Science of the Total Environment 431 (2012) 68–77

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Observation of elevated fungal tracers due to biomass burning in the Sichuan Basin atChengdu City, China

Yihong Yang a, Chuen-yu Chan a,⁎, Jun Tao a,b, Mang Lin a, Guenter Engling c,⁎⁎,Zhisheng Zhang a, Ting Zhang a, Lin Su a

a School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, 510275, Chinab South China Institute of Environmental Sciences, Guangzhou 510655, Chinac Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan

⁎ Correspondence to: C. Chan, School of EnvironmentaYat-sen University, No.135 XingangWest Road, GuangzhTel./fax: +86 20 84110692.⁎⁎ Corresponding author.

E-mail addresses: chzy@mail.sysu.edu.cn (C. Chan),(G. Engling).

0048-9697/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.scitotenv.2012.05.033

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 December 2011Received in revised form 31 March 2012Accepted 10 May 2012Available online 3 June 2012

Keywords:ArabitolMannitolBiomass combustionFine particlesMegacity

Fungal material (i.e., spores and fragments) is an important component of atmospheric aerosols. In order to ex-amine the variability of fungal abundance in fine particles (PM2.5) during a biomass burning season, an intensivemeasurement campaign was conducted in the Sichuan Basin at Chengdu, a megacity in southwest China, inspring 2009. The aerosol samples were analyzed for carbonaceous species, including molecular tracers for bio-mass burning and fungalmaterial, andwater soluble ions. The results were interpretedwith the help of principlecomponent analysis, fire countmaps, and theWRFmodel. Elevated concentrations of arabitol andmannitolwerefound with average concentrations of 21.5±16.6 ng m−3 and 43.9±19.3 ng m−3, respectively, which wereunexpectedly higher than those measured in fine particles in any other study reported previously. Even higherconcentrations were observed in cases with simultaneous enhancements in the biomass burning tracerslevoglucosan and K+. In the case of influence by pollution plumes from biomass burning regions, the fungaltracer concentrations reached maximum values of 79.6 ng m−3 and 121.8 ng m−3, coinciding with peak levelsof levoglucosan and K+. Statistically significant correlations were found between the simultaneously observedfungal tracers (arabitol and mannitol) and biomass burning tracers (levoglucosan and K+), suggesting thatthese species were emitted by co-located sources, and hence the elevated fungal tracers were likely associatedwith biomass burning activities.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Primary biological aerosols (PBAs), which include pollen, fungalspores, bacteria, viruses, and fragments of animals and plants, areubiquitous particles emitted directly from the biosphere to the atmo-sphere (Artaxo and Hansson, 1995; Jaenicke et al., 2007). Severalstudies have shown that PBAs could play important roles in atmo-spheric chemistry and physics, the biosphere and climate (Andreaeand Crutzen, 1997; Ariya and Amyot, 2004; Deguillaume et al.,2008; Després et al., 2012; Georgakopoulos et al., 2009; Mohleret al., 2007; Morris et al., 2011). PBAs can act as cloud condensationnuclei and ice nuclei, thereby influencing precipitation and the energybudget of the Earth (Bauer et al., 2003; Bowers et al., 2009; Christneret al., 2008a; Matthias-Maser et al., 2000; Prenni et al., 2009; Vali,

l Science and Engineering, Sunou, Guangdong 510275, China.

guenter@mx.nthu.edu.tw

rights reserved.

1996). They also influence the spread of biological organisms (Hamiltonand Lenton, 1998), and play an important role in the long-range transportof trace elements and pathogens into and away from specific biomes(Mahowald et al., 2005; Rong, 2000). PBAs have been widely discusseddue to their potential toxicity, allergies, and medical implications, sincethey are associated with allergic respiratory symptoms, decreased lungfunctions, including allergic asthmatics, asthma exacerbation, andasthma-related death (Dales et al., 2000, 2003, 2004; Rutherford et al.,2000; Targonski et al., 1995).

The composition and abundance of PBAs are expected to dependon vegetation characteristics, seasonal and meteorological conditions,geographical location, and human activities (Bowers et al., 2010;Froehlich-Nowoisky et al., 2012; Lacey, 1975, 1981). However, theabundance and origin of PBAs are still poorly understood and quanti-fied (Després et al., 2012; Elbert et al., 2007; Sesartic and Dallafior,2011; Xu et al., 2011). In fact, PBAs can account for a large proportionof aerosol particle mass in ambient air and their abundance varies ona large scale (Bauer et al., 2008b; Elbert et al., 2007; Jaenicke, 2005;Wiedinmyer et al., 2009; Winiwarter et al., 2009; Womiloju et al.,2003; Zhang et al., 2010). Hence, more observations are needed tobetter quantify the contribution of PBAs to aerosols on continentaland global scales.

69Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

Fungal material (i.e., spores and fragments) constitutes an impor-tant part of PBAs (Elbert et al., 2007). It is derived from soil, vegeta-tion, water surfaces, and human activities (Bauer et al., 2002b;Burch and Levetin, 2002; Burge, 2002) and is able to survive evenharsh environmental conditions (such as deserts, hot biomass burn-ing plumes, hailstones, glacial melt water, and deep sea sediments,etc.) (Boreson et al., 2004; Connell et al., 2008; Ma et al., 2000;Mandrioli et al., 1973; Mims and Mims, 2004; Novozhilova andPopova, 1969). It has been demonstrated that together with fungalspores, fragments may be released from moldy surfaces as well(Górny et al., 2002). Furthermore, size-fractionated aerosol data re-veal that arabitol and mannitol are mainly associated with the coarsesize mode and depending on the ambient conditions, such as relativehumidity, may show a tail into the fine size mode, suggesting an en-hanced release of fungal fragments from the biota (e.g., Kourtchevet al., 2009; Claeys et al., 2010). Generally, elevated abundances offungal material are often found in regions with high biological activ-ity, such as rainforests and pastures (Elbert et al., 2007; Grahamet al., 2003; Zhang et al., 2010), although recently, high diversitiesof bacteria and fungi were also observed in urban aerosol (Brodieet al., 2007; Froehlich-Nowoisky et al., 2009).

Because fine particles (with aerodynamic diameters b2.5 μm, PM2.5)can penetrate deeply into human lungs, the measurement of fungalmaterial with aerodynamic diameters less than 2.5 μm would be rela-tively more important in populous urban areas. In fact, although theaerodynamic diameters of fungal spores typically range from 2 to10 μm (Bai et al., 2002; Boekhout, 1991; Burge, 2002; Ingold, 2001;Lin and Li, 1996; Zhang et al., 2010), the diversity of airborne fungidetected in the fine particle fraction is comparable with that in thecoarse fraction. In particular, more human pathogens and allergens(e.g., Candida tropicalis and wallemia spp.) were found in fine particlesthan in coarse particles as revealed by recent DNA analysis studies(Froehlich-Nowoisky et al., 2009; Kaushik and Balasubramanian, 2012).Hence, these facts and new insights highlight the importance of investi-gating the fungal abundance in fine particles in highly populated urbanareas.

In urban areas, biomass burning was proposed as a potentialsource of fungal spores (Griffin et al., 2001; Mims and Mims, 2004).In addition, Mims and Mims (2004) found by traditional incubationand microscopic methods that many viable fungal spores were presentin the smoke from forestfires. The researchers proposed thatmajorfires(which can produce smoke plumes greater than 3 km) could be muchmore effective in launching spores into the troposphere than surfacewind storms, while small burns of diseased plants as well as cookingand heating with diseased firewood might disperse pathogenic sporeson a smaller scale.

The polyols arabitol and mannitol may constitute an importantfraction of the dry weight of fungi (Carlile and Watkinson, 1994),as they are common energy reserve materials in fungi (Lewis andSmith, 1967). Both compounds have been mostly associated withfungal material in aerosols, and were recently proposed as moleculartracers for fungi (Bauer et al., 2008a, 2008b; Claeys et al., 2010; Elbertet al., 2007; Heald and Spracklen, 2009; Zhang et al., 2010). In a recentstudy, Zhang et al. (2010) found high springtime levels of mannitoland arabitol in the coarse fraction of the ambient aerosol in subtropicalsouth China, and suggested that wet emission (the active dischargeof fungal spores, which is accompanied by the emission of aqueousdroplets)may be themajor emissionmechanismbased on good correla-tion between tracer levels and relative humidity. Burshtein et al. (2011),on the other hand, suggested that mannitol and arabitol might not bespecific biomarkers for fungi, as theywere poorly correlatedwith ergos-terol (another fungal tracer) in summer and winter, aside from the factthat these sugar alcohols frequently occur in some plants and algae.Further, the good correlation between arabitol/mannitol and ergosterolobserved in spring and autumnmight be attributed to high levels of veg-etation during spring blossoms and plant decomposition in autumn.

Lang-Yona et al. (2012) compared ergosterol concentrations withDNA-based analyses of airborne fungal spores in the PM10 aerosol frac-tion, and found poor correlations between them throughout the entireyear. They suggested that ergosterol degradation may have contributedto the observed differences and that DNA-based analysesmay be prefer-able for the quantification of fungal spores compared to biomarkermolecules. Considering potential degradation of ergosterol, the resultsof Burshtein et al. (2011) might in that case not be sufficient to provethat arabitol and mannitol cannot be unique tracers of fungal material.

