Environ. Sci. Technol. 2009, 43, 2269–2275
Discovery of Novel Halogenated Polycyclic Aromatic Hydrocarbons in Urban Particulate Matters: Occurrence, Photostability, and AhR Activity T A K E S H I O H U R A , * ,† K E I - I C H I S A W A D A , † TAKASHI AMAGAI,† AND MIHO SHINOMIYA‡ Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, and National Environmental Research and Training Institute, 3-3 Namiki, Tokorozawa, Saitama 359-0042, Japan
Received December 21, 2008. Revised manuscript received February 8, 2009. Accepted February 12, 2009.
Halogenated aromatic compounds (HACs) in the environment have created great concern because of the associated adverse health implications. In this study we investigated the atmospheric behavior and exposure risk of brominated polycyclic aromatic hydrocarbons (BrPAHs) larger than three rings which were associated with particles in the urban air in Japan, and which were discovered as novel HACs in the air. Furthermore, the ambient levels of chlorinated polycyclic aromatic hydrocarbons (ClPAHs) and PAHs, in addition to BrPAHs, were also simultaneously investigated to emphasize the differences. Seven of 11 target BrPAHs were newly detected from the urban air samples in Japan between 2004 and 2005. Of the BrPAHs detected, 5,7-Br2BaA was most abundant (mean concentration, 8.7 pg m-3), followed by 7,12-Br2BaA (6.3 pg m-3) and 6-BrBaP (3.3 pg m-3). The mean concentrations of total BrPAHs, ClPAHs, and PAHs detected were 8.6 pg m-3, 15.2 pg m-3, and 1.2 ng m-3, respectively, which showed that concentrations of such halogenated PAHs (Br-/Cl-PAHs) tended to be approximately 100-fold lower than PAHs. The BrPAHs had photolysis rates that were relatively faster than the corresponding ClPAHs. Comparing the ambient profiles among the PAH congeners suggested that ambient BrPAHs that came from the specific local emission sources differed from ClPAHs and PAHs, and/or could be driven by various seasonal factors, including photodecay processes. Most of the BrPAHs used showed inherent AhR-mediated activities. Toxic equivalents based on the relative potencies of each AhR activity and the ambient concentrations showed that either BrPAHs or ClPAHs accounted for a smaller proportion (∼1%) of the total.
Introduction Airborne particulate matter is an analytically complex matrix that contains both organic and inorganic substances, and these are an important health concern, especially with respect * Corresponding author phone: +81 54 264 5789; fax: +81 54 264 5798; e-mail:
[email protected]. † University of Shizuoka. ‡ National Environmental Research and Training Institute. 10.1021/es803633d CCC: $40.75
Published on Web 03/03/2009
2009 American Chemical Society
to a number of chronic respiratory diseases (1-3). The estimates of the exposure risk of airborne particulate matter are highly uncertain, partly due to the complex nature of the particles and the lack of knowledge about the levels of toxicants that may be present in small amounts. Of the particle-associated contaminants, polycyclic aromatic compounds have studied extensively, and have prompted great concern about the adverse human health effects because of carcinogenicity and mutagenicity (4-6). Despite trace levels compared to PAHs, halogenated aromatic compounds (HACs), as typified by polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), are also of great health concern due to their high toxicities (7-9). HACs are mainly produced as byproduct of combustion processes of chlorine-containing materials, resulting in release into the environment (10). Furthermore, brominated aromatic compounds, such as polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs), which are analogues of PCDD/Fs, have also raised concerns due to physicochemical properties, toxicities, and geochemical behaviors in the environment similar to PCDD/ Fs, although studies involving brominated aromatic compounds lag behind PCDD/Fs (11-13). A few possible routes for the release of PBDD/Fs into the environment have been proposed, including formation during the processing of manufacturing brominated flame retardants (BFRs), formation during