Effect of Relative Humidity on Polycyclic Aromatic Hydrocarbon Emissions from Smoldering Incense

Incense was burned in a controlled environment, in order to understand the effect of relative humidity on polycyclic aromatic hydrocarbons (PAHs) emissions from smoldering incense. The concentrations of individual PAHs were determined using the GC/MS analysis method. Incense was burned in a test chamber with a total airflow rate of 6.0 L/min, with relative humidity of 21.3 ± 1.0, 51.0 ± 0.2 and 90.5 ± 0.5%, at a constant temperature (28.9 ± 0.4°C). The results show that the rate at which the incense burns decreases linearly as the relative humidity increases, at the tested airflow rate. The sums of the total PAH and toxic equivalency emission factors for particulate and gas phases were 47467.39–50218.87 and 325.21–358.85 ng/g, for the incense used in this study, and these values also decrease as the relative humidity increases. The carcinogenic potency of PAHs emissions in the particulate phase is approximately 6 times higher than those of the gaseous phase, at the relative humidity tested.


INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) comprise a group of semi-volatile organic pollutants that contain at least two fused aromatic rings.They have been the subject of much attention in recent years, because some (i.e., dibenz [a,h]anthracene, benzo[a]anthracene, benzo [b] fluoranthene, benzo [k]fluoranthene and benzo[a]pyrene, indeno[1,2,3-cd]pyrene) have been recognized as probable and possible human carcinogens (IARC, 1987).
As a common religious ritual in Oriental society, the burning of incense sticks causes air pollution that may result in a higher incidence of lung cancer (MacLennan et al., 1977), childhood leukemia (Lowergard et al., 1987) and brain tumors (Preston-Martin et al., 1982).Additionally, the Ames test has proven the mutagenic effect of incense smoke (Rasmussen, 1987;Sato et al., 1980).
Burning incense, cigarette and woods were a biomass combustion and important indoor source.The cigarette and woods burning has been demonstrated to produce harmful air pollutants (Bari et al., 2011;Ruttanachot et al., 2011;Wang et al., 2102).Incense is always burned in a smoldering, incomplete, slow and lengthy fashion and produces incense smoke that contains gas and particulates.Most existing studies have focused on the characterization of gaseous and particulate pollutants from incense burned indoors, in temples, or in a test chamber.The gaseous pollutants produced include carbon monoxide, carbon dioxide, nitrogen oxides, formaldehyde and volatile organic compounds (Chang et al., 1997;Yang et al., 2007a, b).The particles are typically less than 1 μm in size (Yang et al., 2006) and the particulate surface areas absorb numerous organic compounds, such as polycyclic aromatic hydrocarbons (Lin and Lee, 1998;Yang et al., 2012a, b, c) and aldehydes (Schoental and Gibbard, 1967;Lin and Tang, 1994;Lin and Wang, 1994;Lee and Wang, 2004).Particles also contain heavy metals, acidic materials, elemental compounds (ECs) (Wang et al., 2006) and reactive oxygen species (ROSs) (Kao and Wang, 2002).The adverse effect on health of incense smoke may be related to the extremely high number of fine particulates, as well as organic compounds, which may enhance each other's effect and easily penetrate the respiratory system, deep into the alveolar regions.
The characteristics of incense smoke may vary with the constituents of the incense stick and the conditions of combustion (i.e., airflow, temperature, humidity and oxygen content).The composition of the smoke may be a critical factor to its degree of danger.During the past years, the authors have investigated the formation of PAHs and the distribution of particle sizes from burning incense sticks and found that both parameters vary with the H/C ratios (Yang et al., 2012b, c).Previous studies focused mainly on characterizing the emission of air pollutants from burning various incense types in a large environmental chamber under constant combustion conditions (temperature, 23 ± 0.5°C; relative humidity, 50 ± 5%) (Lee and Wang, 2004;Wang et al., 2006).However, in Taiwan, the climate is usually warm and humid, which is very different to the environments tested in previous studies.Thus, the actual emission characteristics of smoldering incense may not be similar to those reported.However, previous studies of the effects of relative humidity on the emissions from burning incense contain limited data.These reports mainly investigated the formation of incense smoke and the properties of aldehyde emissions in the particulate and gaseous phases, at various levels of relative humidity (Lin and Tang, 1994;Lin and Wang, 1994;Chang et al., 2007).However, these studies failed to elucidate the effects of relative humidity on the total PAH and toxic equivalency emission factors.Hence, this study aims to understand the effect of relative humidity on the emission and carcinogenic potency of polycyclic aromatic hydrocarbons emitted from smoldering incense in the particulate and gaseous phases, thereby enabling better control of indoor pollution from smoldering incense.

