Atmospheric Environment 54 (2012) 9e17

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Atmospheric Environment
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Evaluation of ginkgo as a biomonitor of airborne polycyclic aromatic hydrocarbons

Michio Murakami a, Maho Abe b, Yoriko Kakumoto b, Hiromi Kawano b, Hiroko Fukasawa b, Mahua Saha b, Hideshige Takada b, *
a Wisdom of Water (Suntory), Corporate Sponsored Research Program, Organization for Interdisciplinary Research Projects, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan b Laboratory of Organic Geochemistry (LOG), Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

a r t i c l e i n f o
Article history: Received 8 September 2011 Received in revised form 21 January 2012 Accepted 4 February 2012 Keywords: Diesel emission PAHs Roadside Temporal variation Tissue distribution Tokyo

a b s t r a c t
The utility of ginkgo leaves as biomonitors of airborne polycyclic aromatic hydrocarbons (PAHs) was evaluated. We investigated PAH concentrations among tree species, the effect of variations in leaf position in a tree, tissue distributions, correlations between ginkgo leaves and air, and seasonal variations. Among the ve species examined (Ginkgo biloba L., Zelkova serrata Makino, Liriodendron tulipifera L., Prunus yedoensis Matsum, and Magnolia kobus DC.), ginkgo accumulated the greatest amount of PAHs from roadside air. Most PAHs (w80%) were accumulated in the wax fraction, and most of the remainder (17%) penetrated the inner tissues of the leaves. PAH concentrations in ginkgo leaves decreased with increasing height and distance from the road, reecting the derivation of PAHs from vehicle emissions. Seasonal time-series sampling showed that PAH concentrations in ginkgo leaves increased with time, attributable to the effects of temperature and accumulation through long-term exposure. Concentrations in ginkgo leaves collected from various roads showed a strong and signicant correlation with those in air collected by a high-volume air sampler (r2 0.68, P < 0.01). Ginkgo leaf data clearly showed a dramatic decrease in the ratio of low-molecular-weight (LMW) PAHs to high-molecular-weight PAHs from 2001 or 2002 to 2006, indicating that on-road diesel emission regulations effectively reduced LMW PAH concentrations in air. 2012 Elsevier Ltd. All rights reserved.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are the product of thermal decomposition, and are formed during incomplete combustion of organic materials and geochemical formation of fossil fuels (Blumer, 1976). The distribution of PAHs is diffuse in air and on the earths surface (Murakami et al., 2005; Boonyatumanond et al., 2007). The importance of the study of PAHs lies in the fact that they are highly lipophilic and some are carcinogenic or mutagenic (Perera et al., 1992; IARC, 2011). Collins et al. (1998), for example, developed potency equivalency factors for PAHs, and estimated that they posed a cancer risk of 1.8 105 in the UK. Wickramasinghe et al. (2011) found PAH concentrations around 696 ng m3 in air at heavily trafcked urban locations in Sri Lanka, posing an excess lifetime lung cancer risk of 4.58 103. Since PAH contamination is much heavier in urban areas than in

* Corresponding author. Tel.: 81 423 67 5825; fax: 81 423 60 8264. E-mail address: shige@cc.tuat.ac.jp (H. Takada). 1352-2310/$ e see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2012.02.014

rural areas (Saha et al., 2009; Wickramasinghe et al., 2011), the increased risk to health posed by PAHs, due to rapid urbanization, is a matter of concern. Since trafc is the dominant source of PAHs in the urban atmosphere (Dickhut et al., 2000; Tang et al., 2005; Boonyatumanond et al., 2007), countermeasures aimed at trafcderived PAHs should reduce concentrations and risk. Okuda et al. (2011) monitored PAHs using an active air sampler during the 2008 Beijing Olympics, and found that trafc-control measures reduced PAHs with molecular weight ranging from 252 to 300. Since PAH concentrations in air uctuate daily and seasonally (Fon et al., 2007) for many reasons, frequent monitoring is required to assess their pollution status. However, resource limitations restrict intensive data monitoring. As an alternative, biomonitoring using vegetation is a prospective approach, with the following advantages: (1) it is easy, economical, and non-technical to sample vegetation; and (2) vegetation can reect accumulation through long-term exposure. Simonich and Hites (1994a) found that vegetation signicantly accumulated PAHs. The accumulation mechanisms of PAHs in

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vegetation, and their controlling factors, have been studied as follows. It was demonstrated that vegetation takes up semivolatile organic compounds such as PAHs mostly from air, not soil, and the air quality can be projected (Ryan et al., 1988; McLachlan, 1999; StAmand et al., 2009). In addition, the partitioning of PAHs between air and vegetation (Simonich and Hites, 1994b; Tao and Hornbuckle, 2001), and its temporal change (Howsam et al., 2001; Alfani et al., 2005; Lehndorff and Schwark, 2009a); the varying distribution in tissue (Kaupp et al., 2000; Wild et al., 2006; De Nicola et al., 2008); the uptake pathways of PAHs (e.g., outer cuticle and stomatal pathways) (Franzaring, 1997; Tao and Hornbuckle, 2001); and photolysis on the leaf surface (Niu et al., 2003; Wang et al., 2005); have all been studied. Furthermore, differences in PAH accumulation among different tree species have been investigated (Howsam et al., 2000; Wang et al., 2008). Biomonitoring of PAHs, using plant leaves, has been conducted in order to understand spatial distribution (Lehndorff and Schwark, 2004; Wang et al., 2008; Lehndorff and Schwark, 2009b; Ratola et al., 2010; De Nicola et al., 2011), the impact of specic anthropogenic activities (Sharma and Tripathi, 2009; Sojinu et al., 2010), and seasonal variations (Prajapati and Tripathi, 2008). Though some studies have demonstrated the impact of trafc on airborne PAHs in urban areas (Lehndorff and Schwark, 2004; De Nicola et al., 2011), very few studies have utilized roadside trees to monitor PAHs in the air near heavily trafcked streets (Wang et al., 2008). The present study focused on roadside trees as biomonitors of PAHs in roadside air. Because roadside air is profoundly affected by trafc-derived PAHs, air monitoring is important in assessing human exposure to the respective carcinogenic compounds. We selected ginkgo (Ginkgo biloba L.) as a target species, because ginkgo is a common roadside tree in east Asia, and is grown around the world between 20 and 60 N and between 20 and 50 S (Sogame, 1984). The rst objective of the present study was to assess the utility of ginkgo as a biomonitor of PAHs in roadside air, and to optimize the monitoring protocol. In pursuit of this objective we focused on the following elements: (1) PAH accumulation in ginkgo leaves, in comparison to other common species of roadside trees, (2) PAH distribution among the gingko tissues, (3) variation in PAH accumulation as a function of leaf position in the ginkgo tree, (4) the relationship between PAHs in ginkgo tissue and in the ambient air, (5) seasonal patterns of PAH accumulation in gingko leaves. All these are important factors in optimizing PAH air monitoring using tree
Table 1 Sampling information. Sampling stations St. 1 St. 2 St. 3 St. 4 St. 5 St. 6 St. 7 St. 8 St. 9 St. 10 St. 11 St. St. St. St. 12 13 14 15 Location Fuchu Fuchu Fuchu (Tokyo University of Agriculture and Technology) Hachioji Daidabashi Hatsudai Showa-joshi Kamiuma Sengakuji Umeshima Itabashi-honcho Senju-shinbashi Kameari Kakinokizaka Takao Street Tokyo Route 133 Tokyo Route 14 e e Route 20 and Expressway 4 Route 20 and Expressway 4 Route 246 and Expressway 3 Route 246 and Expressway 3 Route 15 Route 4 Route 17 and Expressway 5 Route 4 Loop 7 Loop 7 Route 20

