Addressing Emerging Risks: Scientific and Regulatory Challenges Associated with Environmentally Persistent Free Radicals.

Airborne fine and ultrafine particulate matter (PM) are often generated through widely-used thermal processes such as the combustion of fuels or the thermal decomposition of waste. Residents near Superfund sites are exposed to PM through the inhalation of windblown dust, ingestion of soil and sediments, and inhalation of emissions from the on-site thermal treatment of contaminated soils. Epidemiological evidence supports a link between exposure to airborne PM and an increased risk of cardiovascular and pulmonary diseases. It is well-known that during combustion processes, incomplete combustion can lead to the production of organic pollutants that can adsorb to the surface of PM. Recent studies have demonstrated that their interaction with metal centers can lead to the generation of a surface stabilized metal-radical complex capable of redox cycling to produce ROS. Moreover, these free radicals can persist in the environment, hence their designation as Environmentally Persistent Free Radicals (EPFR). EPFR has been demonstrated in both ambient air PM2.5 (diameter < 2.5 µm) and in PM from a variety of combustion sources. Thus, low-temperature, thermal treatment of soils can potentially increase the concentration of EPFR in areas in and around Superfund sites. In this review, we will outline the evidence to date supporting EPFR formation and its environmental significance. Furthermore, we will address the lack of methodologies for specifically addressing its risk assessment and challenges associated with regulating this new, emerging contaminant.

Formation of environmentally persistent free radical (EPFR) in iron(III) cation-exchanged smectite clay.

Environmentally persistent free radicals (EPFRs) have been found at a number of Superfund sites, with EPFRs being formed via a proposed redox process at ambient environmental conditions. The possibility of such a redox process taking place at ambient environmental conditions is studied utilizing a surrogate soil system of phenol and iron(III)-exchanged calcium montmorillonite clay, Fe(III)CaM. Sorption of phenol by the Fe(III)CaM is demonstrated by Fourier-transformed infra-red (FT-IR) spectroscopy, as evidenced by the peaks between 1345 cm(-1) and 1595 cm(-1), and at lower frequencies between 694 cm(-1) and 806 cm(-1), as well as X-ray diffraction (XRD) spectroscopy, as shown by an increase in interlayer spacing within Fe(III)CaM. The formation and characterization of the EPFRs is determined by electron paramagnetic resonance (EPR) spectroscopy, showing phenoxyl-type radical with a g-factor of 2.0034 and ΔHP-P of 6.1 G at an average concentration of 7.5 × 10(17) spins per g. EPFRs lifetime data are indicative of oxygen and water molecules being responsible for EPFR decay. The change in the oxidation state of the iron redox center is studied by X-ray absorption near-edge structure (XANES) spectroscopy, showing that 23% of the Fe(III) is reduced to Fe(II). X-ray photoemission spectroscopy (XPS) results confirm the XANES results. These findings, when combined with the EPFR concentration data, demonstrate that the stoichiometry of the EPFR formation under the conditions of this study is 1.5 × 10(-2) spins per Fe(II) atom.

Contribution of aluminas and aluminosilicates to the formation of PCDD/Fs on fly ashes.

Chlorinated aromatics undergo surface-mediated reactions with metal oxides to form Environmentally Persistent Free Radicals (EPFRs) which can further react to produce polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). Previous work using laboratory-made fly ash surrogates composed of transition metal oxides deposited on silica powder has confirmed their ability to mimic fly ash in the production of PCDD/Fs. However, little is known about the propensity of aluminas and aluminosilicates, other components of fly ash, to form PCDD/Fs. A fly ash sample containing both alumina and mullite, an aluminosilicate, was tested for PCDD/F formation ability and compared to PCDD/F yields from the thermal degradation of 2-monochlorophenol (2-MCP) precursor over γ-alumina, α-alumina, and mullite. A packed-bed flow reactor was used to investigate the thermal degradation of 2-MCP over the various catalysts at 200-600 °C. Fly ash gave similar PCDD/F yields to surrogates made with similar transition metal content. γ-alumina, which is thermodynamically unfavorable, was very catalytically active and gave low PCDD/F yields despite a high destruction of 2-MCP. Mullite and α-alumina, the thermodynamically favorable form of alumina, yielded higher concentrations of dioxins and products with a higher degree of chlorine substitution than γ-alumina. The data suggest that certain aluminas and aluminosilicates, commonly found in fly ash, are active catalytic surfaces in the formation of PCDD/Fs in the post-flame cool zones of combustion systems and should be considered as additional catalytic surfaces active in the process.