Although mannitol and arabitol have been found in some plantsand algae, they are more likely associated with fungal material ratherthan plant debris in this study, because burning processes would leadto the thermal degradation of plants (Lobert and Warnatz, 1993).Sugar alcohols (such as arabitol/mannitol) in plants may easily beremoved and degrade during the burning process. In fact, it is lesslikely for these sugar alcohols to be released in their unaltered formfrom vegetation (e.g., leaves) and incorporated into fine aerosol par-ticles. Berg et al. (1982) also found that simple sugars were degradedvery early and at a high rate during decomposition, which supportsthat vegetation may be a relatively less important source of thesesugar alcohols. Meanwhile, sugar alcohols in fungal spores wouldbe protected by the spore walls (Smits et al., 2001), which might“preserve” them during the burning process. Furthermore, Lianget al. (unpublished data of our group from another recent study)found high correlation between the flow cytometry results of ambientfungal spores and the concentrations of arabitol (R2=0.72) in aerosolsamples collected daily over a period of 1 year in an urban area(Beijing). The sampling site (Chengdu) in this study is far away(over 1000 km) from the nearest sea (North Bay of China), and there-fore algae may not be a potential source of these tracers either.Therefore, we speculate that arabitol and mannitol could be suitablemolecular tracers for fungal material in this study.

In this paper, we report and discuss the elevated levels of fungalmolecular tracers (arabitol and mannitol) observed in an intensivemeasurement campaign conducted during the spring biomass burn-ing season (April and May 2009) at Chengdu, the capital of Sichuanprovince, which has a population of over 10 million. The SichuanBasin is one of the main rice and rapeseed producing areas in China(Lin et al., 2005). Thus, the air quality of the basin is strongly affectedby biomass smoke derived from rape and wheat straw burning, whichmake Chengdu City an ideal site for investigating the impacts ofbiomass burning on fungal abundance in the urban atmosphere. Thepossible linkage between biomass burning and enhancement infungal spores will be discussed through interpretation of the relation-ships between the fungal tracers and biomass burning tracers, includ-ing levoglucosan and potassium ion.

2. Experimental details

2.1. Site description and field sampling

Fig. 1 shows the geographical location of Chengdu (30.39 °N,104.00 °E) in the Sichuan Basin. Surrounded by high mountains (withelevations of 1000–3000 m above sea level), the basin is situated onthe eastern edge of the Tibetan Plateau and has an average altitude of500 m above sea level. With a mild climate that permits diversifiedagriculture, the basin is one of China'smain rice and rapeseedproducingareas. Consequently, crop straw burning is a common agricultural prac-tice in suburban and rural areas during the harvest seasons (April -May).

The sampling site was situated on the rooftop of a building at theInstitute Plateau Meteorology (~15 m above ground level), locatedin the downtown area of Chengdu. There are no industrial emissionsources within a 5 km2 range of the site, and only residential emis-sions (home cooking and transportation) in the surrounding area.The air at this site could, therefore, be considered more or less

Fig. 1. Location of the sampling site (Chengdu) in Sichuan Province (shaded area), China, and topography of the Szechuan Basin.

70 Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

representing continental boundary layer urban air of the basin. DailyPM2.5 samples were collected on quartz filters (Whatman Corpora-tion, 47 mm diameter) by a Mini-volume air sampler (Airmetrics,Eugene, OR, USA) that was operated at an air flow rate of 5.0 L min−1

from April 19 to May 17, 2009. The sampling duration was generally24 h and hence the sampled air volume was 7.2 m3.

Quality control procedures included collection of field blanks(one for every week), which were obtained by mounting the filtersin the sampler for 5 min without airflow. The sampler was calibratedbefore sampling by using a calibration orifice. All quartz filters weredecontaminated by baking at 800 °C for 3 h before sampling. All filterswere weighed before and after sampling on an electronic balanceafter conditioning at constant temperature and humidity. After condi-tioning and weighing, all sample filters were stored in petri dishes ina freezer at−18 °C until chemical analysis, in order to prevent loss ofvolatile components due to evaporation and degradation (Ariya et al.,2002).