leaves. However, no previous studies have examined all these aspects for a certain species of tree. The second objective of the present study was to evaluate the effects of the on-road diesel emission regulations introduced by the Tokyo Metropolitan Government in October 2003, through longterm (ve-year) monitoring using ginkgo trees. Diesel vehicles that do not meet the new particulate matter (PM) standard can be replaced or retrotted with a particulate control device (Tokyo Metropolitan Government, 2006; Rutherford and Ortolano, 2008). Although the effects of the regulations on black carbon, PM, and oxides of nitrogen were reported (Kondo et al., 2006; Rutherford and Ortolano, 2008), the effects on PAHs are not well understood. Thus, we collected annual time-series samples of ginkgo leaves for ve years to compare PAH concentrations before and after the regulations. This is the rst application of long-term biomonitoring of PAHs using tree leaves. 2. Materials and methods 2.1. Sampling Six types of sampling procedure were conducted as follows. 2.1.1. Comparison among tree species To compare PAH concentrations among tree species, we sampled leaves from ve species of deciduous treedginkgo (Ginkgo biloba L.), Zelkova (Zelkova serrata Makino), tulip tree (Liriodendron tulipifera L.), cherry (Prunus yedoensis Matsum.), and Kobushi Magnolia (Magnolia kobus DC.)dlocated either on the walkway or the roadway at Station (St.) 1 (Fuchu on Tokyo Route 133; Table 1) in August 2000. 2.1.2. Tissue distribution We examined the tissue distribution (i.e., water-washed, wax, and interior fractions) of PAHs in ginkgo leaves collected from the roadway at St. 1 in October 2000. 2.1.3. Effect of leaf position in a ginkgo tree To examine the effect of the leaf position on PAH concentrations, we collected ginkgo leaves at 2, 3, 4, and 8 m above the walkway, and at 2, 4, and 6 m above the roadway, at St. 2 (Fuchu on Tokyo Route 133; Table 1) in August 2002. The road has six lanes and the

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Trafc density in 1999 (vehicles/12-h) 14,042 26,588 0 0 43,519 61,178 (104,697) 43,519 61,178 (104,697) 44,548 54,620 (84,483) 29,863 54,620 (84,483) 44,489 36,527 46,863 65,791 (112,654) 48,170 31,917 42,205 5588

Trafc density in 2005 (vehicles/12-h) 12,337 24,224 0 0 38,684 58,012 (96,696) 38,684 58,012 (96,696) 40,441 52,184 (92,625) 26,304 52,184 (78,488) 42,898 34,008 46,093 56,095 (102,188) 44,116 33,539 40,642 5657

Heavy vehicle density in 1999 (vehicles/12-h) 2598 3483 0 0 3569 10,094 (13,663) 3569 10,094 (13,663) 6727 15,075 (21,802) 4927 15,075 (20,002) 7118 6977 7826 14,540 (22,366) 8237 9639 9581 782

Heavy vehicle density in 2005 (vehicles/12-h) 2048 2931 0 0 4797 10,036 (14,833) 4797 10,036 (14,833) 6268 13,777 (20,045) 4314 13,777 (18,091) 6306 6428 8988 14,473 (23,461) 8691 9056 8901 639

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tree stood just beside the edge of road, on the same level as the road, with no obstacles to limit air circulation. 2.1.4. Relationship between PAHs in ginkgo tissue and in ambient air To investigate the relationship between PAH concentrations in ginkgo leaves and air samples, we collected samples from St. 3 to St. 6 in October 2001, at St. 7 in September 2002, and from St. 8 to St. 14 in November 2002 (Table 1). A high-volume air sampler (TFIA-2, Staplex, USA) was installed at 1 m above the ground, following Boonyatumanond et al. (2007). The sampler ran at 700 L min1 for 24 h. Particulate samples (>0.3 mm) were trapped on a prebaked quartz ber lter (Palex membrane lter 2500QAT-UP, 200 mm 250 mm). Polyurethane foam (PUF; Shibata 8013-0941A polyurethane for dioxin, 5 cm 6 cm i.d.) was inserted behind the lter to collect the gaseous PAHs. 2.1.5. Seasonal variation We also collected seasonal time-series of ginkgo leaves and air samples at St. 3 (in Fuchu campus of Tokyo University of Agric. & Technol.) from June to November 2001. Sampling methods were the same as those in 2.1.4. 2.1.6. Annual time-series samples Annual time-series samples of ginkgo leaves were collected at St. 3, St. 6, and St. 8 to St. 15 (Table 1), from 2001 to 2006, to evaluate the effects of the diesel emission regulations on air quality. Information on trafc densities was obtained from the Japan Society of Trafc Engineers (2001, 2006). For all the procedures, leaves were collected with a pole pruner at 2e4 m above the ground near a roadway, unless specically stated (2.1.3). Air temperature was recorded at the same time. Collected lters were dried in a desiccator. The leaves, dried lters, and PUF samples were stored at 30  C in a freezer until PAH analysis. 2.2. PAH analysis

