Interactions of Gaseous 2‑Chlorophenol with Fe3+-SaturatedMontmorillonite and Their Toxicity to Human Lung CellsAnping Peng,†,§ Juan Gao,‡ Zeyou Chen,† Yi Wang,† Hui Li,§ Lena Q. Ma,† and Cheng Gu*,†

†State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, P.R. China‡Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing,Jiangsu 210008, P. R. China§Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, Michigan 48824, United States

*S Supporting Information

ABSTRACT: The interactions of gaseous 2-chlorophenol withFe3+-saturated montmorillonite particles in a gas−solid systemwere investigated to simulate the reactions of mineral dustswith volatile organic pollutants in the atmosphere. Resultssuggested that Fe3+-saturated montmorillonite mediated thedimerization of gaseous 2-chlorophenol to form hydroxylatedpolychlorinated biphenyl, hydroxylated polychlorinated di-phenyl ether, and hydroxylated polychlorinated dibenzofuran.The toxicity of Fe3+-montmorillonite particles to A549 humanlung epithelial cells before and after interaction with 2-chlorophenol was examined to explore their adverse impact onhuman health. Based on cell morphological analysis, cytotox-icity tests, and Fourier-transform infrared imaging spectra,surface-catalyzed reactions of Fe3+-montmorillonite with 2-chlorophenol increased the toxicity of montmorillonite particle onA549 cells. This was supported by increased cellular membrane permeability, the release of extracellular lactate dehydrogenase,and cell damages on cellular DNA, proteins, and lipids. Since mineral dusts are important components of particulate matter, ourresults help to understand the interactions of volatile organic pollutants with particulate matter in the atmosphere and theiradverse impacts on human health.

■ INTRODUCTION

Mineral dust, an important component of atmospheric aerosols,is made of soil particles suspended in the atmosphere by windor human activities.1 Depending on sources, mineralcomposition of dust particles is different.2 Montmorillonite,illite, kaolinite, and quartz are the most common componentsin mineral dusts.2 Especially for montmorillonite, it is a 2:1layered aluminosilicate clay mineral often used to representmineral dust particles3 and considered as an importantcondensation nucleus for cloud and rain formation.4,5 Becauseof isomorphic substitution in the tetrahedral Si and/oroctahedral Al layers, montmorillonite generally possessesstructural negative charges that are compensated by exchange-able cations in the interlayer regions, resulting in sometransition metals incorporated in mineral dusts.3 It wasreported that ∼95% of global atmospheric Fe budgets are inmineral aerosols as iron oxide grains, clay-associated iron, andsoluble iron.6 Studies showed that Fe species in mineral dustaerosols can catalyze the oxidative reactions of NO2 and SO2 toform nitrate and sulfate species.7,8 However, limited efforts havefocused on gaseous reactions of volatile organic pollutants withFe-enriched mineral dusts under atmospheric conditions.

Chlorophenols are a class of chlorinated organic pollutantsthat are widely used in industrial and agricultural practice aswood preservatives, herbicides and fungicides.9 Biodegradationof highly chlorinated aromatic compounds by microorganismsor disinfection of drinking water via chlorine can producechlorophenols.10 As a result, chlorophenols have beenfrequently detected in various environmental compart-ments.10,11 In ambient air, chlorophenols are present as vaporscoming from the combustion of wastes, coal, or wood.10 Ingeneral, the concentration of chlorophenol in the air is an effectof local emission sources, and high concentration can be foundin air samples from certain areas. For instance, pentachlor-ophenol has been detected in urban air at levels of 5.7−7.8 ngm−3.12 The gaseous chlorophenol may be exposed to humans,especially for the residents living near the pesticidemanufacturing industries by inhalation, ingestion, and eye anddermal contact, causing potential adverse effects on health,which has attracted much attention.10 However, little

Received: December 26, 2017Revised: April 2, 2018Accepted: April 3, 2018Published: April 3, 2018

Article

pubs.acs.org/estCite This: Environ. Sci. Technol. 2018, 52, 5208−5217

© 2018 American Chemical Society 5208 DOI: 10.1021/acs.est.7b06664Environ. Sci. Technol. 2018, 52, 5208−5217

