Effect of nitrogen oxides and sulfur oxides to triphenyl phosphate degradation and cytotoxicity on surface of different transition metal salts.

Nitrogen oxides and sulfur oxides, as the dominant toxic gases in the atmosphere, can induce severe human health problems under the composite pollutant conditions. Currently the effect of nitrogen or sulfur oxides in atmospheric environment to the degradation and cytotoxicity of triphenyl phosphate (TPhP) on atmospheric particle surfaces still remain poorly understood. Hence, laboratory simulation methods were used in this study to investigate the effect and related mechanism. First, particle samples were prepared with the TPhP coated on MnSO4, CuSO4, FeSO4 and Fe2(SO4)3 surface. The results showed that, when nitrogen or sulfur oxides were present, more significant TPhP degradation on all samples can be observed under both light and dark conditions. The results proved nitrogen oxides and sulfur oxides were the vital influence factors to the degradation of TPhP, which mainly promoted the OH generation in the polluted atmosphere. The mechanism study indicated that diphenyl hydrogen phosphate (DPhP) and OH-DPhP were two main stable degradation products. These degradation products originated from the phenoxy bond cleavage and hydroxylation of TPhP caused by hydroxyl radicals. In addition, no TPhP related organosulfates (OSs) or organic nitrates (ON) formation were observed. Regarding the cytotoxicity, all the particles can induce more significant cellular injury and apoptosis of A549 cells, which may be relevant to the adsorbed nitrogen oxides or sulfur oxides on particles surfaces. The superfluous reactive oxygen species (ROS) generation was the possible reason of cytotoxicity. This research can supply a comprehensive understanding of the promoting effect of nitrogen and sulfur oxides to TPhP degradation and the composite cytotoxicity of atmospheric particles.

The 10th anniversary of MXenes: Challenges and prospects for their surface modification toward future biotechnological applications.

A broad family of two-dimensional (2D) materials - carbides, nitrides, and carbonitrides of early transition metals, called MXenes, became a newcomer in the flatland at the turn of 2010 and 2011 (over ten years ago). Their unique physicochemical properties made them attractive for many applications, highly boosting the development of various fields, including biotechnological. However, MXenes' functional features that impact their bioactivity and toxicity are still not fully well understood. This study discusses the essentials for MXenes's surface modifications toward their application in modern biotechnology and nanomedicine. We survey modification strategies in context of cytotoxicity, biocompatibility, and most prospective applications ready to implement in medical practice. We put the discussion on the material-structure-chemistry-property relationship into perspective and concentrate on overarching challenges regarding incorporating MXenes into nanostructured organic/inorganic bioactive architectures. It is another emerging group of materials that are interesting from the biomedical point of view as well. Finally, we present an influential outlook on the growing demand for future research in this field.

Ceftriaxone-based Schiff base transition metal(II) complexes. Synthesis, characterization, bacterial toxicity, and DFT calculations. Enhanced antibacterial activity of a novel Zn(II) complex against S. aureus and E. coli.

From the reaction of ceftriaxone 1 antibiotic with 2,6-diaminopyridine 2 a ceftriaxone-based Schiff base (H2L,3) was obtained and its transition metal complexes were synthesized. Spectroscopic and physicochemical techniques, namely, UV-visible, FT-IR, 1H NMR, EPR, mass spectrometry, molar conductance, magnetic susceptibility and density functional theory (DFT) calculations, together with elemental and thermal analyses, were used to find out the binding mode and composition of these complexes. The ceftriaxone-based Schiff base 3 behaves as a monoanionic tridentate N,N,O ligand. Spectral and magnetic data suggest an octahedral geometry for all complexes and the general formulae [M(HL)(OAc)(H2O)2] (M(II) = Mn2+4, Co2+5, Ni2+6, Cu2+7, Zn2+8), are proposed for them. All compounds were screened for antibacterial activity using both the agar disc diffusion method and the minimal inhibitory concentration (MIC). It was found that complex 8 exhibited the most promising bactericidal activity against S. aureus (MIC = 0.0048 µmol/ml) and E. coli (MIC = 0.0024 µmol/ml). It is more active than the free ligand 1 (MIC = 0.0560 µmol/ml for S. aureus and 0.0140 µmol/ml for E. coli). These MIC results were compared with those obtained using similar zinc(II) Schiff base complexes, and with the values obtained using ceftriaxone conjugated with silver and gold nanoparticles (NPs), using earlier published data. Synthesized metal complexes exhibited LC50 values >1000 ppm indicating their nontoxicity against brine shrimp nauplii (Artemia Salina).