by two-step silica gel column chromatography with a 5% H2Odeactivated silica gel column and a fully activated silica gel column, as described in Zakaria et al. (2002). 2.2.2. Tissue distribution: successive extraction PAHs in ginkgo leaves were separated into water-washed, wax, and interior fractions. Five to ten sheets of leaves (w5 g) were stirred in 1 L of distilled water at 20  C for 10 min, 3 times. The water was ltered through a glass ber lter (GF/F, Whatman). The ltrate was then passed through an ODS cartridge (Sep-Pak Plus tC18; Millipore) that had been preconditioned with n-hexane followed by DCM, MeOH, and distilled water. The PAHs were eluted with 30 mL of DCM. The glass lters were Soxhlet-extracted with DCM for w12 h. The extracts from the ltrates and glass lters were dehydrated and combined as the water-washed fraction. The leaves were then air-dried and extracted in 400 mL of n-hexane/ DCM (1:1 v/v) by ultrasonication for 2 h, and dehydrated with anhydrous sodium sulfate to yield the wax fraction. The ultrasonic-extracted leaves were further extracted by the Polytron homogenizer, as above, to yield the interior fraction. All the extracts were spiked with the surrogate standards, concentrated, and puried by the two-step silica gel column chromatography, as above. 2.2.3. Ultrasonic extraction For the procedures described in 2.1.3 (Effect of leaf position), 2.1.4 (Comparison with airborne PAHs), and 2.1.5 (seasonal variation), ultrasonic extraction was employed. A weighted (w5 g) ginkgo leaf was extracted in 400 mL of n-hexane/DCM by ultrasonication for 2 h, without pre-wash by water. The extract was dehydrated with anhydrous sodium sulfate, spiked with the surrogate standards, and puried by the aforementioned two-step silica gel column chromatography and open-top gel permeation chromatography. 2.2.4. Pressurized Fluid Extraction (PFE) P.Chi Annual time-series samples of ginkgo leaves (w4 g) were freeze-dried and extracted by PFE with DCM at 175  C in an accelerated solvent extraction system (ASE 200; Dionex). These extraction parameters had been optimized previously, as described in Supplementary Materials. The extracts were spiked with the surrogate standards, and puried by hydrolysis. Extracts were placed in a glass ampoule, and the solvent was evaporated under a nitrogen gas stream. To the ampoule was added 1 M KOH in MeOH, then the ampoule was sealed, and the samples were alkaline-digested at 80  C for 2 h. Following the addition of water and hexane to the digested sample, the PAH fraction was partitioned between hexane and water/MeOH into the hexane layer. The PAH fraction was puried by two-step silica gel column chromatography and open-top gel permeation chromatography, as above. 2.2.5. Air samples PUFs and lters were Soxhlet-extracted with DCM for w12 h. The extracts were spiked with the surrogate standards, concentrated, and puried by two-step silica gel column chromato graphy, as above. 2.2.6. GC-MS measurements All the puried PAH fractions were concentrated and injected into the GC-MS (Hewlett Packard 5890 Series II, with a 5972A detector). Nineteen PAH species with 3 to 7 ringsdphenanthrene (Phe), anthracene (Anth), 3-methylphenanthrene (3-MP), 2methylphenanthrene (2-MP), 9-methylphenanthrene (9-MP), 1methylphenanthrene (1-MP), uoranthene (Fluo), pyrene (Py), benz(a)anthracene (B(a)A), chrysene (Chry), benzo(b)uoranthene

M.Chi

PAHs were measured with a gas chromatograph equipped with mass spectrometer (GC-MS), utilizing two-step silica gel column chromatography for all the procedures, though different extraction methods (i.e., polytron, sonication or pressurized uid extraction) as well as optional purication (i.e., open-top gel permeation chromatography or hydrolysis) were employed depending on the procedure. The extraction and purication techniques for the individual procedures are described below. 2.2.1. Comparison among tree species: polytron extraction Leaf samples (w5 g) for comparison among species were extracted with 120 mL of dichloromethane (DCM) and 30 g of anhydrous sodium sulfate, in a Polytron RT2000 homogenizer (Kinematica). The DCM fraction was isolated by centrifugation at 740 g for 10 min, and dehydrated with anhydrous sodium sulfate. The extraction was repeated twice, and then all the DCM extracts were combined and spiked with 50 mL of surrogate standards (5 ppm each of anthracene-d10, p-terphenyl-d14, benzo(a)anthracene-d12, and perylene-d12). The extracts were concentrated to 4 mL and centrifuged at 740 g for 5 min. The supernatants were decanted through quartz wool to remove solids, then concentrated, dissolved in 0.5 mL of MeOH/DCM (1:1 v/v), and puried by opentop gel permeation chromatography with Sephadex LH-20 beads (Amersham Pharmacia Biotech AB). The rst 12.5 mL of MeOH/ DCM (1:1 v/v) was discarded, and the following 10 mL was collected as the PAH fraction. The PAH fractions were concentrated, dissolved in 1 mL of n-hexane/DCM (1:1 v/v), and further puried