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information is available for the interactions between gaseouschlorophenols with suspended mineral dusts in the atmosphereand their human health effects.Prior studies have manifested that transition metal oxides or

fly ash surface can catalyze chlorophenols to form dioxins athigh temperature.13−15 Whereas, our previous studies foundthat Fe3+-montmorillonite clay could oxidize chlorophenol toform dioxin and dioxin-like compounds through a singleelectron transfer process under environmentally relevantconditions.16,17 Dioxin and dioxin-like compounds are oftenhighly toxic contaminants due to their potency to activate arylhydrocarbon receptor.18 Here, we hypothesized that similarreactions could occur between gaseous chlorophenols andmineral dust surfaces, potentially posing risks to human health.In this study, Fe3+-montmorillonite was used to simulate

mineral dusts since it is one of the most abundant soil minerals.Because of its high vapor pressure (0.308 kPa at 25 °C), 2-chlorophenol was utilized as a model volatile organic pollutant.The gas-phase reactions were conducted in a self-constructedreactor where 2-chlorophenol vapor could interact directly withthe suspended Fe3+-montmorillonite particles under environ-mentally relevant conditions. The reaction process wasmonitored by an in situ diffuse reflectance infrared Fouriertransform spectroscopy (DRIFTS) system. The toxicity ofmontmorillonite particles to human lung cells before and afterreacting with 2-chlorophenol were evaluated based on cellmorphology, cytotoxicity tests, and Fourier transform infraredimaging spectroscopy (FT-IRIS).

■ MATERIALS AND METHODSChemicals and Clay Minerals. Chemicals and cell line

used in this study are described in the Supporting Information.Fe3+- and Na+-montmorillonites (particle size of 20−80 nm)were prepared from commercial montmorillonite clay mineral,and the detailed information for preparation process along withselected physicochemical properties is summarized in the textand Table S1 of the Supporting Information.Heterogeneous Reaction System. The schematic dia-

gram of the heterogeneous reaction system is shown in sectionsI and II of Figure S1. To simulate the evaporation of 2-chlorophenol and water, compressed air streams 1 and 2 wereintroduced into two stainless steel cylinder jars with 40 mm indiameter and 120 mm in height (parts A and B in Figure S1),which contained 1 mL of 2-chlorophenol and 10 mL ofultrapure water, respectively. The air flow rate of stream 1 was0.2 L min−1. The relative humidity of the reaction system wascontrolled via changing the flow rate of air stream 2. Air stream3 was used to balance the total air flow rate of 1.0 L min−1 forthree air streams, which was monitored and controlled by a S4932/MT digital mass flow controller (Horiba Metron Instru-ments, China). The three air streams were homogeneouslymixed in a 150 mL diffusion jar (part C in Figure S1) beforereaching the reaction tube. 0.1 g of Fe3+-montmorillonitepowder was placed in the sample holder that was mounted inthe center of the quartz reaction tube (10 mm in diameter and150 mm in height, part D in Figure S1). The reaction wasinitiated by passing the 2-chlorophenol air mixture through theprepared Fe3+-montmorillonite for 2 h. The preliminaryexperiment showed that montmorillonite particles weresuspended at air flow rate of 1.0 L min−1 in the reactor. Twotandem bottles with 50 mL of 1.0 M NaOH were connected tothe outlet to collect residual 2-chlorophenol (parts E and F inFigure S1). The temperature for all gas lines and the reactor

(sections I and II) was maintained at 25 °C in a constanttemperature chamber. Na+-saturated montmorillonite was usedas experimental control.

In Situ FTIR Analysis. To trace the whole reaction processon the clay surface, real-time infrared (IR) spectra of the Fe3+-montmorillonite particles at different reaction time werecollected by a Bruker tensor 27 FTIR spectrometer (BrukerOptik, Germany) equipped with a DRIFTS chamber (ZnSewindows, Harrick Scientific) and a mercury cadmium telluridedetector. For in situ FTIR measurement, Fe3+-montmorillonitepowder (0.05 g) was loaded into a DRIFTS cell that wasconnected to a similar air feeding system as described above(section III in Figure S1). The air flow rate and the reactiontime were set at 0.5 L min−1 and 320 min, respectively. Themeasured IR frequency region was 400−4000 cm−1, and a totalof 64 scans were collected for each spectrum with a resolutionof 4 cm−1. The interferences from the IR absorption of water(∼1600 cm−1) and montmorillonite (<1250 cm−1) wereeliminated via subtraction of their spectra from the spectrumof the spent montmorillonite using OPUS software 7.0 (BrukerOptics, Germany).19

Identification of Reaction Products. Spent Fe3+-mont-morillonite particles collected from the heterogeneous systemswere extracted with 2 mL of acetone/hexane mixture (1:1 v/v)for 10 h. The extract was further acetylated with 5 mL ofsodium carbonate (0.5 M) and 120 μL of acetic anhydride for 2h, followed by centrifugation at 1250g for 20 min. Thepreliminary experiment indicated that the recoveries of theextraction method for 2-chlorophenol adsorbed on montmor-illonite particles under different humidity conditions are 88.3 ±2.78% to 107 ± 7.71%. The reaction products were identifiedusing a Thermo Fisher 1310 gas chromatograph coupled withan ISQ mass spectrometer (GC-MS) on a full scan mode withthe molecular weight ranging from 40 to 1000 amu. A TR-5 MScapillary column (length = 30 m; internal diameter = 0.25 mm;film thickness = 0.25 μm) was used. The carrier gas is helium ata flow rate of 1.0 mL min−1 with splitless injection at 290 °C.The oven temperature was programmed initially at 60 °C (2min hold), then increasing to 200 °C (15 °C min−1, 5 minhold), and finally to 320 °C (15 °C min−1, 15 min hold). Theconcentrations of Fe2+ and Cl− generated during the reactionprocess were also measured. Further detailed information isdescribed in the Supporting Information.Density functional theory (DFT) calculations were also