2.2. Sample extraction and chemical analyses

A total of 29 quartz filter samples were analyzed for arabitol, man-nitol, levoglucosan, mannosan, inorganic ions, OC, EC, and water-soluble organic carbon (WSOC). For each filter sample, three portions(one 2.1 cm2 round punch, one-fourth of filter, and one-fourth offilter) were sonicated in deionized-distilled water (2.0, 10, and 15 mL

for sugars (arabitol, mannitol, levoglucosan, and mannosan), inorganicions, and WSOC, respectively; resistivity=18.2 MΩ cm−1) for 60 min.Recovery rates from sample preparation (extraction) were assumed tobe 100% based on previous tests of spiked filter samples. These speciesare completely soluble in water, rendering aqueous extraction an idealsample preparation method. The extracts were passed through syringefilters (PTFE, 0.45 μm pore size) to remove insoluble material. All sam-ple aliquots were stored at 4 °C until sample analysis. A total numberof 4 field blank samples were also extracted and analyzed in the sameway as the ambient samples. All of the data reported in this workwere blank corrected using the results from blank filters.

Concentrations of arabitol, mannitol, levoglucosan, and mannosanwere determined by an improved high-performance anion-exchangechromatography (HPAEC) method with pulsed amperometric detec-tion (PAD). This method was developed by Engling et al. (2006)and the data obtained by HPAEC-PAD showed good agreement withthose of derivatization GC/MS or GC/FID methods (Caseiro et al.,2007; Engling et al., 2006). Sample aliquots were injected (100 μLsample loop) without concentration or chemical derivatization intothe ion chromatograph. A more detailed description of the analyticalprocedures as well as quality control and assurance protocols wereoutlined by Linuma et al. (2009). The HPAEC-PAD system utilizedhere was a Dionex ICS-3000 series ion chromatograph (Dionex Corp.,Sunnyvale, CA, USA), equippedwith SP (quaternary pump and degasser),DC (column compartment), and ED (electrochemical detector and gold

Table 1Concentrations of PM2.5, OC, EC, WSOC, arabitol, mannitol, levoglucosan, mannosanand ionic species.

Component Range Meana SDa,b May 13 May 17

PM2.5 (μg m−3) 60.0–300.5 133.2 55.5 118.5 161.8OC (μg m−3) 9.4–41.8 19.1 6.5 27.8 41.8EC (μg m−3) 3.3–9.9 5.7 1.8 4.5 7.6WSOCc (μg m−3) 6.4–21.4 10.4 3.4 17.4 21.4Arabitol (ng m−3) 5.2–79.6 21.5 16.6 53.5 79.6Mannitol (ng m−3) 20.2–121.8 43.9 19.3 72.5 121.8Levoglucosan (ng m−3) 36.0–1820.9 396.5 405.3 1820.9 1519.1Mannosan (ng m−3) 2–100.8 21.9 24.2 95.0 100.8Na+ (μg m−3) 0–2.3 0.5 0.7 0.3 0.60NH4

+ (μg m−3) 0–17.1 4.6 4.6 3.5 5.4K+ (μg m−3) 0.8–8.5 3.0 1.8 6.4 8.5Ca2+ (μg m−3) 0–8.5 2.4 2.0 1.6 2.2F− (μg m−3) 0.2–0.6 0.3 0.1 0.3 0.3Cl− (μg m−3) 0.4–6.9 2.8 1.7 6.0 6.8NO3

− (μg m−3) 3.7–31.7 9.7 6.2 6.1 9.5SO4

2− (μg m−3) 4.5–37.2 15.5 9.0 8.9 14.0

a The number of samples was 29.b SD: standard deviation.c WSOC: water-soluble organic carbon.

71Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

electrode) units. The waveform used for pulsed amperometric detectionwas the standard quadruple potential for carbohydrate analysis. The sep-aration was carried out on a Dionex CarboPac MA1 column (4 mm×250 mm) with a CarboPac MA1 guard column (4 mm×50mm) witha sodium hydroxide solution (400 mM) eluent at a flow rate of0.4 mLmin−1 (Linuma et al., 2009). The standard solutions (10 mL)were prepared freshly at the beginning of each analytical week andstored at 4 °C. A calibration was performed for each analytical sequence.The detection limits of arabitol, mannitol, levoglucosan and mannosanwere 0.001, 0.002, 0.002 and 0.002 mg L−1, respectively.

Ionic species were measured by ion chromatography (DionexDX-500, Dionex Corp., Sunnyvale, CA, USA). Cation (Na+, NH4

+, K+,Mg2+, and Ca2+) concentrations were determined using a CS12Acolumn (Dionex Corp, Sunnyvale, CA, USA) with 20 mM meth-anesulfonic acid (MSA) eluent at a flow rate of 1.0 mL min−1. Anions(SO4

2−, NO3−, NO2

−, Cl−, and F−) were separated by an AS11-HCcolumn (Dionex Corp, Sunnyvale, CA, USA) with 20 mM potassiumhydroxide (KOH) eluent at a flow rate of 1.5 mL min−1. The detectionlimits of Na+, NH4

+, K+, Mg2+, Ca2+, F−, Cl−, NO2−, NO3

−, SO42− were

0.016, 0.002, 0.003, 0.001, 0.002, 0.002, 0.032, 0.033, 0.003 and0.002 μg m−3, respectively. Blank values were subtracted from sam-ple concentrations.