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(B(b)F), benzo(j)uoranthene benzo(k)uoranthene (B(j/k)F), benzo(e)pyrene (B(e)P), benzo(a)pyrene (B(a)P), perylene (Pery), indeno(1,2,3-cd)pyrene (IndP), benzo(ghi)perylene (B(ghi)P), and coronene (Coro)dwere determined in selected-ion monitoring mode by the GC-MS, as described in Zakaria et al. (2001). 2.2.7. Quality assurance and quality control The concentrations of PAHs were corrected by recoveries of the surrogate standards spiked immediately after extraction. The recovery rate of the surrogate standards was more than 65% for all the procedures. The reproducibility of the extraction methods, and the associated purication technique used in the individual procedures, were examined by replicate analysis of the gingko samples in advance of the analysis in each procedure. The relative standard deviations of individual PAHs in the sample extracts were <17% (mostly <10%) by ultrasonic extraction (n 4), PFE extraction (n 4), Polytron extraction (n 6), and Soxhlet extraction (n 3). Because the reproducibility had been previously conrmed, essentially a single analysis was conducted for each sample, unless specically stated. Lipid contents were gravimetrically determined by taking aliquots of the solvent extracts. Different extraction methods were employed in the analysis of the different procedures. However, comparison in Results and discussion section was made among the samples within each procedure, and therefore the differences among these extraction methods do not affect the discussion. To be certain, we compared the respective extraction efciencies of the ultrasonic extraction and the PFE, and found no signicant difference (<10%) between the amounts of extracted PAHs (fresh-weight basis) in either case. 2.3. Statistical analysis

elapsed time, and between the partition coefcient KL (the ratio of P the PAH concentration in the ginkgo leaf [ng mg-lipid1] to that in air [ng m3]) and the reciprocal of temperature. The t-test was used to investigate the differences in log KL and the reciprocal of temperature between June and September. The paired t-test was used to investigate the difference in the densities of heavy vehicles between 1999 and 2005. The paired t-test with Bonferronis correction was used to investigate the differences in PAH concentrations and L/H ratios (ratios of low-molecular-weight (LMW) PAH (i.e., PheePy) to high-molecular-weight (HMW) PAH (i.e., B(a) AeCoro) concentrations) over the years. IBM SPSS Statistics 19 software was used for the statistical analysis. 3. Results and discussion 3.1. Comparison of PAH concentrations among tree species Fig. 1 compares PAH concentrations in the leaves of the ve tree P species studied. PAH concentrations in ginkgo (on a dry-weight basis) were signicantly higher than those in the other species (P < 0.05; Fig. 1a). All the examined species are deciduous trees whose leaves begin to grow in the spring, and thus they are all exposed to airborne PAHs for roughly the same period of time. Since it was reported that PAHs are preferentially partitioned into lipids (Simonich and Hites, 1994b), differences in lipid content among the species were examined. The lipid content in ginkgo (1.63 mg g-dry1) was less than that in the other species, which ranged from 3.16 mg g-dry1 to 6.05 mg g-dry1 (Table S1). On a lipid-weight basis, the PAH concentration in the gingko leaves was 5e39-times greater than in the others leaves (Fig. 1b), suggesting that not only lipid content but also other factors control the PAH concentration in the tree leaves. Projected-area-based PAH concentrations were also highest for gingko (Fig. 1c). Wang et al. (2008) studied the accumulation of PAHs in six species of roadside tree (ginkgo, peach, Japanese pagodatree, purple leaf plum, Pekin lilac, and green spire) in Beijing, and the second-highest accumulation of PAHs was observed for ginkgo. Similarly, differences in leaf PAH concentrations were observed for three deciduous tree species (oak, ash and hazel), and surface structure (i.e., pubescence) was suggested as an important factor in controlling

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An analysis of variance (ANOVA) was employed to assess the P variation in PAH concentrations among tree species. Group differences were investigated by Tukeys test. Pearson correlations were estimated to evaluate the relationship between PAH concentrations in the ginkgo leaves and in air, and between the P PAH concentrations in the ginkgo leaves and respective trafc densities. Spearmans rank correlation coefcients were estimated P to assess the relationship between the PAH concentrations and

a
PAHs (ng/g-dry)

200

b
a

150

c
a

10 8

b
100

100

PAHs (ng/cm2)

150

PAHs (ng/mg-lipid)

a
6 4 2

50

50

Ginkgo biloba L.

Liriodendron tulipifera L.

Prunus yedoensis Matsum

P Fig. 1. Comparison of PAH concentrations among tree species. (a) Concentrations per dry weights of leaves, (b) concentrations per lipid weights of leaves, (c) concentrations per projected areas of leaves. Values are averages of those from the walkway and the roadway for individual species. Error bars represent the variations between those from the walkway and the roadway. Different letters indicate a signicant difference among tree species (P < 0.05).

Prunus yedoensis Matsum

Prunus yedoensis Matsum

Liriodendron tulipifera L.

Liriodendron tulipifera L.

Zelkova serrata Makino

Zelkova serrata Makino

Zelkova serrata Makino

Magnolia kobus DC.

Magnolia kobus DC.

Magnolia kobus DC.

Ginkgo biloba L.

Ginkgo biloba L.

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PAH concentrations in leaves (Howsam et al., 2000). The greater accumulation in the case of ginkgo may be attributable to the wavywalled surface structure of ginkgo leaves, which enlarges the surface area and traps particles (Fig. S3). These results show that ginkgo accumulates far more PAHs than the other species, rendering it a useful tool for roadside biomonitoring of PAHs. 3.2. Tissue distribution of PAHs in leaves

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Gas-phase PAHs are taken up by leaves via two pathways: (1) partition onto outer cuticular wax (outer cuticle pathway), and (2) mass-transfer to inner tissue through stomatal pores (stomatal pathway) (Franzaring, 1997; Tao and Hornbuckle, 2001). Particulate PAHs are also trapped on the outer cuticle, while ne particles may pass through the stomata. Successive extraction provided some insights into the pathways of PAH accumulation in ginkgo leaves. As shown in Fig. 2a, water-washed, wax, and interior fractions accounted for 3.7%, 80%, and 17%, respectively, of the P PAHs. The smaller contribution of the water-washed fraction is consistent with the results of other studies (Kaupp et al., 2000; De Nicola et al., 2008), which suggested migration (or diffusion) of PAHs from the trapped particles to cuticular wax. The presence of HMW PAHs in the wax fraction supports the assumption of their desorption from the particles and migration into wax. LMW PAHs were dominant in the gas phase (Fig. 2b), as reported previously (Possanzini et al., 2004; Araki et al., 2009). LMW PAHs in the wax fraction are explained by partitioning of gas-phase PAHs to cuticular wax. However, HMW PAHs were exclusively present in the particulate phase, and therefore the HMW in the wax fraction cannot be accounted for by gas-phase PAHs, but by diffusion of HMW PAHs from trapped particles to cuticular wax. Among HMW PAHs, compounds with relatively higher molecular weight (e.g., coronene) were distributed more in water-washed fraction than those with relatively lower molecular weight (e.g.,