the processing of BFRs with polymer resin, and incinerating waste which contain BFRs (14-16). With regard to the concentrations in the atmosphere, it has often been observed that the TEQ concentrations of PBDD/Fs are lower (∼10-fold) than PCDD/Fs, whereas the data are very limited (11, 17). To reveal the environmental occurrences and biological effects of such brominated compounds, further study is needed. Recently, Ohura and co-workers have studied atmospheric occurrences, behavior, fate, and aryl hydrocarbon receptor (AhR) ligand-binding activities of chlorinated polycyclic aromatic hydrocarbons (ClPAHs) with 3-5 aromatic rings (18-22). The environmental features of ClPAHs are similar to those of PAHs. For example, the seasonal levels of atmospheric ClPAHs tend to increase in winter and decrease in summer, as shown with PAHs (19, 20). It could be related to the photostabilities that were strongly dependent on the features of the skeleton, i.e., PAHs (20, 23). Results of factor analysis using ambient ClPAH and PAH concentrations have shown that atmospheric ClPAHs are mainly emitted from incinerators, such as PCDD/Fs (22). Indeed, much of ClPAHs have been found in the fly ash and bottom ash from municipal/hazardous/industrial waste incinerators (24), and there is a significant correlation between ClPAH concentrations and dioxin concentrations in urban air (25). These facts imply that the production of bromine-substituted PAHs, brominated PAHs (BrPAHs), is fully expected as in the case of PBDD/Fs. However, there is no study on BrPAHs in atmosphere. Herein we determined the atmospheric levels, photostabilities, and AhR activities of 11 species of BrPAHs with 3-5 rings. Furthermore, those contributions of BrPAHs were compared to ClPAHs and PAHs. This is first report investigating the environmental and biological effects of halogenated PAHs including BrPAHs.
Materials and Methods Chemicals. In light of their environmental and toxicologic relevance, we selected seven PAH species as reference substances, and brominated them as follows. Briefly, Nbromosuccinimide (∼100 mg) was added to each PAH (∼150 mg) in carbonated propylene (10 mL); the mixture was VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2269
FIGURE 1. Structure of BrPAH derivatives. maintained at 100 °C for ∼2 h. The reaction solvent for each PAH was fractionated by HPLC (column, COSMOSIL 5C18AR; eluent, methanol), and the fractions corresponding to the dominant peaks were isolated and analyzed by GC/MS and 1H NMR spectroscopy (500 MHz, CDCl3). Accordingly, six BrPAHs were obtained by the synthesis, and the purities were confirmed by GC/MS by area, resulting in >95%. As other BrPAHs, 2-bromofluorene and 9,10-dibromoanthracene were purchased from Sigma-Aldrich (St. Louis, MO), and 9-bromoanthracene, 9-bromophenanthrene, and 7-benz[a]anthracene were purchased from Tokyo Chemical Industry (Tokyo, Japan). All the purities were also >95%. Consequently, 11 species of BrPAHs with 3-5 rings were used in the current study (Figure 1). As further target compounds, 15 species of ClPAHs and five species of PAHs with 3-5 rings were also prepared. Both 2- and 9-chloroanthracene were purchased from Sigma-Aldrich and Cambridge Isotope Laboratories (Andover, MA), respectively. The other ClPAHs were synthesized by the authors following published procedures (20). All PAHs were of the highest purity available and were purchased from Wako Pure Chemicals (Osaka, Japan). The individual halogenated PAH standards were dissolved in isooctane (Dojindo Laboratories, Kumamoto, Japan), and each standard solution of the target BrPAHs and ClPAHs was prepared by combining the individual BrPAH and ClPAH standard solutions, respectively. The individual PAH standards were dissolved in acetonitrile (HPLC grade; Wako Pure Chemicals), and a standard solution of the target PAHs was prepared by combining the individual PAH standard solutions. The solvents used for extraction and cleanup were residual-pesticide-analysis grade (Wako Pure Chemicals). The 11 BrPAHs used in this study are abbreviated as follows: 2-bromofluorene (2-BrFlu), 9-bromophenanthrene (9-BrPhe), 9-bromoanthracene (9-BrAnt), 9,10-dibromoanthracene (9,10-Br2Ant), 1-bromopyrene (1-BrPy), 7-benz[a]anthracene (7-BrBaA), 4,7-diboromobenz[a]anthracene (4,7-Br2BaA), 5,7-diboromobenz[a]anthracene (5,7-Br2BaA), 7,11-diboromobenz[a]anthracene (7,11-Br2BaA), 7,12-diboromobenz[a]anthracene (7,12-Br2BaA), and 6-bromobenzo[a]pyrene (6-BrBaP). The 16 ClPAHs used in this study are abbreviated as follows: 9-chlorofluorene (9-ClFlu), 9-chlorophenanthrene (9-ClPhe), 1,9-dichlorophenanthrene (1,9Cl2Phe), 3,9-dichlorophenanthrene (3,9-Cl2Phe), 9,10-dichlorophenanthrene (9,10-Cl2Phe), 3,9,10-trichlorophenanthrene (3,9,10-Cl3Phe), 2-chloroanthracene (2-ClAnt), 9-chloroanthracene (9-ClAnt), 9,10-dichloroanthracene (9,10-Cl2Ant), 3-chlorofluoranthene (3-ClFluor), 8-chlorofluoranthene (8ClFluor), 3,4-dichlorofluoranthene (3,4-Cl2Fluor), 3,8-dichlorofluoranthene (3,8-Cl2Fluor), 1-chloropyrene (1-ClPy), 1-chloropyrene (1-ClPy), 6-chlorochrysene (6-ClChry), 6,12dichlorochrysene (6,10-Cl2Chry), 7-chlorobenz[a]anthracene (7-ClBaA), 7,12-dichlorobenz[a]anthracene (7,12-Cl2BaA), and 6-chlorobenzo[a]pyrene (6-ClBaP). The corresponding six parent PAHs of those halogenated PAHs were used in this 2270
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 7, 2009
study (anthracene (Ant), phenanthrene (Phe), fluorene (Flu), pyrene (Py), benz[a]anthracene (BaA), and benzo[a]pyrene (BaP)). Sampling Methodology. Monitoring was conducted at a roof-top location (20 m above ground level) at the University of Shizuoka campus (latitude 34°59′ N, longitude 138°27′ E) situated ca. 1 km from the nearest busy road and ca. 6 km from the city center. Details of the area and its PAHs pollution levels have been given by Kume et al. (26). Samples were collected for 24 h for each of 3 consecutive days for every month between September 2005 and June 2006. Ambient particle samples were collected by using a highvolume air sampler (HV-1000F; Sibata Co. Ltd., Tokyo, Japan) operating at a constant flow rate of ca. 1.0 m3 min-1. The air was drawn through glass fiber filters (GFFs, 20.3 × 25.4 cm, type A/E; Gelman Sciences Inc., Ann Arbor, MI) to collect particles. GFFs were conditioned in a temperature and humidity-controlled atmosphere chamber for 48 h. After sampling, GFFs were wrapped in aluminum foil and sealed, and stored in a freezer at -45 °C until extraction. Extraction and Cleanup. After collection of air samples, the GFFs were extracted by a previously developed procedure (22, 27). Indeed, GFFs were extracted ultrasonically (39 kHz) for 20 min in a cleaned glass tube with 50 mL of dichloromethane containing fluoranthene-d10 (105 ng) and perylened12 (108 ng) as internal standards. After centrifugal separation (10 min at 2000 rpm), the supernatant (30 mL) was removed to a cleaned new glass tube. The supernatant was concentrated to ca. 1 mL under a gentle stream of N2 at 40 °C. The condensed solution obtained from GFFs was cleaned on a glass column (i.d. ) 15 mm) with silica gel (500 mg; Wako Pure Chemicals), and the columns were eluted continuously with 10 mL of n-hexane/dichloromethane (8:2 [v/v]). The eluate was concentrated under a gentle stream of N2 at 40 °C, and dissolved in 1.0 mL of isooctane. Prior to injection, chrysene-d12 (125 ng) was added to the residue as a syringe spike standard. During these operations, the solutions were protected from light to prevent photochemical degradation of the analytes. Analytical Procedure of Halogenated PAHs and PAHs. Analyses were performed on a JMS-700, high-resolution mass spectrometer (JEOL, Tokyo, Japan) equipped with an HP6890 (Agilent Technologies, Palo Alto, CA) gas chromatograph (HRGC-HRMS). Gas chromatographic separation was performed on a fused silica column DB-5MS (60 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific, Folsom, CA), using a splitless injection (split closed for 1.5 min). The injection volume was 1 µL for each sample and the calibration standards. The column oven temperature was programmed from 100 °C (2.0 min) to 280 °C at 5 °C/min and held there for 22 min. Helium was used as the carrier gas, and the flow rate was 1.2 mL/min. Injector