A portion of each filter (0.53 cm2) was analyzed for OC/EC usinga DRI Model 2001 Thermal/Optical Carbon Analyzer. The IMPROVEthermal/optical reflectance (TOR) protocol was used for the carbonanalysis. Water-soluble organic carbon (WSOC) concentrations weremeasured using a Sievers 900 TOC Analyzer (GE Analytical Instru-ments, Boulder, CO, USA). Detailed descriptions of the analytical pro-cedures for OC/EC and WSOC as well as quality control and assuranceprocedures are reported by Cao et al. (2003) and Sun et al. (2011).

2.3. WRF modeling and satellite observations

The Weather Research and Forecast Model (WRF) (Michalakes etal., 2001) developed by the National Center for Atmospheric Research(NCAR) was used in this study to show the air flow patterns of theSichuan Basin on sub-regional scale. The model was run with a hori-zontal resolution of 30 km×30 km in a domain centered in Chengdu(30.30 °N, 104.00 °E). The boundary condition of the model wasused from the National Center for Environmental Prediction (NCEP)assimilated dynamical inputs (1°×1°) in every 1 h. A detailed de-scription on the model analysis work is available in the literature(Michalakes et al., 2005). Fire counts observed by the ModerateResolution Imaging Spectroradiometer (MODIS) were used to identifythe geographical hot spots of biomass burning activities. The fire datawere obtained by FIRMS (Fire information for resource managementsystem) MODIS Archive Download tool (Davies et al., 2009).

2.4. Meteorological observations and statistical analyses

An on-site automatic weather station (Vaisala MAWS 201, VaisalaCo., Helsinki, Finland) provided data on air temperatures, relativehumidity, rainfall, wind direction, and wind speed for each samplingoccasion. All statistical analyses were performed with SPSS softwareversion 16.0 (SPSS Institute, Chicago). Relationships between param-eters were expressed using the Pearson's correlation coefficient as thelinearity between these observed parameters. The results were con-sidered significant if the P-value was≤0.05. To identify possiblesources of the airborne arabitol and mannitol, the dataset was ana-lyzed with principle component analysis (PCA) with varimax rota-tion, which is a frequently used method for source identificationanalysis of ambient aerosols (Seinfeld and Pandis, 1998). The factorloadings of each variable can be interpreted as correlation betweenthe respective variable and the factor (Lee et al., 2007). While the fac-tor loading of a variable greater than 0.7 implies a significant loadingof the variable into the factor, the factor loading of 0.5–0.7 suggests a

strong correlation of the variable into the factor. Thus, variables with-in the same factor indicate that they are correlated and may originatefrom the same sources.

3. Results and discussion

3.1. Elevated aerosol levels and associated fungal spore tracers

Table 1 presents a summary of concentrations of aerosol tracersfor fungal material emissions, namely arabitol and mannitol, and thebiomass burning smoke tracers levoglucosan and K+ associatedwith fine aerosol (PM2.5) measured during the study period. Interest-ingly, the ambient concentrations of arabitol and mannitol in PM2.5

observed in Chengdu were extremely high, ranging from 5.2 to79.6 ng m−3 and from 20.2 to 121.8 ng m−3 with average concentra-tions of 21.5±16.6 and 43.9±19.3 ng m−3, respectively. Thesevalues were notably higher than those reported from other studiesconducted around the globe, even those in rainforests (Table 2). Inother parts of the world, high levels of ambient fungal spores wereusually found in rainforest areas (7.0–19.5 ng m−3 for arabitol and15.2–26.3 ng m−3 for mannitol) because of the intense biologicalactivities (such as in Jianfenglin and Balbina, Table 2). Fungal abun-dance in urban environments was low (0.9–5.2 ng m−3 for arabitoland 0.7–7.8 ng m−3 for mannitol) (such as in Oslo and Hong Kong,Table 2), probably due to less intense biological activities.