Incense
The incenses used in this study, which consisted of bamboo charcoal, plant essential oils and tree bark, were chosen because of their low variation in weight and length, compared to others in a local market.The carbon, hydrogen, and nitrogen composition of the incense was analyzed using an elemental analyzer (Elementar Vario EL-III, Elementar Analysen Syetem GmbH, Germany) at the Precision Instrumentation Center in the National Taiwan University.The incenses were stored in a cabinet and maintained at a controlled relative humidity of 50 ± 2% at 25 ± 1°C, for 10 days prior to use.

Incense Burning Test System
An incense stick was burned in a combustion system that was used in previous studies by the authors (Yang et al., 2012c).The previous combustion system consisted only of a combustion chamber, a sampling chamber and a gas supply unit.Two additional devices were added to the new system, in order to control the relative humidity and temperature of the airflow.Hence, the new system in this study consists of an incense smoke generation (smoldering) unit, in which incense was burnt, a test chamber, an aircleaning train and two devices to control the relative humidity and temperature of the airflow (Fig. 1).
An air-cleaning train, consisting of a high-efficiency particulate air (HEPA) filter set, followed immediately by a silica gel column, an XAD-2 resin bed and a HEPA filter, was used to provide clean air for smoldering.Upon the removal of particles, gaseous impurities and organic substances, using the air-cleaning train, an air compressor supplied two airflows.The flow rates were controlled by two mass flow controllers.One airflow (A) was regulated at 6 L/min to enter into the inner Nafion tube.The other (B), maintained at 12 L/min, was separated into two airflows, one of which (B1) entered a water vapor generator chamber to incorporate water vapor (wet flow), while the other (dry flow, B2) entered directly into a small mixing chamber.Consequently, the relative humidity of airflow B was controlled by adjusting ratio of the flow rates of dry air (B2) and wet air (B1), before being introduced into the outside shell of the multi-Nafion tube.The multi-Nafion tube (Perma Pure, Inc., Toms River, NJ) is a copolymer of tetrafluoroethylene and perfluoro-3,6-dioxa-4-methyl-7octenesolfonic acid, housed within a single large polypropylene tube shell.The water molecules permeate the nafion member because of the force produced by the difference in water vapor content between the airflow inside and outside the Nafion tube.Finally, the relative humidity of the interior and exterior airflows reaches equilibrium.The relative humidity of the airflow inside the Nafion tube is controlled by the relative humidity of the exterior airflow.Finally, the airflow, A, from the inner Nafion tube flows into a heater tube, in order to maintain a constant airflow temperature.The relative humidity and temperature of the airflow, A, was measured using a hygrometer (Hygro Palm 3, Rotronic AG, Switzerland), before it entered into the combustion chamber (2.1 cm ID, 15 cm long).Using this configuration, the airflow, A, can be maintained at low, medium and high levels of relative humidity (21.3 ± 1.0, 51.0 ± 0.2 and 90.5 ± 0.5%) and a constant temperature (28.9 ± 0.4°C).
The combustion unit comprised a 15-cm-high glass cylinder, with an internal diameter of 2.1 cm.Pollutants in both gaseous and particulate phases, produced during incense burning, flowed directly into the sampling chamber (a steel cylinder with an internal diameter of 10 cm and height of 60 cm).Air sampling was performed at the airstream exit of the sampling chamber.For the three typical experimental conditions, the chamber temperature remained relatively constant (30.2 ± 1.2°C) and the relative humidity inside the chamber was relatively constant, at 20.6 ± 0.8, 49.1 ± 0.2 and 80.0 ± 0.2%.