Model System Study of Environmentally Persistent Free Radicals Formation in a Semiconducting Polymer Modified Copper Clay System at Ambient Temperature.

This paper systematically investigates how environmentally persistent free radicals (EPFRs) are formed in a phenol contaminated model soil. Poly-p-phenylene (PPP) modified and copper-loaded montmorillonite (MMT) clays were developed and used as models of soil organic matter and the clay mineral component, respectively, with phenol being employed as a precursor pollutant. The polymer modification of the clays was carried out via surface-confined Kumada catalyst-transfer chain-growth polymerization. The presence and location of the polymer were confirmed by a combination of thermogravimetric analysis (TGA), Raman spectroscopy, and X-ray diffraction data. EPFRs were formed by the Cu(II)-clay (Cu(II)CaMMT) and poly-p-phenylene-Cu(II)clay (PPP-Cu(II)CaMMT) composite systems under environmentally relevant conditions. The g-factor and concentration of EPFRs formed by the Cu(II)CaMMT and PPP-Cu(II)CaMMT systems were found to be 2.0034 and 1.22 × 1017 spins/g and 2.0033 and 1.58 × 1017spins/g, respectively. These g-factors are consistent with the formation of phenoxyl radicals. Extended X-Ray absorption fine structure (EXAFS) analysis shows that there are distinct differences in the local stuctures of the phenoxyl radicals associated with only the Cu(II) redox centers and those formed in the presences of the PPP polymer. X-ray absorption near edge spectroscopy (XANES) results provided evidence for the reduction of Cu(II) to Cu(I) in the EPFR forming process. The 1/e lifetimes of the formed EPFRs revealed a decay time of ~20 h for the Cu(II)CaMMT system and a two-step decay pattern for the PPP-Cu(II)CaMMT system with decay times of ~13.5 h and ~55.6 h. Finally, the generation of reactive oxygen species (hydroxyl radical; •OH) by these clay systems was also investigated, with higher concentrations of •OH detected for the phenol-dosed Cu(II)CaMMT and PPP-Cu(II)CaMMT systems, compared to the non-EPFR containing undosed PPP-Cu(II)CaMMT system.

Speciation of Iron (III) Oxide Nanoparticles and Other Paramagnetic Intermediates during High-Temperature Oxidative Pyrolysis of 1-Methylnaphthalene.

Low Temperature Matrix Isolation - Electron Paramagnetic Resonance (LTMI-EPR) Spectroscopy was utilized to identify the species of iron oxide nanoparticles generated during the oxidative pyrolysis of 1-methylnaphthalene (1-MN). The otherwise gas-phase reactions of 1--MN were impacted by a polypropylenimine tetra-hexacontaamine dendrimer complexed with iron (III) nitrate nonahydrate diluted in air under atmospheric conditions. The EPR fine structure of Fe (III)2O3 nanoparticles clusters, characterized by g-factors of 2.00, 2.28, 3.76 and 4.37 were detected on a cold finger maintained at 77 K after accumulation over a multitude of experiments. Additionally, a high valence Fe (IV) paramagnetic intermediate and superoxide anion-radicals, O2•- adsorbed on nanoparticle surfaces in the form of Fe (IV) --- O2•- were detected from the quenching area of Zone 1 in the gas-phase.