carried out to evaluate the possible reaction site for 2-chlorophenol. The frontier electron densities (FED) of thehighest occupied molecular orbital (HOMO) and the lowestunoccupied molecular orbital (LUMO) of 2-chlorophenol weredetermined by the DFT method (B3LYP)20 in the Gaussian 09program.21 The 6-311G(d,p) basis set was applied to C, H, O,and Cl atoms. The molecular structure of 2-chlorophenol wasconstructed with the GView program based on the optimizedresult.

Cell Culture. To evaluate the toxicity of Fe3+-montmor-illonite particles collected from gas-phase reaction system,human pulmonary epithelial cell line (A549), a widely usedhuman lung cancer cell model for investigating cytotoxicity andgenotoxicity of nanoparticles or organic pollutants, wasused.22,23 A549 cells were cultured in Dulbecco’s modifiedeagle medium (DMEM) containing 10% (v/v) fetal bovineserum and 1% (v/v) antibiotic−antimycotic solution (GIBCO/BRL) in an incubator with 5% CO2 at 37 °C for 2 days forfurther use.

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Exposure Experiment. Before the exposure experiment,500 μL of A549 cell culture suspension was transferred to eachwell of a 24- or 96-well microtiter plate. For selected wells of24-well plate, CaF2 window (11 mm in diameter and 1 mm inthickness) was preloaded at the bottom of the 24-well plate forthe subsequent microscopic examination. The concentration ofA549 cells in each well was 1 × 102 cells μL−1, which wasconfirmed by a hemacytometer. After 48 h cultivation, all A549cells grew at the bottom of wells or on the CaF2 window. Theculture media were then carefully decanted and replaced withthe same volume of fresh DMEM media containing 0.1−500 μgmL−1 of the reacted Fe3+-montmorillonite particles. Beforeexposure to the cells, an intermittent ultrasonic decompositionprocess (2 min with the interval of 30 s) was applied to theDMEM medium containing montmorillonite particles toensure the clay particles evenly distributed in media, whichwas confirmed by an atomic force microscopy (AFM)measurement (Figure S2). For comparison, the same amountof Fe3+-montmorillonite and 0.8−4000 ng mL−1 of 2-chlorophenol were also exposed to A549 cells. The amountof 2-chlorophenol exposed to the cell was the same as the totalresidual 2-chlorophenol on the Fe3+-montmorillonite surfaceafter the reaction. The concentration ranges of montmorilloniteand chlorophenol used in this study covered most of theexposure levels employed in previous studies24−26 and wouldbe appropriate to evaluate the potential health effects. Theexposure experiments for Na+-montmorillonite and thecorresponding spent Na+-montmorillonite were also conductedusing the same methods. All of the experiments were conductedin four replicates.Examination of Cell Morphology. After 24 h exposure,

the cell morphology of A549 cell grown in 24-well plate wasexamined using a Nikon TS-100 inverted microscope (Tokyo,Japan). The cells growing on the CaF2 window were recordedas FT-IRIS image using a Bruker HYPERION 2000 infraredmicroscope (Ettlingen, Germany) with IR spectral range of600−4000 cm−1 at 4 cm−1 resolution and 32 scans per pixel.The spectrum of CaF2 window was used to correct the signalfrom the instrumental and atmospheric background. Thehyperspectral images were analyzed by OPUS 7.0 software.Cytotoxicity Analysis. To determine the cytotoxicity of

the reacted montmorillonite, the viability of A549 cell grown in96-well plate and the corresponding extracellular cytoplasmicenzyme lactate dehydrogenase (LDH) concentration in mediaafter 24 h exposure were measured using Cell Counting Kit-8(CCK-8) kit and LDH kit following the manufacturers’instruction, respectively. For all the analyses, the cell grownin the media without montmorillonite or 2-chlorophenolexposure was considered as control. To eliminate the potentialinterference of montmorillonite particle on the toxicity analysis,the treatment with the corresponding amount of Fe3+-montmorillonite, Na+-montmorillonite, DMEM medium, andCCK-8 or LDH detection solution without A549 cell was set asthe blank control, and the measurement was conducted underthe same conditions.Statistical Analysis. The experimental data were presented

as the mean ± standard deviation of four independentmeasurements and were evaluated by one-way ANOVAfollowed by Fisher’s least significant difference (LSD) test.Significant differences were established at p < 0.05.