benzouoranthenens), despite a similarity in the distribution of HMW PAHs in the particulate phase in air samples. It is likely that the preferential migration of PAHs from particles to the wax fraction depends on hydrophobicity or molecular size, owing to differences in the types of organic matter found in the particles and the wax. Less hydrophobic PAHs (i.e., lower molecular weight compounds) are considered to migrate preferentially to the wax fraction, whereas more hydrophobic PAHs (i.e., higher molecular weight compounds) preferentially remain in the particulate phase. The smaller proportion of the interior fraction indicates a lesser contribution from the stomatal pathway than the outer cuticular pathway. This is not consistent with the model calculations by Tao and Hornbuckle (2001). However, the smaller proportion of the interior fraction was observed for the roadside trees in Beijing (Wang et al., 2008). The proportion of HMW PAHs was smaller than that of LMW PAHs, suggesting that the uptake of ne particles containing HMW PAHs through stomata is less important. In summary, successive extraction indicates the importance of the outer cuticular pathway for gaseous and particulate PAHs, and for migration of HMW PAHs to cuticular wax. Consequently, cuticular wax was suggested as an important fraction for the monitoring of PAHs. Thus, our later investigations focused on the wax fractions, together with the water-washed fraction. 3.3. Effects of leaf position in the tree on PAH accumulation in leaves P On the side of the roadway, the PAH concentrations in ginkgo leaves were the highest at 2 m above the ground, and decreased, from 69 ng mg-lipid1 to 42 ng mg-lipid1, with increasing height (Fig. 3), reecting the derivation of PAHs from vehicle emissions. This result is consistent with a previous report of decreasing PAHs P in air with increasing height (Tao et al., 2007). PAH concentrations in the walkway samples also tended to decrease, from 49 ng mg-lipid1 to 37 ng mg-lipid1, with increasing height. P PAH concentrations in ginkgo leaves from the roadway tended to be higher than those from the walkway, which is consistent with a previous nding that PAH concentrations decreased with increasing distance from a road (Yang et al., 1991). In the present study, ginkgo leaves from walkways were used to evaluate pedestrian exposure to PAHs in air.

80 70

PAHs (ng/mg-lipid)

60 50 40 30 20 10 0 2m 4m 4m 8m 2m 4m 3m 6m

Walkway
Fig. 2. Distributions of PAHs in (a) leaves and (b) air. Fig. 3. Comparison of position.

Roadway

P PAH concentrations in ginkgo leaves among different leaf

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a
PAHs in air (ng/m3)

HMW PAHs in particulate phase (ng/m3)

r2 = 0.68 P < 0.01

LMW PAHs in gas phase (ng/m3)

200

200

c
r2 = 0.66 P < 0.01

20

r2 = 0.52 P < 0.01

15

150

150

100

100

10

50

50

0 0 50 100 150

0 0 40 80 120 LMW PAHs in ginkgo (ng/mg-lipid)

0 0 10 20 30 40 50 60

PAHs in ginkgo (ng/mg-lipid)

HMW PAHs in ginkgo (ng/mg-lipid)

d
2

e
LMW PAHs in particulate phase (ng/m3)
r2 = 0.16 P = 0.2
1.5

40
r2 = 0.43 P < 0.05

HMW PAHs in gas phase (ng/m3)

30

20

0.5

10

0 0 10 20 30 40 50 60

HMW PAHs in ginkgo (ng/mg-lipid)

0 40 80 120 LMW PAHs in ginkgo (ng/mg-lipid)

P Fig. 4. Relationship between PAH concentrations in ginkgo leaves and air. (a) PAHs in ginkgo leaves and air. (b) LMW PAHs in ginkgo leaves and the gas phase. (c) HMW PAHs in ginkgo leaves and the particulate phase. (d) HMW PAHs in ginkgo leaves and the gas phase. (e) LMW PAHs in ginkgo leaves and the particulate phase.

Fig. 5. Seasonal changes in (a)

P P PAHs and L/H ratios in ginkgo leaves, (b) PAHs and L/H ratios in air, and (c) temperature.

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3.4. Relationship between PAH concentrations in ginkgo leaves and air P Generally, PAH concentrations in ginkgo leaves collected from various roads showed a strong and signicant correlation with those in air collected by a high-volume air sampler (r2 0.68, P < 0.01; Fig. 4a). This shows that ginkgo reects PAH pollution in air and is useful as a biomonitoring tool. LMW PAHs in ginkgo leaves were strongly and signicantly correlated with those in the gas phase (r2 0.66, P < 0.01; Fig. 4b), whereas HMW PAHs in ginkgo leaves were correlated with those in the particulate phase (r2 0.52, P < 0.01; Fig. 4c). However, HMW PAHs in the gas phase were not correlated with HMW PAHs in ginkgo leaves (Fig. 4d). The weak but signicant correlation between LMW PAHs in ginkgo leaves and in the particulate phase (r2 0.43, P < 0.05; Fig. 4e) was attributed to partitioning of LMW PAHs from the gas phase to both the ginkgo leaves and the particulate phase. This result is consistent with the above nding that HMW PAHs in ginkgo leaves are attributable to particles. Similar correlations were observed in the studies in Beijing (Wang et al., 2008). 3.5. Seasonal variations in PAHs in ginkgo leaves and air P PAH concentrations in ginkgo leaves signicantly increased with time (Spearmans rank correlation coefcient r 0.87,

P < 0.01; Fig. 5a). On the other hand, there was no clear temporal P trend in PAH concentrations in air (r 0.04, P > 0.20; Fig. 5b); nor P were clear temporal trends observed in PAH concentrations in the gas phase (r 0.23, P > 0.20) or the particulate phase (r 0.26, P P > 0.15) (Fig. S4a, b). Temporal change in PAH concentrations in ginkgo leaves showed a mirror image of air temperature (Fig. 5c). Simonich and Hites (1994b) found that the partitioning of PAHs between air and tree leaves depends on the reciprocal of temperature (expressed as 1000/T). The partition coefcient KL was signicantly correlated with 1000/T (r 0.59, P < 0.01; Fig. S5), indicating that the temporal increase in PAH concentrations in ginkgo is attributable to the decrease in temperature. However, log KL in September was signicantly higher than that in June (0.45 vs 0.084; P < 0.01), whereas there was no signicant difference in 1000/T between the two (both 3.4 K1; P > 0.20). In addition to the effect of temperature, the temporal increase in PAH concentrations in ginkgo is attributable to accumulation through long-term exposure (from spring until leaf fall).

