Oxygen-Containing Polycyclic Aromatic

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Parent, alkylated and sulfur/oxygen-containing polycyclic aromatic hydrocarbons in mainstream smoke from 13 brands of Chinese cigarettes Bo Gao, Xueqing Du, Xinming Wang, Jianhui Tang, Xiang Ding, Yanli Zhang, Xinhui Bi, and Gan Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01108 • Publication Date (Web): 29 Jun 2015 Downloaded from http://pubs.acs.org on July 10, 2015

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Environmental Science & Technology

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Parent, alkylated and sulfur/oxygen-containing polycyclic

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aromatic hydrocarbons in mainstream smoke from 13

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brands of Chinese cigarettes

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Bo Gao,†, ‡ Xueqing Du,† Xinming Wang,†, §,* Jianhui Tang,ǁ Xiang Ding,†

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Yanli Zhang, † Xinhui Bi, † Gan Zhang†

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†

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Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China

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‡

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Protection, Guangzhou 510655, China

State Key Laboratory of Organic Geochemistry, Guangzhou Institute of

South China Institute of Environmental Sciences, Ministry of Environmental

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§

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Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou

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510640, China

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ǁ

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Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shandong

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264003, China

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*Corresponding author:

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Dr. Xinming Wang

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State Key Laboratory of Organic Geochemistry

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Guangzhou Institute of Geochemistry, Chinese Academy of Sciences

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Tel: +86-20-85290180; Fax: +86-20-85290706

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Email: [email protected]

Guangdong Key Laboratory of Environmental Protection and Resources Utilization,

Shandong Provincial Key Laboratory of Coastal Environmental Processes, Yantai

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ACS Paragon Plus Environment


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Abstract

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China has the world’s largest population of smokers with serious health consequences,

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yet we know a very limited spectrum of hazardous chemicals in cigarette smoke even

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for carcinogenic polycyclic aromatic hydrocarbons (PAHs). Here we chose 13

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popular cigarette brands sold in China markets, collected particulate matters in

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mainstream smoke using filter pads and an auto-smoking machine, and analyzed 56

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PAHs, including 31 parent, 18 alkylated and 7 sulfur/oxygen-containing PAHs (S/O

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PAHs). The 56 PAHs in mainstream smoke totaled from 244.2±28.5 ng cig-1 to

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10254.8±481.5 ng cig-1; and parent, alkylated and S/O PAHs shared 16-23%, 64-74%

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and 6-18%, respectively. Benzo[a]pyrene (BaP) ranged 1.1-40.3 ng cig-1 while BaP

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equivalent concentrations (BaPeq) ranged 3.6-120.2 ng cig-1, but contributions to

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BaPeq by individual carcinogenic PAH species varied with cigarette brands. When

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these cigarette smoke source profiles were pooled together with those of other

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combustion ones available in literature, we found widely used diagnostic ratios of

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parent PAHs failed to distinguish cigarette smoke from other combustion sources,

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except

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benzo[g,h,i]perylene) can largely separate cigarette smoke from vehicular emissions,

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and that the ratio of Retene/(Retene + chrysene) can further discriminate cigarette

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smoke from coal combustion when alkylated PAHs are involved.

that

the

ratio

indeno[1,2,3-cd]pyrene

/(indeno[1,2,3-cd]pyrene

2


+

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Introduction

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Polycyclic aromatic hydrocarbons (PAHs) are known to cause cancer in humans.

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They are ubiquitous in ambient air largely from incomplete combustion of fossil fuels

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and/or biomass burning, yet environmental tobacco smoke (ETS), either mainstream

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or side-stream, contributes substantially to PAHs in indoor micro-environments, 1, 2

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particularly in smoker’s homes.3 As PAHs are among the most potent carcinogens in

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cigarette smoke and tobacco smoke-derived PAHs bear a large burden in human

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exposure to this class of carcinogens,4 characterizing compositions and levels of

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PAHs in cigarette smoke is of wide and enduring concern for the protection of public

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health.