■ RESULTS AND DISCUSSION

Heterogeneous Surface Reactions on Fe3+-Montmor-illonite. After the reaction between 2-chlorophenol vapor andFe3+-montmorillonite, the color of montmorillonite particleschanged from yellow to dark green, indicating that reactionsoccurred on the Fe3+-montmorillonite surfaces. Similar colorchanges were also reported after reactions of polyaromatichydrocarbons and pentachlorophenol on Fe3+-montmorillonitesurfaces.16,27

The GC-MS chromatogram and the mass spectra of thereaction products at reaction humidity of 10% are shown inFigure 1. Except for the residual 2-chlorophenol peak, noobvious product was found in the control of 2-chlorophenol +Na+-montmorillonite (Figure S3). Three acetylated reactionproducts with molecular ion clusters at mass-to-charge ratios

Figure 1. GC-MS chromatogram and mass spectra of acetylatedacetone/hexane (1:1, v/v) extract of Fe3+-montmorillonite afterreaction with 2-chlorophenol: (A) GC-MS chromatogram (retentiontime 30−45 min); mass spectra of (B) HO-PCB (retention time 44.86min), (C) HO-PCDE (retention time 35.33 min), and (D) HO-PCDF(retention time 40.70 min). Experimental conditions: 0.1 g of Fe3+-montmorillonite reacted with gaseous 2-chlorophenol for 2 h at 25 °Cunder 10% humidity. Insets are the proposed structure for eachreaction product.

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(m/z) of 338, 296, and 294 were obtained in the 2-chlorophenol + Fe3+-montmorillonite system, and each ioncluster has chlorine isotope ratio of 9:6:1 in the relativeabundance (Figure 1). Considering that the natural abundanceratio of 35Cl and 37Cl of 3:1, the observed isotope distributionpattern indicates that these three products all contain two −Clgroups,28 which might result from dimerization of 2-chlorophenol.29 Typically, the radical−radical coupling reactioncould form the products with the molecular weights followingthe pattern of nM0 − 2(n − 1), where M0 is the molecularweight of 2-chlorophenol and n is a natural number.30 Herein,the peak with m/z value of 338 could correspond to theacetylation of dimerization product of two 2-chlorophenolmolecules (128 + 128 − 2 + 42 (acetyl group) × 2). Twochlorinated phenyl radicals are linked by C−C bond to formthis product, leaving the two phenolic groups intact. The peakwith m/z value of 296 might be identified as the acetylation ofdimerization product of 2-chlorophenoxy radical and chlori-nated phenyl radical linked by the C−O−C bond, leaving onephenolic group intact (128 + 128 − 2 + 42). The third productwith the m/z value of 294 (128 + 128 − 2 − 2 + 42) may resultfrom acetylation of dibenzofuran to form a central furan ring,leaving one phenolic group intact. Previous studies showed thatthe position of hydroxyl group might affect the chemicalproperties and toxicities of the hydroxylated compounds.31,32

Therefore, the exact structures of the reaction products need tobe identified. Although the primary mass spectrum is notcapable of differentiating isomers,33 theoretical calculationswere employed to provide more information to determine thestructure of the reaction products.34 The calculated FED resultfor each atom of 2-chlorophenol is listed in Table S2.According to the frontier orbital theory, the position withhigher 2FED2

HOMO value in 2-chlorophenol molecule would bethe possible reaction site,35,36 where one electron is extractedby Fe3+, forming the corresponding 2-chlorophenol radicalcation. Compared to the 2FED2

HOMO values for other positionsin 2-chlorophenol (except for C1 and Cl atoms, which cannotderive a radical site), the hydroxyl O and C6 atoms have higher2FED2

HOMO values, so it is expected that the reaction wouldoccur at hydroxyl O and C6 sites (Table S2). Finally, along withthe mass fragmentation pattern of each product and thereaction products reported in similar studies,37,38 the threeproducts are proposed as 3,3′-dichloro-2,2′-dihydroxybiphenyl(HO-PCB), 2′,3-dichloro-2-hydroxydiphenyl ether (HO-PCDE), and 2′,3-dichloro-2-hydroxydibenzofuran (HO-PCDF), and their structures are shown in Figure 1. This isalso consistent with the calculated energy profiles by Pan et al.,who found that the self-coupling of phenoxy radicals toproduce HO-PCB and the coupling between the oxygen-centered radical mesomer to produce HO-PCDE have lowerreaction energies.39 Formation of polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofurans (PCDD/Fs) on thesurface of transition metal oxides from chlorophenols havebeen widely studied, and these reactions generally occur at hightemperature.14,15 However, in this research, no PCDD/Fsformation was observed probably due to the mild gas-phasereaction conditions, in which no further condensation reactionproceeds. Furthermore, the negligible amount of Cl− wasreleased during the reaction between 2-chlorophenol and Fe3+-montmorillonite under different humidity (Figure S4),indicating that dechlorination reaction could be negligible dueto lower chlorine substitution in 2-chlorophenol molecule.Compared to highly chlorinated chlorophenols, fewer chlorine