3.6. Secular trends in PAH concentrations in ginkgo, and evaluation of on-road diesel emission regulations

Hoa

P PAH concentrations in ginkgo leaves were strongly and signicantly correlated with trafc densities (r2 0.71, P < 0.01;

a
PAHs in ginkgo (ng/mg-lipid)

30

LMW PAHs in ginkgo (ng/mg-lipid)

2001/ 2003 2004 2005 2006 2002 Sep. Oct. Oct. Oct. Nov.

40

30

20

20

10

10

2001/ 2003 2004 2005 2006 2002 Sep. Oct. Oct. Oct. Nov.

c
HMW PAHs in ginkgo (ng/mg-lipid)

20

4 St. 3 St. 6 St. 8 2 St. 9 St. 10 1 St. 11 St. 12

10

L/H ratio in ginkgo

15

0
2001/ 2003 2004 2005 2006 2002 Sep. Oct. Oct. Oct. Nov.

2001/ 2003 2004 2005 2006 2002 Sep. Oct. Oct. Oct. Nov.

St. 13 St. 14 St. 15

Fig. 6. Secular changes in PAH concentrations and L/H ratios in ginkgo leaves. (a)

P PAHs, (b) LMW PAHs, (c) HMW PAHs, (d) L/H ratios.

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M. Murakami et al. / Atmospheric Environment 54 (2012) 9e17

Fig. S6), conrming that ginkgo is a useful biomonitoring tool reecting the accumulation of PAHs derived from trafc. There was no signicant difference in the densities of heavy vehicles between 1999 and 2005 (Table 1; P > 0.20). On the other hand, the number of diesel vehicles that did not meet the regulations in Tokyo decreased from 202,000 in March 2002 to 135,000 in March 2003, 44,000 in September 2004, and 7000 in December 2005 (Tokyo P Metropolitan Government, 2006). PAH concentrations in ginkgo leaves decreased signicantly from 2001 or 2002 to 2003e2006 (Fig. 6a; P < 0.01 for 2001e02 vs. 2004 and 2006; P < 0.05 for 2001e02 vs. 2003 and 2005). LMW PAH concentrations also decreased signicantly from 2001 or 2002 to 2003e2006 (Fig. 6b; P < 0.01 for 2001e02 vs. 2004 and 2006; P < 0.05 for 2001e02 vs. 2003 and 2005), whereas HMW PAH concentrations did not (Fig. 6c; P 0.09 for 2001e02 vs. 2003; P > 0.20 for 2001e02 vs. 2004e2006). LMW PAHs are abundant in diesel vehicle exhaust particles, whereas HMW PAHs are abundant in gasoline vehicle exhaust particles (Rogge et al., 1993; Okuda et al., 2003; Boonyatumanond et al., 2007). Since PAH concentrations can be affected by time of year and monthly temperature, which differed over the years (especially 2003; Table S2), we used L/H ratios to reduce the effects of accumulation and temperature in investigating the change of load from diesel vehicle exhaust particles from 2001e02 to 2006. By year, the L/H ratios decreased signicantly from 2001e02 to 2005e2006 (Fig. 6d; P > 0.20 for 2001e02 vs. 2003; P 0.07 for 2001e02 vs. 2004; P < 0.01 for 2001e02 vs. 2005e2006). The average L/H ratio in ginkgo leaves in 2006 was 1.3, less than half that in 2001e02 (2.8). This result shows that the on-road diesel emission regulations effectively reduced LMW PAH concentrations in air. Leaves were collected in different months in different years, though in the same season (autumn). However, seasonal time-series sampling demonstrated that the L/H ratios remained nearly constant throughout the seasons (Fig. 5a). Thus, the temporal decreasing trend in the L/H ratio is signicant and gives rm evidence of a reduction in LMW PAHs. 4. Conclusions Among ve typical roadside trees (Ginkgo biloba L., Zelkova serrata Makino, Liriodendron tulipifera L., Prunus yedoensis Matsum, and Magnolia kobus DC.) in Tokyo, ginkgo accumulated the greatest amount of PAHs. PAH concentrations in ginkgo leaves decreased with increasing height above ground and distance from the road, reecting the derivation of PAHs from vehicle emissions. Approximately 80% of PAHs were present in the wax fraction of the ginkgo leaves. The distribution of the water-washed fraction increased with increasing molecular weight, because HMW PAHs, which derive from the particulate fraction, are preferentially retained in the P particles. There was a strong correlation between the PAH concentrations in the ginkgo leaves and in air. LMW PAHs in ginkgo leaves were correlated with those in the gas phase, whereas HMW PAHs were correlated with those in the particulate phase. PAH concentrations in ginkgo leaves increased from spring to autumn. This increase is attributable to the effects of temperature and to accumulation through long-term exposure. Analysis of annual timeseries ginkgo samples showed that the on-road diesel emission regulations effectively reduced LMW PAH concentrations in air; however, the regulations were not effective enough to reduce HMW PAHs. Because HMW PAHs are more important in terms of carcinogenicity, identication of the sources of airborne HMW PAHs, as well as their regulation, are requisite elements of future initiatives. Acknowledgements We wish to express special thanks to Ms. Kakurai for her arrangement of the sampling. We thank Dr. Takeshi Izuta for his