A simple, low-cost and green method for controlling the cytotoxicity of MXenes.

MXene phases are a member of the intriguing 2D material family, beyond graphene. They are good candidates for many applications, however, their potential toxicity is of crucial importance for future development. Herein, we present a simple, low-cost and fully green approach for controlling the potential cytotoxicity of 2D MXenes after delamination by harnessing the interactions that occur between the surface of MXene phases and natural biomacromolecule - collagen. We also demonstrate that the step-by-step adsorption and desorption of collagen from the surface of 2D MXenes is easily controlled using in situ zeta potential measurements coupled with dynamic light scattering (DLS) method. The obtained results demonstrated that the electrostatically driven unprecedented susceptibility of the MXenes' surfaces to collagen. Surface-modification reduces toxicity of MXenes in vitro i.e. adjust cells' viabilities as well as reduce their oxidative stress. This indicates enhanced biocompatibility of 2D Ti3C2 and Ti2C MXenes surface-modified with collagen, which is involved in many bio-interactions as important building blocks in the human body. The presented study opens new avenues for designing MXenes with defined surface properties and paves the way for their future successful management in nano-medicinal applications.

Recent advances in two-dimensional transition metal dichalcogenides for biological sensing.

Layered transition metal dichalcogenides (TMDs) are important members in the family of two-dimensional (2D) materials. The large surface-to-volume ratio, combined with the fascinating tunable electronic and optical properties, low toxicity, unique van der Waals layered structure, and engineerable surface structure, renders 2D TMDs highly valuable for next-generation biosensing applications. Herein, the recent progress in the development of 2D TMDs-based biosensors is comprehensively reviewed, with special focus on the implementation of the structural, electronic and optical properties of 2D TMDs in the realization of high-performance biosensors with different configurations for a wide spectrum of bioanalytes and bio-species. In addition, the comparison on biosensing performances with graphene as the currently most studied 2D candidate is critically discussed. Finally, future perspectives are provided along the development progress of 2D TMDs-based biosensors which are currently undergoing an intense study. This work will lead researchers to explore more novel sensing candidates within the category of TMDs with exotic chemical composition, structure, morphologies, dimensionalities, and properties.

Summer-winter differences of PM2.5 toxicity to human alveolar epithelial cells (A549) and the roles of transition metals.

Atmospheric fine particulate matters (PM2.5) induce adverse human health effects through inhalation, and the harmful effects of PM2.5 are determined not only by its air concentrations, but also by the particle components varied temporally. To investigate seasonal differences of the aerosol toxicity effects including cell viability and membrane damage, cell oxidative stress and responses of inflammatory cytokines, the human lung epithelial cells (A549) were exposed to PM2.5 samples collected in both summer and winter by the in vitro toxicity bioassays. Toxicological results showed that, the PM2.5 led to the cell viability decrease, cell membrane injury, oxidative stress level increase and inflammatory responses in a dose-dependent manner. Temporally, the cytotoxicity of winter PM2.5 was higher than summer of this studied industrial area of Nanjing, China. According to the different contents of heavy metals accumulated in PM2.5, the transition metals such as Cu might be an important contributor to the aerosol cell toxicity.

Cytotoxicity of Group 5 Transition Metal Ditellurides (MTe2 ; M=V, Nb, Ta).