substitution would decrease the electron density of thearomatic-π system, thus lowering the tendency to lose Cl−

for mono- or dichlorophenol.40

The presence of water in the reaction chamber suppressesthe surface reaction of 2-chlorophenol, which is confirmed bydecreased reaction products and Fe2+ formation with increasinghumidity (Figure 2A,B). As relative humidity increases from 10

to 50%, the peak areas of HO-PCB, HO-PCDE, and HO-PCDF decrease by 90%, 85%, and 91%, respectively (Figure2A), and the Fe2+ from Fe3+ reduction decreases from 5.88 to1.90 mg g−1 montmorillonite (Figure 2B). Under highermoisture, water molecules may occupy the adsorption sites onmontmorillonite, thereby reducing the adsorption of chlor-ophenol.41 As such, the adsorption of 2-chlorophenol bymontmorillonite decreases from 5.2 to 0.8 mg g−1 as humidityincreases from 10 to 50% (Figure S5). In addition, watermolecules could also form the strong complex with Fe3+, whichimpedes the direct electron transfer to Fe3+ from 2-chlorophenol.42

Figure 2. Reaction products formation (A) and Fe2+ release (B) afterreactions of gaseous 2-chlorophenol with Fe3+-montmorillonite underdifferent humidity conditions. Experimental conditions: Fe3+-mont-morillonite 0.1 g, reaction time 2 h, temperature 25 °C. (C) Real-timeFTIR monitoring of the reaction of gas phase 2-chlorophenol withFe3+-montmorillonite at different duration. Experimental conditions:Fe3+-montmorillonite 0.05 g, total gas flow rate 0.5 L min−1, reactiontemperature 25 °C, and humidity 10%. Error bars are the standarddeviations of quadruplicate analyses.

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As shown in Figure 2C, in situ IR spectra during the reactionwere collected in a DRIFTS system, with the IR peakssummarized in Table S3. The specific IR peaks of 2-chlorophenol at 1587, 1484 cm−1 (CC stretching of benzenering), 1339 cm−1 (O−H deformation and bending vibration),1288 cm−1 (C−O stretch vibration), 1159 cm−1 (C−H in-planebending vibration of benzene ring), and 1055 cm−1 (chlorine-sensitive vibration) all increase during the first 20 min ofreaction (Figure 2C), indicating that 2-chlorophenol isgradually adsorbed onto montmorillonite surface.43 As thereaction proceeds, three new peaks at 756, 839, and 1244 cm−1

appear. The IR bands at 756 and 839 cm−1 are attributed to theC−H out-of-plane bending and ring puckering vibrations of1,2,3-substituents and 1,2,3,4-substituents of the benzene ring,respectively.44,45 The band at 1244 cm−1 is assigned to theasymmetric stretching mode of the diphenyl ether group.46 Theappearance of these new bands further confirms the formationof HO-PCB, HO-PCDE, and HO-PCDF. Furthermore, thebands for 2-chlorophenol at 1484 and 1587 cm−1 due to CCstretching of the aromatic ring exhibit blue-shifts to 1490 and1595 cm−1, respectively, suggesting that more electrophilicfunctional groups (e.g., phenoxy structure or halogen group)are attached to the benzene ring.47 This is also consistent withthe formation of HO-PCB, HO-PCDE, and HO-PCDF.Toxicity of Montmorillonite Particles after Reaction

with 2-Chlorophenol. Generally, hydroxylated polybromi-nated biphenyls (HO-PBB) and hydroxylated polybrominateddiphenyl ethers (HO-PBDE) are much more toxic than theirparent compounds (PBB and PBDE),48 since they could exert arange of toxic effects that are not shown by their parentcompounds, e.g., the inhibition of mitochondrial respiration,damage to DNA, estrogenic activity, etc.31,32,49 Therefore, it isexpected that the reactions of 2-chlorophenol on the Fe3+-montmorillonite surface could affect the toxicity of montmor-illonite. To evaluate the potential risks for human exposure tomontmorillonite after reaction with 2-chlorophenol, humanlung cell A549 was used to measure the toxicity of Fe3+-montmorillonite before and after reaction with 2-chlorophenol.The montmorillonite samples collected after the reaction at