The concentrations of fungal tracers and biomass burning smoketracers (levoglucosan and K+) showed gradual increases from thebeginning to the end of the study period, coinciding with the gradualchange from rainy days (April 19–29), with intense daily rainfall, tothe dry period after May 8 (Fig. 2). Arabitol and mannitol were signif-icantly correlated (R=0.69, pb0.01), but the variations of arabitoland mannitol were not in the same phase in some cases (such as onApril 22 and April 26) (Fig. 2), which are worthwhile to further inves-tigate in the future. We also noted that the enhancements of fungaltracers and biomass burning tracers were almost always coincidentwith each other, with exceptions only on April 24. The elevatedarabitol, mannitol and Ca2+ concentrations on April 24, when ex-tremely high concentrations of PM2.5 (300.5 μg m−3) were observed,however, coincided with low levoglucosan concentrations. This phe-nomenon was associated with a dust storm, but a detailed analysisof this episode is beyond the scope of this paper. Other than this

Table 2Overview of arabitol and mannitol concentrations in PM2.5 observed at different locations and seasons.

Arabitol (mean) (ng m−3) Mannitol (mean) (ng m−3) Location Land type Time Reference

21.5 a 43.9 a Chengdu (China) Megacity Spring This study5.2 7.8 Averio (Potugal) City Summer (Pio et al., 2008)0.9 0.7 Hong Kong (China) City Summer (Hu et al., 2008)1.7 2.0 Helsfyr (Norway) City Fall (Yttri et al., 2007)1.0 1.6 Oslo (Norway) City Fall4.3 2.8 Elverum (Norway) Suburban Winter2.0 2.0 Summer0.4 0.3 Birkenes (Norway) Rural Annual4.8 5.3 K-puszta (Hungary) Rural Summer (Ion et al., 2005)7.0 16.0 Jiangfengling (China) Rainforest Spring (Zhang et al., 2010)13.8 15.2 Balbina (Brazil) Rainforest July (Graham et al., 2003)19.5 26.3 Rondonia (Brazil) Pasture October (Graham et al., 2002)19.0 22.3 Rainforest16.7 21.9 Rondonia (Brazil) Pasture Dry period (Decesari et al., 2006)9.9 20.2 Pasture Transition8.9 18.0 Pasture Wet period15.2 13.5 Juelich (German) Mixed forest July (Kourtchev et al., 2008)6.9 30 Changbai Mountain (China) Boreal forest July (Wang et al., 2008)0.7 1.9 Chongming island (China) Temperate forest June2.8 5.0 Dinghu Mountain (China) Subtropical forest August15 16 Jianfengling (China) Rainforest November

aThe number of samples was 29.

72 Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

case, enhancements of arabitol and mannitol were consistentlyaccompanied by those of levoglucosan and K+.

It is noteworthy that the average PM2.5 concentration measured inChengdu city (133.2±55.5 μg m−3) was higher than those reportedfor other megacities in China, such as Guangzhou (91 μg m−3)(Wang et al., 2006) and Beijing (115 μg m−3) (He et al., 2001) andsignificantly above the 24-h National Ambient Air Quality Standardof the U.S.A. (35 μg m−3). This is partly due to the fact that theatmospheric dispersion capability was relatively weak in the Chengduarea, as the city is located at the bottom of the Sichuan Basin, and lowdaily wind speeds were frequently recorded during the study period(Fig. 2). This was also reflected in the regional wind flow patternssimulated by the WRF model (Fig. 5). Therefore, the elevated fungaltracer abundance observed in this region could also be a consequenceof the accumulation of PM2.5.

The rough quantitative estimates of fungal concentrations and theircontributions to PM2.5mass and organic carbon in PM2.5 (OC2.5, Table 3)were obtained by using three conversion factors (arabitol andmannitolconcentration to spore number concentration, spore number concen-tration to spore fresh mass concentration, spore fresh mass concentra-tion to organic carbon concentration of spores) suggested by Baueret al. (2002a, 2008a, 2008b). The numbers of fungal spores in PM2.5 dur-ing this study ranged from 4300 to 66300 spores m−3 and 11900 to71600 spores m−3, which were comparable with the concentrationsreported for AAS andABS (range from103 to 104 m−3, with exceptionalmaximumvalues up to ~106 m−3) from tropical regions bymicroscopicinvestigations (Elbert et al., 2007). However, the estimate of fungal con-tributions to PM2.5 (0.3–1.7%) in this study was notably lower than theestimate (range from 2.3% to 18.2%, mean value 7.9%) of Mspore to PM10

in a tropical rainforest region (Zhang et al., 2010), while the contribu-tion of fungal spores to OC2.5 mass (0.8–3.0%) was comparable to theestimate (3.6%) for a mixed forest site using the samemannitol conver-sion factor (Kourtchev et al., 2008). This is likely due to the fact thatcoarse mode PM measurements were performed in the study ofZhang et al. (2010) or there were significant anthropogenic emissionscontributing to the mass concentration of aerosol in the urban area ofChengdu. Nevertheless, caution should be applied when interpretingthese data, as the conversion factors were obtained based on measure-ments at suburban and urban sites in Vienna, Austria, where ambienttemperatures, relative humidity and land cover were different fromthose in this study.