Phenols from pyrolysis and co-pyrolysis of tobacco biomass components.

Phenol and its derivatives (phenol, o-, m-, p-cresols, catechol, hydroquinone, methoxy substituted phenols, etc. referred to as phenolic compounds or phenols) are well-known toxicants that exist in the environment and affect both human and natural ecosystems. This study explores quantitatively the yields of phenolic compounds from the thermal degradation (pyrolysis and oxidative pyrolysis) of common tobacco biomass components (lignin, tyrosine, ethyl cellulose, sodium alginate, and laminarin) as well as some mixtures (lignin/tyrosine, ethyl cellulose/tyrosine and sodium alginate/tyrosine) considered important in high temperature cooking, tobacco smoking, and forest fires. Special attention has been given to binary mixtures including those containing tyrosine-pyrolysis of binary mixtures of tyrosine with lignin and ethyl cellulose results in significant reductions in the yields of majority phenols relative to those from the thermal degradation of tyrosine. These results imply that the significant reductions of phenol yields in mixtures are not only dependent upon the mass fractions of the components but also the synergetic inhibition effect of biomass components on the thermal degradation of tyrosine. A mechanistic description of this phenomenon is suggested. The results may also be implied in tobacco industry that the cigarette paper (as ethyl cellulose derivative) may play a critical role in reducing the concentration of phenolic compounds released during tobacco burning.

PCDD/PCDF ratio in the precursor formation model over CuO surface.

The discrepancies between polychlorinated dibenzo-p-dioxin to polychlorinated dibenzofuran (PCDD to PCDF) ratios in laboratory and field studies in the exhaust of combustion sources are not fully explained by available formation models. In this paper we present the results of experimental studies of the surface mediated formation of PCDD/F at the conditions mimicking the combustion cool zone from a mixture of 1,2-dichlorobenzene (1,2-DCBz) and 2-monochlorophenol (2-MCP) over a model surface consisting of 5% CuO/Silica. The PCDD to PCDF ratio was found to be strongly dependent on the ratio of chlorinated benzenes to chlorinated phenols and oxygen content. The higher the 1,2-DCBz to 2-MCP ratio, the lower the PCDD to PCDF ratio. PCDFs are formed predominantly from chlorinated benzenes, while chlorinated phenols are responsible for majority of PCDDs. These laboratory results are in general agreement with full-scale measurement and can be used to improve predictive models of PCDD/F formation.

Environmentally persistent free radicals (EPFRs). 3. Free versus bound hydroxyl radicals in EPFR aqueous solutions.

Additional experimental evidence is presented for in vitro generation of hydroxyl radicals because of redox cycling of environmentally persistent free radicals (EPFRs) produced after adsorption of 2-monochlorophenol at 230 °C (2-MCP-230) on copper oxide supported by silica, 5% Cu(II)O/silica (3.9% Cu). A chemical spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), in conjunction with electron paramagnetic resonance (EPR) spectroscopy was employed. Experiments in spiked O(17) water have shown that ∼15% of hydroxyl radicals formed as a result of redox cycling. This amount of hydroxyl radicals arises from an exogenous Fenton reaction and may stay either partially trapped on the surface of particulate matter (physisorbed or chemisorbed) or transferred into solution as free OH. Computational work confirms the highly stable nature of the DMPO-OH adduct, as an intermediate produced by interaction of DMPO with physisorbed/chemisorbed OH (at the interface of solid catalyst/solution). All reaction pathways have been supported by ab initio calculations.

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes.

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 µm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 µm in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition.

Combustion By-Products and their Health Effects--combustion engineering and global health in the 21st century: issues and challenges.