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Mainstream smoke (MS), referring specifically to the smoke that a smoker inhales

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and then exhales, represents the direct health hazard to smokers though voluntary

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inhalation. Previous studies have revealed the presence of PAHs and other pollutants

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in MS,1, 5-7 and among them benzo[a]pyrene (BaP) was the most studied species due

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to its ability to induce lung tumors.8 Besides BaP, some other PAHs within the

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USEPA’s 16 priority PAHs are also identified as probably or possibly carcinogenic to

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humans by agencies like the International Agency for Research on Cancer (IARC).1

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Consequently, the 16 priority PAHs or their subsets in ETS were investigated

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concerning their levels, compositions

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smoking conditions and cigarette design on the chemical delivery7, 10, 11. However, as

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cigarette smoke is a complex mixture with multiple classes of chemical compounds,

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more PAHs, such as alkylated and sulfur/oxygen (S/O) PAHs, also exist in cigarette

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smoke,12,

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carcinogenicity of other PAHs6. Previous studies have evidenced that some alkylated

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and S/O PAHs were more toxic than their parent ones.14, 15 Hence, accurate evaluation

13

1, 6, 9

and toxicity5, as well as the influence of

either being possible human carcinogens or promoters for the

3



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of the toxicity of cigarette smoke requires more PAH species to be measured.

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Unfortunately, there is little quantitative data about these additional PAHs. Only a few

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studies

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methylphenanthrenes/methylanthracenes

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5-methylchrysene16 in the MS particulates.

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China is the world’s leading tobacco producer and has a third of the world’s tobacco

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consumers.4 However, most of the published researches focused on PAHs in reference

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cigarettes and/or commercial cigarette brands outside of China. Only a few studies are

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available for cigarettes smoke PAHs in China4, 17, 18, and the target compounds were

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still limited within the 16 priority PAHs. To our best knowledge, only one study

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published in English reported 9 priority PAHs and other harmful chemicals in 20

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brands of Chinese cigarettes.4 In this study, we measured the levels of 56 PAHs,

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including 31 parent, 18 alkylated and 7 S/O PAHs, in the MS particulates from 13

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popular Chinese cigarette brands so as to complement these deficiencies. PAH levels

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in reference cigarettes were also measured for comparison with previous studies. The

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BaP equivalent (BaPeq) concentrations were further calculated to indicate carcinogenic

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potency of PAHs using our current data. As diagnostic ratios have been widely used to

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identify PAH sources19, 20 and utilizing the alkylated and S/O analogues may facilitate

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source discrimination,21, 22 here we finally evaluate whether PAH (including parent,

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alkylated and S/O PAHs) ratios can distinguish cigarette smoke from other sources,

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using published source profiles for combustion sources together with our data.

reported

the

concentrations and

of

methylnaphthalenes7,

methylpyrenes,12

and

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Experimental Section

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Materials

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16 priority PAHs and deuterated PAHs (naphthalene-d8, acenaphthene-d10,

4


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phenanthrene-d10, chrysene-d12, perylene-d12) were obtained from Ultra Scientific,

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Inc. (North Kingstow, RI, USA). A standard mixture solution of 10 PAHs

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(1-methylnaphthalene, 2-methylnaphthalene, biphenyl, 2,6-dimethylnaphthalene,

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2,3,6-trimethylnaphthalene,

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3,6-dimethylphenanthrene, benzo[e]pyrene and perylene) was obtained from

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AccuStandards, Inc. (New Haven, CT, USA). A standard mixture solution of 8 S/O

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PAHs

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4-methyldibenzothiophene, 2-methyldibenzothiophene, 3-methyldibenzothiophene,

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benzonaphtho[2,1-d]thiophene, benzonaphtho[2,3-d]thiophene) was purchased from

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Chiron AS (Trondheim, ST, Norway). Hexamethylbenzene and coronene were

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purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Reference

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cigarettes, 1R3F and 2R4F, were obtained from the University of Kentucky

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(Lexington, KY). Domestic commercial cigarettes were purchased from various retail

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sources in China. Cambridge filter pads (CFPs) used to collect MS particulate matter

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were obtained from Whatman (Maidstone, United Kingdom).