Sampling Process
In order to ensure experimental reliability, each experiment was performed in at least triplicate and three representative samples were collected.The sampling process was as follows: An accurately weighed incense stick was placed in the combustion unit.The exit airflow rate was maintained at 6 L/min.At this rate of flow, a dynamic equilibrium was achieved in approximately 4 minutes, according to the results of a calculation using a single-compartment mass balance model (Fan and Zhang, 2001).
After the incense had been burned for 4 minutes, a sampling train, consisting of a two-piece filter cassette (255-2050LF, SKC, USA) with a 37-mm quartz membrane filter (7201, Pall, USA), followed by a XAD-2 sorbent tube (8 mm o.d., 110 mm length glass tube, 200/400 mg XAD-2 resin, Cat.No. 226-30-06, S.K.C., USA) connected in series, was used to collect particulate and gaseous PAHs at a flow rate of 2 L/min, for 15 min.After sampling, the incense was burned for another minute, at which point the incense was extinguished, using a stream of nitrogen gas.The remaining incense was weighed.
The carbon background was removed by maintaining the quartz filters at > 450°C, for high-temperature roasting, for at least 5 hr before the experiment.The change in the weight of the filters before and after sampling was measured using a microbalance (CP2P-F; Sartorius AG, Goettingen, Germany), in order to determine the mass of the particles.A soap-bubble gas flow-meter (Gilian Instrument Co., NJ, USA) was used to calibrate the sampling rates, both before and after sampling.Before and after burning, individual incense samples were weighed using a precision balance (AG135; Mettler Toledo, Inc., Switzerland), in order to determine the burning rate.

Extraction of PAHs
The PAH extraction procedure is described as follows: To isolate the organic compounds from the particulate and gas portions, the filter and the front and back XAD-2 were inserted into 50, 8 and 8-mL brown sampling vials, followed by extraction using 25, 5 and 5-mL of dichloromethane (UN1539; Merck, Germany).The mixtures were ultrasonicated (DC400H; Delta, Taiwan) for 15 min, followed by a rest period of 5 min (Karthikeyan et al., 2006;Orecchio, 2007;Mannino and Orecchio, 2008).This extraction procedure was repeated three times, to ensure complete extraction.The resultant solutions were filtered through a glass syringe with a 13-mm disposable syringe filter unit (0.5 μm; Advantec, Inc., Japan).The filtered solutions of the particulate portion were concentrated using a rotary evaporator (N-1000S, Eyela, Japan) with a vacuum controller (NVC-2100, Eyela, Japan), at 650 hPa, and a thermostatic bath (OSB-2100, Eyela, Japan) at 33°C.The final volume, about 1 mL, was further reduced to 0.8 mL using a gentle stream of nitrogen.In addition, the filtered solution of the gas portion was directly reduced to 4.5 mL using a gentle stream of nitrogen.After the particulate and gas sample had spiked at 0.1 and 0.5 mL internal standards (Supelco, USA) the solution volumes were adjusted to 1 and 5 mL, respectively, by adding pure dichloromethane.