3.2. Biomass burning source of fungal tracers

Levoglucosan is emitted as a predominant and specific componentof smoke particles during thermal decomposition of cellulose, andtherefore it has been often used as chemical tracer for biomass burn-ing emissions (Bergauff et al., 2009; Simoneit, 2002; Simoneit andElias, 2001; Simoneit et al., 1999; Ward et al., 2011). In addition, K+

has been widely utilized as tracer for biomass burning as well(Andreae and Merlet, 2001; Fine et al., 2001; Yamasoe et al., 2000).The average levoglucosan concentration measured (396.5 ng m−3)in this study was comparable to those reported for mainland Chinesecities such as Beijing (307 ngm−3) (Zhang et al., 2008) and Guangzhou(200–600 ng m−3) (Wang et al., 2007), yet higher than those levelsobserved in Taiwan (262 ng m−3, biomass burning season) (Lee et al.,2008) and Hong Kong (170 ng m−3) (Wan and Yu, 2007). However,compared with sites in other countries, such as Valencia, Spain(74 ngm−3, rice straw burning) (Viana et al., 2008), Washington andIdaho, U.S.A. (78 ng m−3, agricultural burning) (Jimenez et al., 2006),and several European background sites (50–430 ng m−3) (Puxbaumet al., 2007), the average levoglucosan concentration in this study wassignificantly higher. This tends to suggest that there are predominantsources (i.e., biomass/biofuel burning emissions) of levoglucosan inChina.

In this study, we used levoglucosan and K+ as tracers of biomassburning emissions to investigate the possible linkage between fungalmaterial and biomass burning emissions. Total K+ ion instead of non-sea salt K+ was used here, as the Sichuan Basin is far away from theinfluence of the sea.We found a statistical correlation between arabitol,mannitol and levoglucosan (R=0.82, pb0.01 and R=0.62, pb0.01,respectively) and K+ (R=0.78, pb0.01 and R=0.46, pb0.05, respec-tively), while the correlations between arabitol and mannitol, andother molecular tracers such as Ca2+ (tracer for soil emissions), SO4

2−

(indicator for secondary aerosol), and Na+ (tracer of sea salt emissions)are not significant (not shown).

The correlation between biomass burning emissions and fungaltracers observed in our study further supports the results of a fewprevious studies which found that fungal spores could be emittedthrough biomass burning activities. For instance, burning of crops,brush, timber and waste may disperse large numbers of fungi, whilesmall burns of diseased plants as well as cooking and heating withdiseased firewood might disperse spores on a smaller scale (Pisaric,

Fig. 2. Variation of daily average arabitol, mannitol, levoglucosan, K+ and PM2.5 concentrations, as well as relative humidity, temperature and precipitation throughout the samplingperiod.

73Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

2002). Pisaric (2002) also found that agitation of leaves caused byturbulent convection may dislodge spores, which would then becarried upward with the warm plume. Furthermore, turbulent airrushing in to feed the flames may also carry spores into the smokeplume, and plant pathogens might be spread considerably fartherthan surrounding fields (Kendrick, 2000; Pisaric, 2002). Moreover,fungal spores could also be transported over long distances in smokefrom biomass fires (Mims and Mims, 2004). Therefore, based on theresults presented above, we suggest that biomass burning emissionswere the primary source of mannitol and arabitol in this area.

In fact, principle component analysis using the measured chemicalspecies revealed that arabitol, mannitol, levoglucosan and water-soluble potassium (K+) were grouped within the first componentcontributing to the aerosol mass, which accounted for 36.9% of thetotal variance with the factor loadings of arabitol (0.8), mannitol(0.5), levoglucosan (0.9), K+ (0.9) (Table 4). Arabitol and mannitol,however, showed poor correlation with relative humidity and windspeed (not shown), although active release ofwet discharged ascospores

and basidiospores is known to occur in most ascomycetes and inhymenomycetes (the largest order of basidiomycetes) (Ingold andHudson, 1993). This phenomenon implies that the sources of fungalmaterial at Chengdu city may not be based on natural discharge.

Calcium (Ca2+) was loaded in the component 2, together withsodium (Na+) and magnesium (Mg2+). Generally, the soluble Na+

and Mg2+ (which are the first and second most abundant cations inseawater) are usually associated with sea salt. However, Chengdu isan inland basin city which is over 1000 km away from the nearestsea (North Bay of China). Therefore, we suggested that component2 was not affected by the sea salt source. In contrast, enrichmentof Ca2+ is usually associated with crustal material with enrichedCaCO3, so we inferred that component 2 was related with calcium-rich dust. Sulfate, nitrate and ammonium were loaded in component3. Because sulfate, nitrate and ammonium are mostly derived fromanthropogenic sources in a megacity (such as from coal burning,vehicle emissions, and livestock), we inferred that component 3was related with local emissions of air pollutants in Chengdu.