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Smoke Collection

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Prior to smoking, the cigarettes and CFPs were conditioned at constant relative

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humidity (60%) and temperature (22 °C) for no less than 48 h using a temperature and

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humidity chamber (WTC binder, Germany). MS total particulate matter (TPM)

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generated following US Federal Trade commission (FTC)/International Standard

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Organization (ISO) conditions (60 s puff interval, 2 s puff duration, and 35 mL puff

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volume) was collected on individual CFPs using a Borgwaldt (Germany) RM200

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20-port auto-smoking machine. The cigarettes were smoked to a butt length of 23 mm

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or the length of the filter overwrap plus 3 mm, whichever was longer, using the

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industry-standard Cambridge filter pad holder. Three replicate samples were collected

(dibenzofuran,

2-methylphenanthrene,

1-methyldibenzofuran,

5


1-methylphenanthrene,

dibenzothiophene,


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per cigarette type with 20 cigarettes smoked for each sample. Table S1 presents the

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specification and average puff count for each type of cigarette.

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Sample preparation and GC/MS analysis

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The analytical procedure used for extraction, separation, and measurement of PAHs

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was detailed elsewhere23,

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extraction, each CFP was spiked with the deuterated PAH standards as surrogates, and

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then Soxhlet-extracted with dichloromethane for 72 h. The extracts were concentrated

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to 1 ml, solvent-exchanged to redistilled hexane and then separated into two fractions

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using a 1:2 alumina/silica column chromatography. The first fraction containing

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nonpolar compounds was eluted by 30 mL of hexane. The second fraction, containing

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PAHs, was eluted by 70 mL of dichloromethane/ hexane (3:7 v/v), concentrated to ~1

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mL and blown to ~200 µL under a gentle stream of nitrogen. 8 µL of 50 µg mL-1

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hexamethylbenzene internal standard was added to the samples before instrumental

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analysis.

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The samples were analyzed using an Agilent GC-MS (6890-5973N) equipped with a

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HP-5 capillary column (50 m×0.32 mm×0.17 µm). The column temperature was

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initiated at 80 °C (held for 5 min) and increased to 290 °C at 3 °C min-1 (held for 30

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min). An aliquot of 1 µL was injected in split mode with a solvent delay of 6 min.

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Parent, alkylated and S/O PAHs were identified based on their mass spectra and

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retention times. For those with authentic standards, quantitation was performed using

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the internal calibration method with the five-point calibration curves; and for

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alkylated PAHs without authentic standards, their amounts were approximated with

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their isomers/homologues or parent PAHs closest in retention times as the alternative

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standards.

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Recoveries were evaluated by the deuterated surrogates spiked to the samples. The

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, and only a brief description is given here. Before

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mean recoveries were 76±20%, 89±13%, 98±15%, 89 ±16% and 82±16%,

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respectively, for naphthalene-d8, acenaphthalene-d8, phenanthrene-d10, chrysene-d12

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and perylene-d12. The relative standard deviations (RSDs) of individual PAHs except

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for naphthalene were all within 15% among the three replicate samples collected per

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cigarette brand.