Chemical Analysis of PAHs
A total of 16 PAH organic pollutants produced from burning of incenses were identified and quantified using gas chromatography/mass spectrometry (GC/MS) (GCMS-QP2010; Shimadzu, Japan).The GC/MS conditions were as follows: (a) The capillary column was a DB-5MS (Agilent J&W, USA), with an inner diameter of 0.25 mm, length of 30 m and film thickness of 0.25 µm.(b) The initial temperature of the oven was 70°C for 3 min, which was then increased to 300°C, at a rate of 10 °C/min, and maintained at 300°C for 20 min.The analytical period for each cycle was 46 min.(c) The temperatures of the injection port, ion source, and interface were set at 280°C, 230°C and 290°C, respectively.(d) The carrier gas flow rate was 1.0 mL/min, corresponding to a linear velocity of 36.7 cm/sec.(e) The injection mode was splitless, with an injection volume of 1.0 μL.Analysis was conducted in selective ion monitoring (SIM) modes.The process of the quantification ion and confirmation ion for the SIM mode were based on that of Orecchio (2011).
The , were analyzed for concentrations of 5-1400 ng/mL, in order to establish the standard calibration curves and to determine the concentration of each of the 16 PAHs.All correlation coefficients for the calibration curves were > 0.998.The detection limits of 8.5-56.9ng/m 3 were determined using a threefold standard deviation of seven measurements of the lowest concentration of a calibration curve.Blank filters were used in each experiment.The background values for the blank filter and the front and back XAD-2 were subtracted for all samples.A known amount of PAH was added to a cleaned blank filter and XAD-2, in order to determine the recovery yields.The mean recovery yields for particulate and gaseous PAHs, using this procedure, were 76.6-118.9%and 77.2-91.8%,respectively.No breakthrough occurred for any gaseous PAH samples in this study.

Emission Rates and Factors of Individual Pollutants
According to the calculated results using the diffusion loss formula (Hinds, 1982), particle diffusion losses in the combustion and sampling chamber, from 0.010 to 10 µm, were less than 1.00 and 2.48%, respectively.Because the mass of the small particles lost was very little, in comparison with the total particulate mass, the effect of the subsequent emission factor calculation was very small.Thus, the estimated emission factors in the particulate phase do not take account of the loss of the "wall effect".The emission rates and the emission factors of each pollutant were calculated using the conservation of mass, as follows: where V (m 3 ) is the sampling chamber volume (4.7 L), C i (ng/m 3 ) is the concentration of each pollutant at a given time, R (g/hr) is the incense burning rate, E f (ng/g) is the emission factor of each pollutant and Q (6 L/min) is the airflow rate; dCi/dt = 0, when the generation and removal of the pollutant are in dynamic equilibrium.Eq. ( 1) can be rewritten as follows: In Eq. ( 2), the emission rate (E r ) is calculated by multiplying flow rate by the equilibrium concentration of the pollutants.The emission factor, E f , is the specific emission rate normalized to the incense burning rate.

Characteristic Incense and Particle Mass Emission Factor
The carbon, hydrogen and nitrogen composition of the tested incenses were 54.03 ± 0.29, 3.70 ± 0.05 and 0.42 ± 0.008%, respectively.The H/C atomic ratios were 0.82 ± 0.02.The burning rates for the incense at a relative humidity of 21.3, 51.0 and 90.5% were 1.10 ± 0.01 (average ± S.D., n = 3), 1.04 ± 0.01 (n = 3) and 0.99 ± 0.01 (n = 3) g/hr, respectively (Fig. 2).The water vapor pressure was calculated according to the relative humidity and temperature.The water vapor pressures for low, medium and high levels of relative humidity of the supply airflow were 4.16, 15.2 and 24.4 mmHg, respectively.Since the water molecules absorb Fig. 2. The burning rate and particulate mass emission factors due to the burning of incense at various levels of relative humidity (Bar: mean ± standard deviation).the heat released from the burning tip of the incense, the temperature of the burning tip decreases, so the rate at which incense is burned in an environment with high relative humidity is lower than that for an environment with low relative humidity.
However, the mass emission factors of the total particulates generated by burning incense at 21.3, 51.0 and 90.5% relative humidity were 18.18 ± 0.70 (n = 3), 17.48 ± 0.1 (n = 3) and 16.54 ± 0.23 (n = 3) mg/g, respectively (Fig. 2).The particulate mass emission factors differ significantly with relative humidity (F-Test, P = 0.01).This result demonstrates that the total particulate mass emission factors generated from burning incense substantially decrease when the relative humidity is increased.