and transfer line temperatures were all at 280 °C. The mass spectrometer was operated in electron impact ionization mode at a resolution of R > 10 000 (10% valley) and the electron energy was 38 eV. The BrPAHs were determined by selected ion monitoring at the two most intensive ions of the molecular ion cluster. The ions monitored for the identification of individual BrPAHs and internal standards, and the chromatograph are given in Table S1 and Figure S1 of the Supporting Information, respectively. For the analysis of ClPAHs, the extracted samples were used again, and performed on a HRGC-HRMS with a specific method developed previously (18). Concerning PAHs analysis, we performed a highly sensitive and multicomponent analysis method developed previously (28). Quality Assurance. The detection limits of individual BrPAHs, defined at a signal-to-noise ratio of 3 of the peak of a dilute standard solution, ranged from 0.056 ng (9,10-Br2Ant) to 2.4 ng (6-BrBaP); these values convert to air concentrations of 0.014-0.60 pg m-3 based on air volumes of 4000 m3. Prior
to sampling, the amounts of the BrPAHs targeted in the GFFs were confirmed to be below the detection limits. Analytical recoveries of the reference BrPAHs in the GFFs (n ) 6) ranged from 81% for 2-BrFlu to 112% for 4,7-, and 5,7-Br2BaA, indicating that the current analytical method was appropriate for atmospheric BrPAHs. The recoveries of each BrPAH estimated by the developed analytical procedure are summarized in Table S1 of the Supporting Information. Details regarding the detection limits and the recoveries of ClPAHs and PAHs have already been reported (18, 28). Photodegradation Experiments. Photodegradation experiments were performed with a turntable photoreactor (Ace Glass Inc., Vineland, NJ) using a 450 W high-pressure mercury lamp (Ushio Co., Ltd., Tokyo, Japan) as the light source, and a quartz immersion well with circulating water. The immersion well was surrounded by a Pyrex sleeve to filter out high-energy UV bands (λ < 290 nm). Although this setup does not duplicate the spectral distribution of the actinic flux, it does reproduce the wavelengths normally encountered in the atmosphere. The photoreactor was positioned in a water bath with constant water circulation and the temperature in the bath was maintained at 25 ( 1 °C. The solutions were irradiated in 13 × 100 mm quartz reaction tubes. Each synthesized BrPAH was dissolved in toluene at a concentration of ∼0.15 mM. Samples were removed with a pipet, transferred to amber glass chromatography vials, and analyzed with a GC-MS system in the scan mode to identify the photolysis rates and products. AhR Activity in Yeast Assay and Calculation of TEQs. Saccharomyces cerevisiae YCM3, which expresses human AhR and Arnt, and carries a lacZ reporter plasmid containing the aryl hydrocarbon response element (XRE) system (29), were provided by Dr. C. A. Miller, III (Tulane University, New Orleans, LA). The assay procedure was described in elsewhere (21). In the YCM3 cell assay system, the intensity of the AhRassociated toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was 60-fold higher than that of BaP (30). On the basis of these relative potency values, we estimated toxic equivalents (TEQs) concentrations of a mixture of halogenated PAHs/PAHs using the following equation: TEQ )
∑ [C ] × REP i
BaP,i /60
(1)
where Ci represents the concentration of an individual halogenated PAH/PAH.
Results Ambient Levels of BrPAHs, ClPAHs, and PAHs. Measurements of the targeted 11 BrPAHs, 15 ClPAHs, and 5 PAHs were performed in the particulate phase in urban air (Shizuoka, Japan) between September 2005 and June 2006. The detection rates, mean concentrations and standard deviation, the maximum and minimum concentrations, and the detection limits (S/N ) 3) for the target compounds are summarized in Table 1. The concentrations of the target compounds in each sampling term are represented in Table S2 of the Supporting Information. Seven of the 11 targeted BrPAHs were detected over the sampling term, whereas 7-BrBaA, 7,12-Br2BaA, and 4,7Br2BaA were seldom detected with a detection rate