Much research effort has been put in to study layered compounds with transition metal dichalcogenides (TMDs) being one of the most studied compounds. Due to their extraordinary properties such as excellent electrochemical properties, tuneable band gaps, and low shear resistance due to weak van der Waals interactions between layers, TMDs have been found to have wide applications such as electrocatalysts for hydrogen evolution reactions, supercapacitors, biosensors, field-effect transistors (FETs), photovoltaics, and lubricant additives. In very recent years, Group 5 transition metal ditellurides have received an immense amount of research attention. However to date, little has been known of the potential toxicities posed by these materials. As such, we conducted the cytotoxicity study by incubating various concentrations of the Group 5 transition metal ditellurides (MTe2 ; M=V, Nb, Ta) with human lung carcinoma epithelial A549 cells for 24 hours and the remaining cell viabilities after treatment was measured. Our findings indicate that VTe2 is highly toxic whereas NbTe2 and TaTe2 are deemed to exhibit mild toxicities. This study constitutes an exemplary first step towards the understanding of the Group 5 transition metal ditellurides' toxicity effects in preparation for their possible future commercialisation.

Synergistic Antimicrobial Effects of Silver/Transition-metal Combinatorial Treatments.

Due to the emergence of multi-drug resistant strains, development of novel antibiotics has become a critical issue. One promising approach is the use of transition metals, since they exhibit rapid and significant toxicity, at low concentrations, in prokaryotic cells. Nevertheless, one main drawback of transition metals is their toxicity in eukaryotic cells. Here, we show that the barriers to use them as therapeutic agents could be mitigated by combining them with silver. We demonstrate that synergism of combinatorial treatments (Silver/transition metals, including Zn, Co, Cd, Ni, and Cu) increases up to 8-fold their antimicrobial effect, when compared to their individual effects, against E. coli and B. subtilis. We find that most combinatorial treatments exhibit synergistic antimicrobial effects at low/non-toxic concentrations to human keratinocyte cells, blast and melanoma rat cell lines. Moreover, we show that silver/(Cu, Ni, and Zn) increase prokaryotic cell permeability at sub-inhibitory concentrations, demonstrating this to be a possible mechanism of the synergistic behavior. Together, these results suggest that these combinatorial treatments will play an important role in the future development of antimicrobial agents and treatments against infections. In specific, the cytotoxicity experiments show that the combinations have great potential in the treatment of topical infections.

Transition and heavy metals compared to oxidative parameter balance in patients with deep vein thrombosis: A case-control study.

Our study compared copper (Cu), zinc (Zn) and manganese (Mn), crucial for human normal physio-logy maintenance, and lead (Pb) levels as environmental pollutant, in subjects suffering of deep vein thrombosis of lower limbs (DVTs) vs. healthy subjects. Furthermore, we evaluated oxidative stress parameters, the thiobarbituric acid reactive substances (TBARS) as the sum of malondialdehyde or 1,1,3,3-tetraethoxypropane (MDA) and 4-hydroxynonenal 4-HNE) and cytosolic superoxide dismutase (Cu/Zn-SOD) concentrations in both groups. We recruited 24 DVT cases and 46 healthy subjects as controls. Questionnaire with socio­demographic, habits and lifestyle were collected. Hair concentrations of Zn, Mn, Cu and Pb were measured by ICP-MS, plasma concentrations of MDA and 4-HNE were measured by HPLC and SOD plasma concentrations were detected by ELISA test. A quantitative and qualitative variables comparison between cases and controls group was made by Mann-Whitney U test and Pearson's Chi-square test, respectively. We found low concentrations of Zn, Mn and Cu vs. high Pb concentrations in DVTs subjects. TBARS were found higher in the cases group, conversely, SOD concentrations were found lower in cases with respect to controls. Furthermore, we found the diet of pathological subjects significantly deficient in vegetables. These results are indicative of a lower enzymatic activity in patients, related to low transition metal levels in the DVTs and high levels of Pb, coupled with an unbalanced diet.

Role of transition metals present in air particulate matter on lung oxygen metabolism.