comments on this study. This research was supported by the Environment Research and Technology Development Fund (B0904) of the Ministry of the Environment, Japan, and Nippon Life Insurance Foundation. Several graduates and undergraduates in LOG provided welcomed assistance with the eldwork. Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atmosenv.2012.02.014. References
Alfani, A., De Nicola, F., Maisto, G., Prati, M.V., 2005. Long-term PAH accumulation after bud break in Quercus ilex L. leaves in a polluted environment. Atmos. Environ. 39, 307e314. Araki, Y., Tang, N., Ohno, M., Kameda, T., Toriba, A., Hayakawa, K., 2009. Analysis of atmospheric polycyclic aromatic hydrocarbons and nitropolycyclic aromatic hydrocarbons in gas/particle phases separately collected by a high-volume air sampler equipped with a column packed with XAD-4 resin. J. Health Sci. 55, 77e85. Blumer, M., 1976. Polycyclic aromatic compounds in nature. Sci. Am. 234, 34e45. Boonyatumanond, R., Murakami, M., Wattayakorn, G., Togo, A., Takada, H., 2007. Sources of polycyclic aromatic hydrocarbons (PAHs) in street dust in a tropical Asian mega-city, Bangkok, Thailand. Sci. Total Environ. 384, 420e432. Collins, J.F., Brown, J.P., Alexeeff, G.V., Salmon, A.G., 1998. Potency equivalency factors for some polycyclic aromatic hydrocarbons and polycyclic aromatic hydrocarbon derivatives. Regul. Toxicol. Pharm. 28, 45e54. De Nicola, F., Claudia, L., MariaVittoria, P., Giulia, M., Anna, A., 2011. Biomonitoring of PAHs by using Quercus ilex leaves: source diagnostic and toxicity assessment. Atmos. Environ. 45, 1428e1433. De Nicola, F., Maisto, G., Prati, M.V., Alfani, A., 2008. Leaf accumulation of trace elements and polycyclic aromatic hydrocarbons (PAHs) in Quercus ilex L. Environ. Pollut. 153, 376e383. Dickhut, R.M., Canuel, E.A., Gustafson, K.E., Liu, K., Arzayus, K.M., Walker, S.E., Edgecombe, G., Gaylor, M.O., MacDonald, E.H., 2000. Automotive sources of carcinogenic polycyclic aromatic hydrocarbons associated with particulate matter in the Chesapeake Bay region. Environ. Sci. Technol. 34, 4635e4640. Fon, T., Noriatsu, O., Hiroshi, S., 2007. Polycyclic aromatic hydrocarbons (PAHs) in the aerosol of Higashi Hiroshima, Japan: pollution scenario and source identication. Water Air Soil Pollut. 182, 235e243. Franzaring, J., 1997. Temperature and concentration effects in biomonitoring of organic air pollutants. Environ. Monit. Assess. 46, 209e220. Howsam, M., Jones, K.C., Ineson, P., 2000. PAHs associated with the leaves of three deciduous tree species. I e Concentrations and proles. Environ. Pollut. 108, 413e424. Howsam, M., Kevin, J.C., Philip, I., 2001. PAHs associated with the leaves of three deciduous tree species. II: uptake during a growing season. Chemosphere 44, 155e164. IARC, 2011. http://monographs.iarc.fr/ENG/Classication/index.php (accessed 19.08.11). Japan Society of Trafc Engineers, 2001. Road Trafc Census in FY1999 (in Japanese). Japan Society of Trafc Engineers, 2006. Road Trafc Census in FY2005 (in Japanese). Kaupp, H., Blumenstock, M., McLachlan, M.S., 2000. Retention and mobility of atmospheric particle-associated organic pollutant PCDD/Fs and PAHs in maize leaves. New Phytol. 148, 473e480. Kondo, Y., Komazaki, Y., Miyazaki, Y., Moteki, N., Takegawa, N., Kodama, D., Deguchi, S., Nogami, M., Fukuda, M., Miyakawa, T., Morino, Y., Koike, M., Sakurai, H., Ehara, K., 2006. Temporal variations of elemental carbon in Tokyo. J. Geophys. Res. Atmos. 111, D12205. Lehndorff, E., Schwark, L., 2009a. Biomonitoring airborne parent and alkylated three-ring PAHs in the Greater Cologne Conurbation I: Temporal accumulation patterns. Environ. Pollut. 157, 1323e1331. Lehndorff, E., Schwark, L., 2009b. Biomonitoring airborne parent and alkylated three-ring PAHs in the Greater Cologne Conurbation II: Regional distribution patterns. Environ. Pollut. 157, 1706e1713. Lehndorff, E., Schwark, L., 2004. Biomonitoring of air quality in the Cologne Conurbation using pine needles as a passive sampler-Part II: polycyclic aromatic hydrocarbons (PAH). Atmos. Environ 38, 3793e3808. McLachlan, M.S., 1999. Framework for the interpretation of measurements of SOCs in plants. Environ. Sci. Technol. 33, 1799e1804. Murakami, M., Nakajima, F., Furumai, H., 2005. Size- and density-distributions and sources of polycyclic aromatic hydrocarbons in urban road dust. Chemosphere 61, 783e791. Niu, J., Chen, J., Martens, D., Quan, X., Yang, F., Kettrup, A., Schramm, K.-W., 2003. Photolysis of polycyclic aromatic hydrocarbons adsorbed on spruce [Picea abies (L.) Karst.] needles under sunlight irradiation. Environ. Pollut. 123, 39e45. Okuda, T., Matsuura, S., Yamaguchi, D., Umemura, T., Hanada, E., Orihara, H., Tanaka, S., He, K., Ma, Y., Cheng, Y., Liang, L., 2011. The impact of the pollution control measures for the 2008 Beijing Olympic Games on the chemical composition of aerosols. Atmos. Environ. 45, 2789e2794.