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Results and discussion

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PAH levels in reference cigarettes

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We measured 56 PAHs in MS for 1R3F and 2R4F Kentucky reference cigarettes

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using our method (Table S2). Individual PAHs for 1R3F cigarettes showed levels

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1.1-2.5 times that for 2R4F ones, and the total PAH levels in 1R3F (4208.8±746.7 ng

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cig-1) were 1.6 times that in 2R4F (2564.3±134.8 ng cig-1). However, TPM

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normalized PAH levels (PAHs/TPM) for 1R3F (303.9±51.6 ng mg-1) were just

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slightly higher than that for 2R4F (272.9±5.3 ng mg-1). Considering 1R3F is close to

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2R4F in tobacco blend, higher PAH yields for 1R3F may be due to its lower filter

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ventilation than 2R4F. 2R4F is designed as the substitute of 1R4F25 and both of them

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are examples of American blended cigarettes with filler consisting of bright, burley,

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oriental, and reconstituted tobaccos.1 Literature data of these two types of reference

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cigarettes smoked under FTC/ISO conditions were listed in Table 1 for comparison.

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Among the PAH levels for 2R4F determined by previous studies1, 16, 26, the TPM level

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was only reported by Roemer et al.26, and it was almost the same as that measured in

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our study (Table 1) although the PAHs emitted per cigarette in our study were much

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higher. Compared to the amounts of PAHs emitted per cigarette by Ding et al.1, our

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results were lower for most PAHs except for BaA, BkF, BeP and BaP. However,

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levels of BbF and IcdP quite approximated that reported by Ding et al.16. The

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differences in measured PAH levels for 2R4F between studies might be partly caused

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by the numbers of cigarettes being smoked: only 1-3 cigarettes were smoked by Ding

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et al.1, 16 whereas 20 or 30 cigarettes were smoked by Roemer et al.26 and 20 cigarettes

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were smoked in this study. Different pretreatment and instrumental determination

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procedures could also be reasons for the different levels measured for the same

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species. As can be seen in Table 1, none of previous studies reported all the listed 18

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PAH species for 2R4F or 1R4F as we did. Comparison of our 2R4F results with those

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reported for 1R4F also revealed similar TPM levels while the individual PAHs fell

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into three categories: the levels of NaP, Acey, Ace and Fl were lower in 2R4F than in

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1R4F; the levels of BaA, BkF, BeP, BaP and IcdP in 2R4F were higher in 2R4F than

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in 1R4F; and the levels of remaining PAHs in 2R4F fell into the range of literature

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data for 1R4F (Table 1).

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PAH levels in different commercial cigarettes

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Levels of individual PAHs in the MS of 13 commercial cigarettes were listed in Table

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S2 in comparison with previous particle-phase data for various cigarettes smoked

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using FTC/ISO conditions1, 4-7, 9-11, 16, 27, 28. The ranges for 6 parent PAHs, namely

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BaA, BbF, BkF, BaP, DahA, and IcdP, were wider than those reported in the IARC

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monograph30, in which the data were obtained more than 30 years ago.

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Emission of individual PAH compounds varied from 0.1 to 1123.1 ng cig-1, and also

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exhibited large variations across different brands, despite that they fell into the broad

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range of literature data, which covered 1-3 orders of magnitude (Table S2). The

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average RSD for individual species ranged from 21% for dibenzothiophene (DBT) to

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182% for benzo[c]phenanthrene, with an average of 52%. The variations in the

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compositions of PAHs could be associated with many factors including tobacco types

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and blends; ingredient types; filter types, size and ventilation; manufacturing

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processes and even analytical procedures.1 Notably, PAHs generated from Kent were

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one order of magnitude less than those from others probably due to higher ventilation.

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The average TPM yield for the cigarettes tested was 15.7±4.7 mg cig-1, with a less

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variation (RSD~30%) compared to PAHs. These TPM levels were similar with that of

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15.5±3.0 mg cig-1 reported by Akpan et al.4 for 20 brands of Chinese cigarettes, and

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close to those for some custom made blended cigarettes (8.61-11.01 mg cig-1)10 and

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blended US market cigarettes (2-28.6 mg cig-1)5, but much lower than the mean value

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determined by Moir et al. (46.9 mg cig-1)7. A Pearson correlation analysis indicated

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most PAHs were strongly correlated with each other (r=0.514-0.996, p

Oxygen-Containing Polycyclic Aromatic

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