Distribution of PAHs
The total PAH mass concentrations in the particulate and gaseous phases generated by burning incense at various levels of relative humidity were 10736.30-14908.68 and 119676.95-139064.35ng/m 3 , respectively.The mass percentages of two-(Nap), three-(AcPy, Acp, Flu, Phen, Ant), four-(FL, Pyr, BaA, Chr), five-(BbF, BkF, BaP, DBA), and six-ring PAHs (INP, BghiP) in the particulate phase, for incense burned at various levels of relative humidity, were N.D.-0.06, 24.04-29.04, 54.81-57.48, 11.62-13.44 and 4.47-5.04%,respectively (Table 1).The four-ring PAHs predominate in the total particulate masses.Fig. 3 shows that the major species are Phen, FL and Pyr and that only trace amounts of Nap in the particulate phase result from the burning of incense under the three tested conditions.

Emission Factors of PAHs
The total PAH emission factors for the total particulate mass generated by burning incense at a relative humidity of 21.3, 51.0 and 90.5% were 4863.82 ± 129.39 (n = 3), 4224.48 ± 270.05 (n = 3) and 3907.35 ± 48.10 (n = 3) ng/g, respectively.The gaseous PAH emission factors for the three levels of relative humidity were 45355.05± 1615.51 (n = 3), 44503.44 ± 77.45 (n = 3) and 43560.05± 1228.81 (n = 3) ng/g, respectively (Table 1).The sums of the total PAH emission factors for particulate and gaseous phases were 47467.39-50218.87ng/g for incense tested at each relative humidity.All of the particulate, gaseous and total PAHs emission factors during the smoldering of incense at various levels of relative humidity are classified in the following order: 90.5% < 51.0% < 21.3%.This analysis shows that a relative humidity of 90.5% minimizes the particulate, gaseous and total PAH emission factors.In addition, the various partitions of PAHs in the gaseous and particulate phases were in the ranges, 90.30-91.77%and 8.23-9.70%,for the incense type, at the relative humidity used (Table 2).Hence, the PAHs generated by burning incense mainly occur in the gaseous phase, for the levels of relative humidity tested.

Degree of Carcinogenicity of PAHs
The method used to estimate the carcinogenic potency of the emission of a given PAH compound in this study is described as follows: The Benzo[a]pyrene equivalent (BaPeq) emission factors of individual PAHs are the toxic equivalent factors (TEFs) of individual PAHs (Nisbet and LaGoy, 1992) multiplied by the amount of the individual PAHs emission factors (Eq.( 3)).The toxic equivalency (TEQ) emission factors of the 16 PAHs were calculated using Eq. ( 4).The TEQ emission factors of total the particulate mass generated by burning incense at the levels of relative humidity investigated were 311.94 ± 12.34 (n = 3), 299.48 ± 26.64 (n = 3) and 279.58 ± 1.55 (n = 3) ng/g, respectively (Fig. 4).In terms of the TEQ contribution by individual PAHs in the particulate phase, BaP (57.00-59.69%),BaA (13.13-14.32%),BbF (9.00-10.09%)and DBA (8.02-11.75%)predominate.The gaseous TEQ emission factors at the levels of relative humidity used were 46.90 ± 1.73 (n = 3), 45.88 ± 0.16 (n = 3) and 45.63 ± 1.51 (n = 3) ng/g, respectively (Fig. 4).Nap accounted for 85.64-86.61% of the TEQ emission factor in the gaseous phase, for the incense.The total sums of the TEQ emission factors for the particulate and gaseous phases were 325.21-358.85ng/g for the incense, at the levels of relative humidity used (Fig. 4).The major TEQ contribution for individual PAHs in the total PAHs are BaP (49.37-51.88%),BaA (11.37-12.31%),Nap (11.33-12.01%),BbF (7.80-8.67%)and DBA (6.98-10.22%).All of the particulate, gaseous and total TEQ emission factors during the smoldering of incense at various levels of relative humidity are classified in the following order: 90.5% < 51.0% < 21.3%.This analysis shows that a relative humidity of 90.5% minimizes the particulate, gaseous and total TEQ emission factors.
The various partitions of TEQ emission factor of PAHs in the gaseous and particulate phases were in the range, 13.09-14.03%and 85.97-86.91%,for the incense type and levels of relative humidity used (Table 3).The total TEQ emission factors of PAHs, generated by burning incense,  mainly consisted of the particulate phase (> 85%); i.e., the carcinogenic potency of PAHs emission in the particulate phase was approximately 6 times higher than that of the gaseous phase, at the various levels of relative humidity.This result is explained by the fact that the low carcinogenic potency of Nap is the dominant PAH in the gaseous phase, while high molecular weight PAHs with high carcinogenic potency are dominant in the particulate phase, due to the burning of incense.Table 3.The mean carcinogenic potency of the PAH fractions of the particulate and gaseous phases in the total carcinogenic potency of PAH from the burning of incense at various levels of relative humidity.