Several epidemiological studies have shown a positive correlation between daily increases in airborne particulate matter (PM) concentration and the occurrence of respiratory and cardiovascular diseases. Transition metals present in air PM were associated with adverse health effects after PM exposure. The aim of this work was to study lung O2 metabolism after an acute exposure to transition metal-coated nanoparticles (NPs). Female Swiss mice (25g) were intranasally instilled with a suspension of silica NP containing Ni (II), Cd (II), Fe (III), or Cr (VI) at 0, 0.01, 0.05, 0.1, and 1.0mg metal/kg body weight. Lung O2 consumption was found to be significantly increased after the exposure to most doses of Ni-NP and Fe-NP, and the 0.05mg metal/kg body weight dose of Cr-NP, while no changes were observed for Cd-NP. Lucigenin chemiluminescence (as an indicator of NADPH oxidase (NOX) activity) was evaluated in lung homogenates. Only Ni-NP and Fe-NP have shown the ability to induce a significant increase in lucigenin chemiluminescence. In order to establish the possible occurrence of pulmonary oxidative stress, TBARS levels and the GSH/GSSG ratio were determined. The higher doses of Ni-NP and Fe-NP were able to induce an oxidative stress condition, as shown by changes in both TBARS levels and the GSH/GSSG ratio. Taken together, the present results show differential effects for all the metals tested. These findings emphasize the importance of transition metals present air PM in PM adverse health effects, and contribute to the understanding of the pathological mechanisms triggered by the exposure to environmental PM.

ROS-generating/ARE-activating capacity of metals in roadway particulate matter deposited in urban environment.

In this study we investigated the possible causal role for soluble metal species extracted from roadway traffic emissions in promoting particulate matter (PM)-induced reactive oxygen species (ROS) production and antioxidant response element (ARE) promoter activation. To this end, these responses have been evaluated in alveolar macrophage and epithelial lung cells that have been exposed to 'Unfiltered', 'Filtered' and 'Filtered+Chelexed' water extracts of PM samples collected from the roadway urban environments of Thessaloniki, Milan and London. Except for Thessaloniki, our results demonstrate that filtration resulted in a minor decrease in ROS activity of the fine PM fraction, suggesting that ROS activity is attributed mainly to water-soluble PM species. In contrast to ROS, ARE activity was mediated predominantly by the water-soluble component of PM present in both the fine and coarse extracts. Further removal of metals by Chelex treatment from filtered water extracts showed that soluble metal species are the major factors mediating ROS and ARE activities of the soluble fraction, especially in the London PM extracts. Finally, utilizing step-wise multiple-regression analysis, we show that 87% and 78% of the total variance observed in ROS and ARE assays, respectively, is accounted for by changes in soluble metal concentration. Using a statistical analysis we find that As, Zn and Fe best predict the ROS-generating/ARE-activating capacity of the near roadway particulate matter in the pulmonary cells studied. Collectively, our findings imply that soluble metals present in roadside PM are potential drivers of both pro- and anti-oxidative effects of PM in respiratory tract.

Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity.

The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells. Prnp-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

Selenite reduction by the obligate aerobic bacterium Comamonas testosteroni S44 isolated from a metal-contaminated soil.

BACKGROUND: Selenium (Se) is an essential trace element in most organisms but has to be carefully handled since there is a thin line between beneficial and toxic concentrations. Many bacteria have the ability to reduce selenite (Se(IV)) and (or) selenate (Se(VI)) to red elemental selenium that is less toxic. RESULTS: A strictly aerobic bacterium, Comamonas testosteroni S44, previously isolated from metal(loid)-contaminated soil in southern China, reduced Se(IV) to red selenium nanoparticles (SeNPs) with sizes ranging from 100 to 200 nm. Both energy dispersive X-ray Spectroscopy (EDX or EDS) and EDS Elemental Mapping showed no element Se and SeNPs were produced inside cells whereas Se(IV) was reduced to red-colored selenium in the cytoplasmic fraction in presence of NADPH. Tungstate inhibited Se(VI) but not Se(IV) reduction, indicating the Se(IV)-reducing determinant does not contain molybdenum as co-factor. Strain S44 was resistant to multiple heavy and transition metal(loid)s such as Se(IV), As(III), Cu(II), and Cd(II) with minimal inhibitory concentrations (MIC) of 100 mM, 20 mM, 4 mM, and 0.5 mM, respectively. Disruption of iscR encoding a transcriptional regulator negatively impacted cellular growth and subsequent resistance to multiple heavy metal(loid)s. CONCLUSIONS: C. testosteroni S44 could be very useful for bioremediation in heavy metal(loid) polluted soils due to the ability to both reduce toxic Se(VI) and Se(IV) to non-toxic Se (0) under aerobic conditions and to tolerate multiple heavy and transition metals. IscR appears to be an activator to regulate genes involved in resistance to heavy or transition metal(loid)s but not for genes responsible for Se(IV) reduction.

