZVI did not transport through the sand medium. In contrast, the 5.5nm CMC-nZVI transported very well without much deposition. The 75nm CMC-nZVI transports significantly as well but breakthrough curves from column experiments indicates a higher extent of particle aggregation and deposition. The effects of nZVI particle concentration, ionic strength and flow rate of the flushing solution on transport will be presented. Altered Gene Expression Profiles in Murine Brains
Following Exposure to Inhaled Nickel The respiratory tract is the primary target for inhaled nanoparticles (NPs). However, there is evidence that once deposition occurs, these particles can escape clearance mechanisms and target secondary organs like the brain. The aim of this study was to examine the transcriptional response of three regions (olfactory bulb, midbrain, and cerebellum) in the mouse brain following exposures to inhaled nickel nanoparticles (Ni NPs). Utilizing a whole-body exposure system, C57 male mice inhaled either 100 µg/m3 of spark generated Ni NPs or filtered air for 5h/d, 5d/w, for up to 5m. The three regions were collected 1w, 3m, and 5m, post exposure (24h) for gene expression analyses (n=4/group). Since studies suggest that NPs damage tissue through oxidative stress and inflammatory mediated pathways, PCR profiling systems for both pathways were used to evaluate the change in expression of 168 genes in each region as compared to controls. A change of 3-fold or higher was considered important. After 1w of exposure, the expression of approximately 50 oxidative stress and inflammatory related genes were found to be different from the controls for all regions. Following 3 and 5m of exposure, more changes in gene expression were observed in the olfactory bulb (85 and 72, respectively) followed by the cerebellum (35 and 45, respectively) and the midbrain (21 and 28, respectively). To confirm these results, individual real time RT-PCR was performed on selected genes: Mcp-1, Tnfα, IL-1β, Ho-1, Gpx-1, and GFAP. Brain deposition of inhaled Ni NPs was determined for all regions, using graphite furnace atomic absorption spectroscopy. A significant difference was only observed for the olfactory bulb (n=8, P<0.05). These results suggest that all three regions are affected by exposures of inhaled Ni NPs via oxidative stress- inflammatory mediated pathways; but, the olfactory bulb may be a more sensitive target to adverse effects. Additional studies are underway to investigate major routes of translocation utilizing intravenous injections and intranasal instillations. These studies should contribute to understanding the changes in genes expression and should provide a link toward delineating mechanisms of toxicity.
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