The 13th International Congress on Combustion By-Products and their Health Effects was held in New Orleans, Louisiana from May 15 to 18, 2013. The congress, sponsored by the Superfund Research Program, National Institute of Environmental Health Sciences, and National Science Foundation, brought together international academic and government researchers, engineers, scientists, and policymakers. With industrial growth, increased power needs and generation and coal consumption and their concomitant emissions, pernicious health effects associated with exposures to these emissions are on the rise. This congress provides a unique platform for interdisciplinary exchange and discussion of these topics. The formation, conversion, control, and health effects of combustion by-products, including particulate matter and associated heavy metals, persistent organic pollutants, and environmentally persistent free radicals, were discussed during the congress. This review will summarize and discuss the implications of the data presented.

Effect of copper oxide concentration on the formation and persistency of environmentally persistent free radicals (EPFRs) in particulates.

Environmentally persistent free radicals (EPFRs) are formed by the chemisorption of substituted aromatics on metal oxide surfaces in both combustion sources and superfund sites. The current study reports the dependency of EPFR yields and their persistency on metal loading in particles (0.25, 0.5, 0.75, 1, 2, and 5% CuO/silica). The EPFRs were generated through exposure of particles to three adsorbate vapors at 230 °C: phenol, 2-monochlorophenol (2-MCP), and dichlorobenzene (DCBz). Adsorption resulted in the formation of surface-bound phenoxyl- and semiquinoine-type radicals with characteristic EPR spectra displaying a g value ranging from ∼ 2.0037 to 2.006. The highest EPFR yield was observed for CuO concentrations between 1 and 3% in relation to MCP and phenol adsorption. However, radical density, which is expressed as the number of radicals per copper atom, was highest at 0.75-1% CuO loading. For 1,2-dichlorobenzene adsorption, radical concentration increased linearly with decreasing copper content. At the same time, a qualitative change in the radicals formed was observed--from semiquinone to chlorophenoxyl radicals. The two longest lifetimes, 25 and 23 h, were observed for phenoxyl-type radicals on 0.5% CuO and chlorophenoxyl-type radicals on 0.75% CuO, respectively.

Hydroxyl radical generation from environmentally persistent free radicals (EPFRs) in PM2.5.

Hydroxyl radicals were generated from an aqueous suspension of ambient PM2.5 and detected utilizing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap coupled with electron paramagnetic resonance (EPR) spectroscopy. Results from this study suggested the importance of environmentally persistent free radicals (EPFRs) in PM2.5 to generate significant levels of ·OH without the addition of H2O2. Particles for which the EPFRs were allowed to decay over time induced less hydroxyl radical. Additionally, higher particle concentrations produced more hydroxyl radical. Some samples did not alter hydroxyl radical generation when the solution was purged by air. This is ascribed to internal, rather than external surface associated EPFRs.

Assessment of environmentally persistent free radicals in soils and sediments from three Superfund sites.

We previously reported the presence of environmentally persistent free radicals (EPFRs) in pentachlorophenol (PCP) contaminated soils at a closed wood treatment facility site in Georgia. The reported EPFRs were pentachlorophenoxyl radicals formed on soils under ambient conditions via electron transfer from PCP to electron acceptors in the soil. In this study, we present results for soil and sediment samples from additional Superfund sites in Montana and Washington. Paramagnetic centers associated with different chemical environments were characterized by distinct g-factors and line widths (ΔHp-p). EPFR concentrations in contaminated samples were ~30×, ~12×, and ~2× higher than background samples at the Georgia, Montana, and Washington sites, respectively. EPR signals in the Montana contaminated soils were very similar to those previously observed for pentachlorophenol contaminated soils at the Georgia site, i.e., g = 2.00300 and ΔHp-p = 6.0 G, whereas signals in the Washington sediment samples were similar to those previously observed for other PAH contaminated soils, i.e., g = 2.00270 and ΔHp-p = 9.0 G. Total carbon content measurements exhibited direct correlation with EPFR concentration. The presence of radicals in sites contaminated a decade to a century ago suggests continuous formation of EPFRs from molecular contaminants in the soil and sediment.