Low modulus Ti-Nb-Hf alloy for biomedical applications.

ß-Type titanium alloys with a low elastic modulus are a potential strategy to reduce stress shielding effect and to enhance bone remodeling in implants used to substitute failed hard tissue. For biomaterial application, investigation on the mechanical behavior, the corrosion resistance and the cell response is required. The new Ti25Nb16Hf alloy was studied before and after 95% cold rolling (95% C.R.). The mechanical properties were determined by tensile testing and its corrosion behavior was analyzed by potentiostatic equipment in Hank's solution at 37°C. The cell response was studied by means of cytotoxicity evaluation, cell adhesion and proliferation measurements. The stress-strain curves showed the lowest elastic modulus (42GPa) in the cold worked alloy and high tensile strength, similar to that of Ti6Al4V. The new alloy exhibited better corrosion resistance in terms of open circuit potential (EOCP), but was similar in terms of corrosion current density (iCORR) compared to Ti grade II. Cytotoxicity studies revealed that the chemical composition of the alloy does not induce cytotoxic activity. Cell studies in the new alloy showed a lower adhesion and a higher proliferation compared to Ti grade II presenting, therefore, mechanical features similar to those of human cortical bone and, simultaneously, a good cell response.

The ColRS signal transduction system responds to the excess of external zinc, iron, manganese, and cadmium.

BACKGROUND: The ColRS two-component system has been shown to contribute to the membrane functionality and stress tolerance of Pseudomonas putida as well as to the virulence of Pseudomonas aeruginosa and plant pathogenic Xanthomonas species. However, the conditions activating the ColRS pathway and the signal(s) sensed by ColS have remained unknown. Here we aimed to analyze the role of the ColRS system in metal tolerance of P. putida and to test whether ColS can respond to metal excess. RESULTS: We show that the ColRS system is necessary for P. putida to tolerate the excess of iron and zinc, and that it also contributes to manganese and cadmium tolerance. Excess of iron, zinc, manganese or cadmium activates ColRS signaling and as a result modifies the expression of ColR-regulated genes. Our data suggest that the genes in the ColR regulon are functionally redundant, as several loci have to be deleted to observe a significant decrease in metal tolerance. Site-directed mutagenesis of ColS revealed that excess of iron and, surprisingly, also zinc are sensed by a conserved ExxE motif in ColS's periplasmic domain. While ColS is able to sense different metals, it still discriminates between the two oxidation states of iron, specifically responding to ferric and not ferrous iron. We propose a signal perception model involving a dimeric ColS, where each monomer donates one ExxE motif for metal binding. CONCLUSIONS: Several transition metals are essential for living organisms in certain amounts, but toxic in excess. We show that ColRS is a sensor system which detects and responds to the excess of physiologically important metals such as zinc, iron and manganese. Thus, the ColRS system is an important factor for metal homeostasis and tolerance in P. putida.

Metallobiology of Tuberculosis.

Transition metals are essential constituents of all living organisms, playing crucial structural and catalytic parts in many enzymes and transcription factors. However, transition metals can also be toxic when present in excess. Their uptake and efflux rates must therefore be carefully controlled by biological systems. In this chapter, we summarize the current knowledge about uptake and efflux systems in Mycobacterium tuberculosis for mainly three of these metals, namely iron, zinc, and copper. We also propose questions for future research in the field of metallobiology of host-pathogen interactions in tuberculosis.

Nutritional immunity: transition metals at the pathogen-host interface.