10% humidity were used for the toxicity tests due to the higherreaction efficiency. Cell membrane damage is an indicator ofthe toxicity of montmorillonite particles to cells. When cellmembrane integrity is damaged or ruptured, LDH is oftenreleased into the surrounding extracellular space, so LDHactivity in the culture medium can be used to determine theextent of cell membrane damage.50 Compared to non-montmorillonite-exposed control, no significant change inLDH activity was observed for cells exposed to 1−50 μgmL−1 of Fe3+-montmorillonite particles regardless of whether itreacts with 2-chlorophenol or not (Figure 3A). However,higher concentrations of Fe3+-montmorillonite before and afterreaction with 2-chlorophenol (100−500 μg mL−1) bothsignificantly impair the membrane integrity of A549 cells; e.g.,when the cell was exposed to 100 μg mL−1 of Fe3+-montmorillonite, the LDH released from A549 is 1.3 timeshigher than that in the nonexposed controls (Figure 3A),indicating that Fe3+-montmorillonite could cause damage tocytomembrane at high concentrations. A similar inhibitionphenomenon was also observed for the survival rate of A549(Figure 3C). For example, as Fe3+-montmorillonite concen-trations increase from 0.1 to 500 μg mL−1, the survival rate ofA549 cell decreases from 101 to 17% (Figure 3C). Studies havedemonstrated that fine particles can be adsorbed on the cell

membrane and interact with lipid or protein components tolower cell membrane permeability, integrity, and cellsurvival.51,52 Compared to Fe3+-montmorillonite, spent Fe3+-montmorillonite particles show higher toxicity as indicated byboth extracellular LDH activity and cell viability tests (Figure3A,C). For example, at the spent Fe3+-montmorilloniteexposure concentration of 10 μg mL−1, the cell viabilitydecreases by 4.7%, while the extracellular LDH activityincreases by 18% compared to Fe3+-montmorillonite. Thedifference becomes even more significant with increasingmontmorillonite concentration; e.g., at 200 μg mL−1, the cellviability decreases 28% and LDH activity increases 20%compared to Fe3+-montmorillonite. Furthermore, as shown inFigure S6, the LC50 values for Fe

3+-montmorillonite and spentFe3+-montmorillonite are 82.7 and 52.4 μg mL−1, respectively.Therefore, these results collectively demonstrate that Fe3+-montmorillonite becomes more toxic to lung cells after reactionwith 2-chlorophenol. However, after the reaction, some 2-chlorophenol still remains on the surface. To evaluate thetoxicity of adsorbed 2-chlorophenol, we exposed A549 cells to2-chlorophenol alone at the concentration range of 8 ng mL−1−4 μg mL−1; the selected 2-chlorophenol concentrations are thesame as the residual 2-chlorophenol on 0.01−50 μg (themontmorillonite amount used in the exposure experiment) ofspent Fe3+-montmorillonite after 2 h reaction. As shown inFigure 3B, the LDH activities after 24 h exposure to 8 ngmL−1−4 μg mL−1 of 2-chlorophenol alone show no significantdifference (p < 0.05) from the nonexposed control. The cell

Figure 3. Concentration-dependent cytotoxicity to A549 cells of 2-chlorophenol, Fe3+-montmorillonite, and spent Fe3+-montmorilloniteafter reaction with 2-chlorophenol. (A, B) LDH assay; (C, D) CCK-8assay. The exposure concentrations of Fe3+-montmorillonite and spentFe3+-montmorillonite were 1−500 μg mL−1 and 0.1−500 μg mL−1 inLDH and CCK-8 assay, respectively. The exposed 2-chlorophenolamounts to the cells were same with the total residual 2-chlorophenolon the different exposure amounts of Fe3+-montmorillonite surfacesafter the reaction, which were 0.008−4 μg mL−1 and 0.0008−4 μgmL−1 in LDH and CCK-8 assay, respectively. The asterisk representsthe significant difference (p < 0.05) between Fe3+-montmorillonite andspent Fe3+-montmorillonite treatments. There is a significant differ-ence between none-Fe3+-montmorillonite-exposed control and 100−500 μg mL−1 of Fe3+-montmorillonite in LDH assay, as well betweennone-Fe3+-montmorillonite-exposed control and 20−500 μg mL−1 ofFe3+-montmorillonite in CCK-8 assay (p < 0.05).

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viability obtained from CCK-8 assay also verifies that 2-chlorophenol at the tested concentration range does not causetoxic effect on cell survival (Figure 3D). The toxicity profilefrom the Agency for Toxic Substances and Disease Registryshows that low concentration of 2-chlorophenol does notinduce adverse effect on rats, which might be due to its low Kow

value, rendering it difficult to accumulate in tissues.53

Moreover, in order to distinguish the different toxic effect,Na+-montmorillonite was used as control, since preliminaryresults have verified that there is no reaction between 2-chlorophenol and Na+-montmorillonite. Our results show thatno significant difference was observed for Na+-montmorilloniteand the spent Na+-montmorillonite treatment (Figure S7),

indicating that the adsorbed 2-chlorophenol on montmor-illonite does not affect its toxicity to A549. This result furthermanifests that the reaction products formed by the reactionbetween 2-chlorophenol and Fe3+-montmorillonite are respon-sible for the enhanced toxicity of clay particles. These productsare highly hydrophobic and easy to bind on cell surface toactivate the aryl hydrocarbon receptor, finally inducing cellapoptosis and inhibiting cell growth.54−56 In addition, thereaction time would significantly affect the surface reaction andsubsequent toxicity. Even after 4 h reaction, no observable newreaction products appear, whereas the peak areas of the threemajor products increase as the reaction time extends from 0.5to 4 h (Figure S8), indicating the accumulation of reaction