Model combustion-generated particulate matter containing persistent free radicals redox cycle to produce reactive oxygen species.

Particulate matter (PM) is emitted during thermal decomposition of waste. During this process, aromatic compounds chemisorb to the surface of metal-oxide-containing PM, forming a surface-stabilized environmentally persistent free radical (EPFR). We hypothesized that EPFR-containing PM redox cycle to produce ROS and that this redox cycle is maintained in biological environments. To test our hypothesis, we incubated model EPFRs with the fluorescent probe dihydrorhodamine (DHR). Marked increases in DHR fluorescence were observed. Using a more specific assay, hydroxyl radicals ((•)OH) were also detected, and their level was further increased by cotreatment with thiols or ascorbic acid (AA), known components of epithelial lining fluid. Next, we incubated our model EPFR in bronchoalveolar lavage fluid (BALF) or serum. Detection of EPFRs and (•)OH verified that PM generate ROS in biological fluids. Moreover, incubation of pulmonary epithelial cells with EPFR-containing PM increased (•)OH levels compared to those in PM lacking EPFRs. Finally, measurements of oxidant injury in neonatal rats exposed to EPFRs by inhalation suggested that EPFRs induce an oxidant injury within the lung lining fluid and that the lung responds by increasing antioxidant levels. In summary, our EPFR-containing PM redox cycle to produce ROS, and these ROS are maintained in biological fluids and environments. Moreover, these ROS may modulate toxic responses of PM in biological tissues such as the lung.

Role of the Filters in the Formation and Stabilization of Semiquinone Radicals Collected from Cigarette Smoke.

The fractional pyrolysis of Bright tobacco was performed in nitrogen atmosphere over the temperature range of 240 - 510 °C in a specially constructed, high temperature flow reactor system. Electron paramagnetic resonance (EPR) spectroscopy was used to analyze the free radicals in the initially produced total particular matter (TPM) and in TPM after exposure to ambient air (aging). Different filters have been used to collect TPM from tobacco smoke: cellulosic, cellulose nitrate, cellulose acetate, nylon, Teflon and Cambridge. The collection of the primary radicals (measured immediately after collection of TPM on filters), the formation and stabilization of the secondary radicals (defined as radicals formed during aging of TPM samples on the filters) depend significantly on the material of the filter. A mechanistic explanation about different binding capability of the filters decreasing in the order: cellulosic < cellulose nitrate < cellulose acetate < nylon ~ teflon is presented. Different properties were observed for the Cambridge filter. Specific care must be taken using the filters for identification of radicals from tobacco smoke to avoid artifacts in each case.

Molecular products from the thermal degradation of glutamic acid.

The thermal behavior of glutamic acid was investigated in N2 and 4% O2 in N2 under flow reactor conditions at a constant residence time of 0.2 s, within a total pyrolysis time of 3 min at 1 atm. The identification of the main pyrolysis products has been reported. Accordingly, the principal products for pyrolysis in order of decreasing abundance were succinimide, pyrrole, acetonitrile, and 2-pyrrolidone. For oxidative pyrolysis, the main products were succinimide, propiolactone, ethanol, and hydrogen cyanide. Whereas benzene, toluene, and a few low molecular weight hydrocarbons (propene, propane, 1-butene, and 2-butene) were detected during pyrolysis, no polycyclic aromatic hydrocarbons (PAHs) were detected. Oxidative pyrolysis yielded low molecular weight hydrocarbon products in trace amounts. The mechanistic channels describing the formation of the major product succinimide have been explored. The detection of succinimide (major product) and maleimide (minor product) from the thermal decomposition of glutamic acid has been reported for the first time in this study. Toxicological implications of some reaction products (HCN, acetonitrile, and acyrolnitrile), which are believed to form during heat treatment of food, tobacco burning, and drug processing, have been discussed in relation to the thermal degradation of glutamic acid.

Environmentally persistent free radicals and their lifetimes in PM2.5.