M. Murakami et al. / Atmospheric Environment 54 (2012) 9e17 Okuda, T., Takada, H., Naraoka, H., 2003. Thermodynamic behavior of stable carbon isotopic compositions of individual polycyclic aromatic hydrocarbons derived from automobiles. Polycycl. Aromat. Comp. 23, 219e236. Perera, F.P., Hemminki, K., Gryzbowska, E., Motykiewicz, G., Michalska, J., Santella, R.M., Young, T.L., Dickey, C., Brandtrauf, P., Devivo, I., Blaner, W., Tsai, W.Y., Chorazy, M., 1992. Molecular and genetic-damage in humans from environmental-pollution in Poland. Nature 360, 256e258. Possanzini, M., Di Palo, V., Gigliucci, P., Scian, M.C.T., Cecinato, A., 2004. Determination of phase-distributed PAH in Rome ambient air by denuder/GC-MS method. Atmos. Environ. 38, 1727e1734. Prajapati, S.K., Tripathi, B.D., 2008. Biomonitoring seasonal variation of urban air polycyclic aromatic hydrocarbons (PAHs) using Ficus benghalensis leaves. Environ. Pollut. 151, 543e548. Ratola, N., Amigo, J., Alves, A., 2010. Levels and sources of PAHs in selected sites from Portugal: Biomonitoring with Pinus pinea and Pinus pinaster needles. Arch. Environ. Contam. Toxicol. 58, 631e647. Rogge, W.F., Hildemann, L.M., Mazurek, M.A., Cass, G.R., Simoneit, B.R.T., 1993. Sources of ne organic aerosol. 2. Noncatalyst and catalyst-equipped automobiles and heavy-duty trucks. Environ. Sci. Technol. 27, 636e651. Rutherford, D., Ortolano, L., 2008. Air quality impacts of Tokyos on-road diesel emission regulations. Transport Res. D-Tr. E 13, 239e254. Ryan, J.L., Bell, R.M., Davidson, J.M., OConnor, G.A., 1988. Plant uptake of non-ionic organic chemicals from soils. Chemosphere 17, 2299e2323. Saha, M., Togo, A., Mizukawa, K., Murakami, M., Takada, H., Zakaria, M.P., Chiem, N.H., Tuyen, B.C., Prudente, M., Boonyatumanond, R., Sarkar, S.K., Bhattacharya, B., Mishra, P., Tana, T.S., 2009. Sources of sedimentary PAHs in tropical Asian waters: differentiation between pyrogenic and petrogenic sources by alkyl homolog abundance. Mar. Pollut. Bull. 58, 189e200. Sharma, A., Tripathi, B., 2009. Assessment of atmospheric PAHs prole through Calotropis gigantea R.Br. leaves in the vicinity of an Indian coal-red power plant. Environ. Monit. Assess. 149, 477e482. Simonich, S.L., Hites, R.A., 1994a. Importance of vegetation in removing polycyclic aromatic hydrocarbons from the atmosphere. Nature 370, 49e51. Simonich, S.L., Hites, R.A., 1994b. Vegetation-atmosphere partitioning of polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 28, 939e943. Sogame, Y., 1984. A study on the distribution of ginkgo biloba in the world. J. Koshien Junior College 4, 1e14 (in Japanese). Sojinu, O.S., Sonibare, O.O., Ekundayo, O., Zeng, E.Y., 2010. Biomonitoring potentials of polycyclic aromatic hydrocarbons (PAHs) by higher plants from an oil exploration site, Nigeria. J. Hazard. Mater. 184, 759e764.

17

St-Amand, A.D., Mayer, P.M., Blais, J.M., 2009. Modeling PAH uptake by vegetation from the air using eld measurements. Atmos. Environ. 43, 4283e4288. Tang, N., Hattori, T., Taga, R., Igarashi, K., Yang, X., Tamura, K., Kakimoto, H., Mishukov, V.F., Toriba, A., Kizu, R., Hayakawa, K., 2005. Polycyclic aromatic hydrocarbons and nitropolycyclic aromatic hydrocarbons in urban air particulates and their relationship to emission sources in the Pan-Japan Sea countries. Atmos. Environ. 39, 5817e5826. Tao, S., Wang, Y., Wu, S.M., Liu, S.Z., Dou, H., Liu, Y.N., Lang, C., Hu, F., Xing, B.S., 2007. Vertical distribution of polycyclic aromatic hydrocarbons in atmospheric boundary layer of Beijing in winter. Atmos. Environ. 41, 9594e9602. Tao, Z., Hornbuckle, K.C., 2001. Uptake of polycyclic aromatic hydrocarbons (PAHs) by broad leaves: analysis of kinetic limitations. Water Air Soil Pollut.: Focus 1, 275e283. Tokyo Metropolitan Government, 2006. Environmental White Paper in Tokyo 2006 (in Japanese). Wang, D., Chen, J., Xu, Z., Qiao, X., Huang, L., 2005. Disappearance of polycyclic aromatic hydrocarbons sorbed on surfaces of pine [Pinua thunbergii] needles under irradiation of sunlight: volatilization and photolysis. Atmos. Environ. 39, 4583e4591. Wang, Y.Q., Tao, S., Jiao, X.C., Coveney, R.M., Wu, S.P., Xing, B.S., 2008. Polycyclic aromatic hydrocarbons in leaf cuticles and inner tissues of six species of trees in urban Beijing. Environ. Pollut. 151, 158e164. Wickramasinghe, A.P., Karunaratne, D.G.G.P., Sivakanesan, R., 2011. PM10-bound polycyclic aromatic hydrocarbons: Concentrations, source characterization and estimating their risk in urban, suburban and rural areas in Kandy, Sri Lanka. Atmos. Environ. 45, 2642e2650. Wild, E., Dent, J., Thomas, G.O., Jones, K.C., 2006. Visualizing the air-to-leaf transfer and within-leaf movement and distribution of phenanthrene: further studies utilizing two-photon excitation microscopy. Environ. Sci. Technol. 40, 907e916. Yang, S.Y.N., Connell, D.W., Hawker, D.W., Kayal, S.I., 1991. Polycyclic aromtic hydrocarbons in air, soil and vegetation in the vicinity of an urban roadway. Sci. Total Environ. 102, 229e240. Zakaria, M.P., Okuda, T., Takada, H., 2001. Polycyclic aromatic hydrocarbon (PAHs) and hopanes in stranded tar-balls on the coasts of Peninsular Malaysia: applications of biomarkers for identifying sources of oil pollution. Mar. Pollut. Bull. 42, 1357e1366. Zakaria, M.P., Takada, H., Tsutsumi, S., Ohno, K., Yamada, J., Kouno, E., Kumata, H., 2002. Distribution of polycyclic aromatic hydrocarbons (PAHs) in rivers and estuaries in Malaysia: a widespread input of petrogenic PAHs. Environ. Sci. Technol. 36, 1907e1918.