Figure 4. A549 cell morphology observed by inverted microscope (magnification 200×; left panels A−D) after exposure to 2-chlorophenol (0.008,0.08, 0.8, or 4 μg mL−1), Fe3+-montmorillonite and spent Fe3+-montmorillonite (1, 10, 100, or 500 μg mL−1), along with A549 cell morphologyobserved by FT-IRIS (magnification 400×; right panel E) after exposure to 0.8 μg mL−1 of 2-chlorophenol, 100 μg mL−1 of Fe3+-montmorillonite,and spent Fe3+-montmorillonite (with 0.8 μg mL−1 of 2-chlorophenol adsorbed on the surface).

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products on clay surface. Similarly, the toxicity of the spentmontmorillonite also increases as the increase of reaction time(Table S4).The toxicity of Fe3+-montmorillonite after reaction with 2-

chlorophenol was also examined using the inverted microscopicand FT-IRIS images (Figure 4). The regular A549 cells exist ascell clusters with pebble-like shape and smooth surface (Figure4A). After they were exposed to 2-chlorophenol at 0.008−4 μgmL−1, little impact on cell morphology was found (Figure 4B),which is consistent with the results of LDH activity and cellviability tests (Figure 3). The presence of Fe3+-montmorilloniteinduces damage on the cell membrane, which is positivelyrelated to its content in the suspension (Figure 4C). The Fe3+-montmorillonite after reaction with 2-chlorophenol causesmore damages on cells (Figure 4D). The FT-IRIS images ofA549 cells after exposure to 0.8 μg mL−1 of 2-chlorophenol,100 μg mL−1 of spent Fe3+-montmorillonite containing 0.8 μgmL−1 of 2-chlorophenol, and 100 μg mL−1 of Fe3+-montmorillonite are shown in Figure 4E. Compared to controlA549 cells, 2-chlorophenol exposure does not change its shapeor structure, while Fe3+-montmorillonite before or afterreaction with 2-chlorophenol damages the membrane integrityof A549 cells, with more destructive effect for spent Fe3+-montmorillonite. Characteristic band alterations identified byFT-IRIS spectra for A549 cells are shown in Figure 5, and themajor infrared absorption peaks corresponding to different cellcomponents are listed in Table S5. The IR spectroscopicchanges within the range of 900−1300 cm−1 indicate thedamage of DNA in A549 cells (Figure 5A). The bands at 979

and 1065 cm−1 appeared in the control or 2-chlorophenoltreatment represent the stretching vibration of C−C/C−O indeoxyribose sugar moiety and phosphodiester linkage in DNA,respectively.57,58 However, these two bands decrease ordisappear after the cell was exposed to Fe3+-montmorilloniteor spent Fe3+-montmorillonite, indicating DNA damage inA549 cell. Similar results were also found in the study ofAhmed et al.57 The peak at 2947 cm−1 (C−H antisymmetricstretching vibration in CH2 of fatty acyl chains in the cellularlipid) corresponding to the membrane fluidity of the cell red-shifts to 2942 cm−1 after interacting with spent Fe3+-montmorillonite compared to the control (Figure 5B),revealing that the presence of spent Fe3+-montmorillonitecould reduce the cell membrane fluidity of A549.59,60 It wasreported that lowered cell membrane fluidity usually implies theincrease of cell viscosity and the decrease of transport functionof the membrane, which could destroy membrane structure andeven cause the cell death.60 Studies have shown that there aretwo typical FTIR spectral signatures to indicate cell death.57,61

One is the change of overall protein conformational state in thecell, which is indicated by the amide I peak band (a major IRpeaks of protein secondary structure in a cell) shifting to lowerfrequency, inducing the disturbance of normal cellularfunctions;62 the other is the appearance of a new carbonyl(CO) peak that is associated with the CO stretching modeof non-hydrogen-bonded ester carbonyl within the phospho-lipid due to the formation of lipid-rich vesicles during cellapoptosis.63 In this study, the IR peak intensity of amide I at1700 cm−1 decreases and shifts to a lower frequency (1695

Figure 5. FT-IRIS spectra in the range of (A) 900−1200 cm−1 corresponding to DNA; (B) 2940−2950 cm−1 corresponding to cellular lipids; (C)1650−1670 cm−1 corresponding to amide I (protein); (D) FT-IRIS images assembled according to the integrated area of 1650−1719 cm−1 (amideI) of A549 cells after exposure to 0.8 μg mL−1 2-chlorophenol, 100 μg mL−1 Fe3+-montmorillonite, and spent Fe3+-montmorillonite after 24 hreaction. The color changes from the heliotrope to blue, indicating the increase of cell damage.