For the first time, an expansive study into the concentration and extended decay behavior of environmentally persistent free radicals in PM2.5 was performed. Results from this study revealed three types of radical decay-a fast decay, slow decay, and no decay-following one of four decay patterns: a relatively fast decay exhibiting a 1/e lifetime of 1-21 days accompanied by a slow decay with a 1/e lifetime of 21-5028 days (47% of samples); a single slow decay including a 1/e lifetime of 4-2083 days (24% of samples); no decay (18% of samples); and a relatively fast decay displaying an average 1/e lifetime of 0.25-21 days followed by no decay (11% of samples). Phenol correlated well with the initial radical concentration and fast decay rate. Other correlations for common atmospheric pollutants (ozone, NOx, SO2, etc.) as well as meteorological conditions suggested photochemical processes impact the initial radical concentration and fast decay rate. The radical signal in PM2.5 was remarkably similar to semiquinones in cigarette smoke. Accordingly, radicals inhaled from PM2.5 were related to the radicals inhaled from smoking cigarettes, expressed as the number of equivalent cigarettes smoked. This calculated to 0.4-0.9 cigarettes per day for nonextreme air quality in the United States.

Tar balls from Deep Water Horizon oil spill: environmentally persistent free radicals (EPFR) formation during crude weathering.

Tar balls collected from the Gulf of Mexico shores of Louisiana and Florida after the BP oil spill have shown the presence of electron paramagnetic resonance (EPR) spectra characteristic of organic free radicals as well as transition metal ions, predominantly iron(III) and manganese(II). Two types of organic radicals were distinguished: an asphaltene radical species typically found in crude oil (g = 2.0035) and a new type of radical resulting from the environmental transformations of crude (g = 2.0041-47). Pure asphaltene radicals are resonance stabilized over a polyaromatic structure and are stable in air and unreactive. The new radicals were identified as products of partial oxidation of crude components and result from the interaction of the oxidized aromatics with metal ion centers. These radicals are similar to semiquinone-type, environmentally persistent free radicals (EPFRs) previously observed in combustion-generated particulate and contaminated soils.

Molecular products from the pyrolysis and oxidative pyrolysis of tyrosine.

The thermal degradation of tyrosine at a residence time of 0.2s was conducted in a tubular flow reactor in flowing N2 and 4% O2 in N2 for a total pyrolysis time of 3min. The fractional pyrolysis technique, in which the same sample was heated continuously at each pyrolysis temperature, was applied. Thermal decomposition of tyrosine between 350 and 550°C yielded predominantly phenolic compounds (phenol, p-cresol, and p-tyramine), while decomposition between 550 and 800°C yielded hydrocarbons such as benzene, toluene, and ethyl benzene as the major reaction products. For the first time, the identification of p-tyramine, a precursor for the on of formation of p-tyramine and its degradation to phenol and p-cresol, and toxicological discussion of some of the harmful reaction products is also presented.

Particulate matter containing environmentally persistent free radicals and adverse infant respiratory health effects: a review.

The health impacts of airborne particulate matter (PM) are of global concern, and the direct implications to the development/exacerbation of lung disease are immediately obvious. Most studies to date have sought to understand mechanisms associated with PM exposure in adults/adult animal models; however, infants are also at significant risk for exposure. Infants are affected differently than adults due to drastic immaturities, both physiologically and immunologically, and it is becoming apparent that they represent a critically understudied population. Highlighting our work funded by the ONES award, in this review we argue the understated importance of utilizing infant models to truly understand the etiology of PM-induced predisposition to severe, persistent lung disease. We also touch upon various mechanisms of PM-mediated respiratory damage, with a focus on the emerging importance of environmentally persistent free radicals (EPFRs) ubiquitously present in combustion-derived PM. In conclusion, we briefly comment on strengths/challenges facing current PM research, while giving perspective on how we may address these challenges in the future.