Characteristic Ratios
The characteristic ratios of polycyclic aromatic hydrocarbon, based on parent PAHs, have often been use as a tool for the identification of pollution sources.For the burning of incense at various levels of relative humidity, performed in this study, the Ant/(Ant + Phen) ratios for particulate, gaseous and total (gaseous and particulate) phases were 0.22 ± 0.001, 0.14 ± 0.001 and 0.17 ± 0.001, respectively (Table 4).The Ant/(Ant + Phen) ratios for particulate and total (gaseous and particulate) phases differ significantly for various levels of relative humidity (F-Test, all P < 0.10), except for the Ant/(Ant + Phen) ratio for gaseous phases.The overall mean and standard deviations of the FL/(FL + Pyr), BaA/(BaA + Chr) and INP/(INP + BghiP) ratios for the particulate phase at various levels of relative humidity were 0.47 ± 0.001, 0.49 ± 0.001 and 0.53 ± 0.001, respectively.These ratios of FL/(FL + Pyr), BaA/ (BaA + Chr) and INP/(INP + BghiP) for the particulate phase are the same as that of the total (gaseous and particulate) phases, because the FL, Pyr, BaA, Chr, INP and BghiP are not found in the gaseous phase.These characteristic ratios of the PAHs for particulate, total (gaseous and particulate) phases do not differ significantly for various levels of relative humidity (F-Test, all P > 0.10).Finally, since the relative humidity of the environment does not affect the Ant/(Ant + Phen) ratio for the gaseous phase, the FL/(FL + Pyr), BaA/ (BaA + Chr) and INP/(INP + BghiP) ratios for the particulate phase, generated from incense smoke, are regarded as characteristic ratios for burning incense that consists of bamboo charcoal, plant essential oils and tree bark.

CONCLUSIONS
In this study, the ranges of the total mass, gaseous and particulate PAH emission factors were 16.54-18.18mg/g, The incense burning-rate decreases as the level of relative humidity increases.The total mass and particulate PAH emission factors for incense smoke depend on the relative humidity and decrease when the rate at which the incense is burned decreases.The carcinogenic potency of PAH emissions in the particulate phase (279.58-311.94ng/g) was approximately 6 times higher than that of the gaseous phase (45.63-46.90ng/g), for the levels of relative humidity investigated.This result indicates that the control of particulate PAH emissions is more important than the control of the gaseous PAH emissions, in terms of human health.In terms of the TEQ contribution of individual PAHs in the total PAHs of particulate and gaseous phases, BaP (49.37-51.88%),BaA (11.37-12.31%)and Nap (11.33-12.01%)contribute most.This study concludes that the burning of incense at a high level of relative humidity may minimize the production of polycyclic aromatic hydrocarbons.

Fig. 1 .
Fig. 1.A schematic diagram of the incense combustion testing system.

Fig. 3 .
Fig. 3.The mass percentages of individual PAH in the particulate and gaseous phases due to the burning of incense at various levels of relative humidity (Bar: mean ± standard deviation).

Fig. 4 .
Fig. 4. The total emission factors and toxic equivalency emission factors of PAHs in the particulate and gaseous phases due to the burning of incense at various levels of relative humidity (Bar: mean ± standard deviation).

Table 2 .
The mean PAH fractions of the particulate and gaseous phases in the total PAHs, due to the burning of incense at various levels of relative humidity.