Transition metals occupy an essential niche in biological systems. Their electrostatic properties stabilize substrates or reaction intermediates in the active sites of enzymes, and their heightened reactivity is harnessed for catalysis. However, this heightened activity also renders transition metals toxic at high concentrations. Bacteria, like all living organisms, must regulate their intracellular levels of these elements to satisfy their physiological needs while avoiding harm. It is therefore not surprising that the host capitalizes on both the essentiality and toxicity of transition metals to defend against bacterial invaders. This Review discusses established and emerging paradigms in nutrient metal homeostasis at the pathogen-host interface.

Chemical properties of air pollutants and cause-specific hospital admissions among the elderly in Atlanta, Georgia.

BACKGROUND: Health risks differ by fine particle (aerodynamic diameter ≤ 2.5 µm) component, although with substantial variability. Traditional methods to assess component-specific risks are limited, suggesting the need for alternative methods. OBJECTIVES: We examined whether the odds of daily hospital admissions differ by pollutant chemical properties. METHODS: We categorized pollutants by chemical properties and examined their impacts on the odds of daily hospital admissions among Medicare recipients > 64 years of age in counties in Atlanta, Georgia, for 1998-2006. We analyzed data in two stages. In the first stage we applied a case-crossover analysis to simultaneously estimate effects of 65 pollutants measured in the Aerosol Research and Inhalation Epidemiology Study on cause-specific hospital admissions, controlling for temperature and ozone. In the second stage, we regressed pollutant-specific slopes from the first stage on pollutant properties. We calculated uncertainty estimates using a bootstrap procedure. We repeated the two-stage analyses using coefficients from first-stage models that included single pollutants plus ozone and meteorological variables only. We based our primary analyses on exposures on day of admission. RESULTS: We found that 24-hr transition metals and alkanes were associated with increased odds [0.26%; 95% confidence interval (CI), 0.02-0.48; and 0.37%; 95% CI, 0.04-0.72, respectively] of hospital admissions for cardiovascular disease (CVD). Transition metals were significantly associated with increased hospital admissions for ischemic heart disease, congestive heart failure, and atrial fibrillation. Increased respiratory-related hospital admissions were significantly associated with alkanes. Aromatics and microcrystalline oxides were significantly associated with decreased CVD- and respiratory-related hospital admissions. CONCLUSIONS: The two-stage approach showed transition metals to be consistently associated with increased odds of CVD-related hospital admissions.

ST depression, arrhythmia, vagal dominance, and reduced cardiac micro-RNA in particulate-exposed rats.

Recently, investigators demonstrated associations between fine particulate matter (PM)-associated metals and adverse health effects. Residual oil fly ash (ROFA), a waste product of fossil fuel combustion from boilers, is rich in the transition metals Fe, Ni, and V, and when released as a fugitive particle, is an important contributor to ambient fine particulate air pollution. We hypothesized that a single-inhalation exposure to transition metal-rich PM will cause concentration-dependent cardiovascular toxicity in spontaneously hypertensive (SH) rats. Rats implanted with telemeters to monitor heart rate and electrocardiogram were exposed once by nose-only inhalation for 4 hours to 3.5 mg/m(3), 1.0 mg/m(3), or 0.45 mg/m(3) of a synthetic PM (dried salt solution), similar in composition to a well-studied ROFA sample consisting of Fe, Ni, and V. Exposure to the highest concentration of PM decreased T-wave amplitude and area, caused ST depression, reduced heart rate (HR), and increased nonconducted P-wave arrhythmias. These changes were accompanied by increased pulmonary inflammation, lung resistance, and vagal tone, as indicated by changes in markers of HR variability (increased root of the mean of squared differences of adjacent RR intervals [RMSSD], low frequency [LF], high frequency [HF], and decreased LF/HF), and attenuated myocardial micro-RNA (RNA segments that suppress translation by targeting messenger RNA) expression. The low and intermediate concentrations of PM had less effect on the inflammatory, HR variability, and micro-RNA endpoints, but still caused significant reductions in HR. In addition, the intermediate concentration caused ST depression and increased QRS area, whereas the low concentration increased the T-wave parameters. Thus, PM-induced cardiac dysfunction is mediated by multiple mechanisms that may be dependent on PM concentration and myocardial vulnerability (this abstract does not reflect the policy of the United States Environmental Protection Agency).