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cm−1) after exposure to spent Fe3+-montmorillonite, whereasonly small decrease of the signal was observed when the cellswere exposed to 2-chlorophenol or Fe3+-montmorillonite(Figure 5C), verifying the occurrence of cell death.Furthermore, compared to Fe3+-montmorillonite and 2-chlorophenol, a new carbonyl CO peak at 1763 cm−1

appears when the cells interact with spent Fe3+-montmorillonite(Figure 5C), which also demonstrates that there are more deadcells after the exposure.57 In addition, 100 random samplingpoints on CaF2 window were selected to conduct FTIRanalysis; the integrated area of the amide I peak (1600−l700cm−1) was determined, and the values were converted to a heatmap to better demonstrate the damage of amide I band. Thehigher values (corresponding to the less damage on amide I)are represented in heliotrope, while the lower values(corresponding to the greater damage on amide I) are inblue. As shown in Figure 5D, compared to the normal A549cells, more and more heliotrope areas are shifted to green; i.e.,increasing cell damage appears when the cells were respectivelyexposed to 2-chlorophenol, Fe3+-montmorillonite, and spentFe3+-montmorillonite. These results further corroborate thehigher toxicity of spent Fe3+-montmorillonite after reactionwith gaseous 2-chlorophenol.Environmental Implications. Air pollution by particulate

matter (PM) is of a global concern because inhalation andingestion of PM can cause adverse effects on human health.52,64

PM often carries harmful chemicals, and its long-distancetransportation in the atmosphere can lead to the widespread ofpollutants on a global scale.1 In this study, toxic organicpollutants HO-PCB, HO-PCDE, and HO-PCDF are formedvia the heterogeneous reaction of gaseous 2-chlorophenol withFe3+-montmorillonite particles (an important component inPM) in a gas−solid reaction system under environmentallyrelevant conditions, and the products formed on montmor-illonite surfaces enhance its toxicity to human lung cell A549.Clay mineral usually (but not necessarily) has the small particlesize (<2 μm), which can easily enter the human lung byinhalation. Therefore, the results of this study indicate thatvolatile organic pollutants adsorbed on PM particles could betransformed to more toxic chemicals after interactions with PMsurfaces. Meanwhile, the humidity studied in this researchcovered a wide range related to human life; the results wouldindicate that the reactions on natural clay could have a seriousadverse impact on human health, especially for arid andsemiarid regions. Furthermore, more mineral dust could begenerated under dry conditions, which also increases thepossibility for the interaction of volatile compounds withmineral dust. Overall, the results obtained in this studydemonstrate that PM surface-mediated reaction should beincorporated into the risk assessment to adequately evaluate itsenvironmental exposure. This is because not only the PMparticles themselves but also the chemicals adsorbed and theresulted products on PM surfaces would pose potential threatsto human health.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.7b06664.

Additional details of chemicals and reagents in this study,preparation of Na+- or Fe3+-montmorillonite clay, clayparticle size analysis, Cl− and Fe2+ analysis, schematic

diagram of the constructed gas-phase reactor (FigureS1), AFM of clay particle in culture media (Figure S2),GC-MS chromatogram of Na+- montmorillonite (FigureS3), Cl− released during the reaction (Figure S4), 2-chlorophenol adsorption on clay particle (Figure S5),LC50 analysis of Fe3+-montmorillonite and the spentFe3+-montmorillonite (Figure S6), cytotoxicity of Na+-montmorillonite to A549 cells (Figure S7), products atdifferent reaction time (Figure S8), chemical composi-tions of montmorillonite (Table S1), frontier electrondensities of 2-chlorophenol (Table S2), FTIR bandfrequencies of 2-chlorophenol, reaction products, and cellcomponents (Tables S3 and S5), and time-dependentcytotoxicity of Fe3+-montmorillonite to A549 (Table S4)(PDF)

■ AUTHOR INFORMATIONCorresponding Author*(C.G.) E-mail chenggu@nju.edu.cn; Ph/Fax +86-25-89680636.

ORCIDHui Li: 0000-0003-3298-5265Lena Q. Ma: 0000-0002-8463-9957Cheng Gu: 0000-0002-6939-7101NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was financially supported by National Key BasicResearch Program of China (2014CB441102), NationalScience Foundation of China (21477051 and 21777066), andthe Collaborative Innovation Center for Regional Environ-mental Quality. We thank Dr. Ping Xiang and Rongyan Liu fortheir help with the A549 cells cultivation. We also appreciateDr. Chao Wang for assistance in density functional theorycalculations.

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