Fig. 3. Arabitol and mannitol concentrations during biomass burning days and non-biomass burning days.

Table 3Estimates of spore concentrations, spore mass concentrations, spore organic carbonconcentrations and contributions of spore mass to PM, spore organic carbon to OC2.5

and WSOC.

Concsporea

spores m−3Mspore

b

μgm−3OCsporec

μg m−3Mspore/PMd

%OCspore/OC2.5%

OCspore/WSOC%

Arabitol (n=29)e

Average 18256 0.60 0.24 0.46 1.17 2.19Std. 13941 0.46 0.18 0.30 0.65 1.49Max. 66333 2.19 0.86 1.35 3.56 8.48Min. 4346 0.14 0.06 0.13 0.37 0.69

Mannitol (n=29)Average 25810 0.85 0.34 0.73 1.83 3.35Std. 11346 0.37 0.15 0.38 0.63 1.22Max. 71647 2.36 0.93 1.65 2.97 6.11Min. 11903 0.39 0.15 0.29 0.79 1.40

aConcspore=number concentration of spores; 1.2 pg arabitol spore−1; 1.7 pg mannitolspore−1 (Bauer et al., 2008a).bMspore=mass of fungal spores; 33 pg fresh mass spore−1 (Bauer et al., 2008b).cOCspore=organic spore carbon of fungal spores; 13 pg OC spore−1 (Bauer et al., 2002a).dPM=PM2.5 mass concentration.en=number of samples.

74 Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

3.3. Biomass burning events and their impacts on fungal tracerenhancements

Eight sampling days (April 29, May 5, 8, 10–13 and 17) were cat-egorized as biomass burning days, by defining an episode event as theone with levoglucosan and K+ concentrations simultaneously higherthan their average concentrations (396.5 ng m−3 and 3.0 μg m−3, re-spectively, Fig. 2), while the other days were defined as non-biomassburning days. Concentrations of arabitol and mannitol on biomassburning days (37.3±19.8 ng m−3 and 56.0±28.8 ng m−3, respec-tively) were found to be significantly higher than those on non-biomass burning days (15.1±9.7 ng m−3 and 40.0±11.7 ng m−3,respectively) (Fig. 3). The high levels of arabitol and mannitol on bio-mass burning days were mainly associated with the samples of May13 and 17. The enhancement in arabitol and mannitol concentrations(Fig. 2) were 2.5 and 1.7 (May 13) and 3.7 and 2.8 (May 17) times theaverage tracer concentrations, respectively, while the concentrationsof levoglucosan on May 13 and 17 were 4.6 and 3.8 times higherthan the average concentration. In addition, mannosan, WSOC, K+,Cl− and OC were also elevated (at least>45%) in these episodes(Table 1). The enhancements of these species are also apparent forall the episode cases, as shown in Fig. 4, which displays the normal-ized species concentrations of both episode and non-episode cases

Table 4Results of principle component analysis on the molecular tracers and ions.

Factor Component

1 2 3

Arabitol 0.79 0.45 0.00Mannitol 0.54 0.50 −0.32Levoglucosan 0.92 0.24 −0.13Na+ −0.27 0.73 0.52NH4

+ 0.46 −0.63 0.60K+ 0.86 0.16 −0.09Mg2+ −0.24 0.72 0.44Ca2+ −0.38 0.76 0.34F− −0.30 0.45 0.69Cl− 0.79 0.28 0.40NO3

− 0.39 −0.53 0.70SO4

2− 0.31 −0.52 0.74% of total variance 36.89 26.53 21.31Eigenvalue 4.79 3.45 2.77Cumulative % 36.89 63.42 84.74

against the overall average concentrations. The two cases (May 13and May 17) are discussed in the subsequent paragraph.

The concentrations of arabitol (79.6 ng m−3) and mannitol(121.8 ng m−3) on May 17 were the highest during the entire studyperiod, coinciding with the highest concentration of K+ (8.5 μg m−3)and second highest concentration of levoglucosan (1519.1 ng m−3).We found that there were several active fires present in the Chengduregion during these days (Fig. 5b). Indeed the hot spots of fireswere identified at 50 km distance away from Chengdu city. Inten-sive agricultural residue burning activities (especially rape andwheat straw burning) occur on small scales across the SichuanBasin during each harvest season (typically in April and May),although straw burning is officially prohibited by the regional gov-ernment. However, many of these burns may not be detected bysatellite, because the fires may be too small (i.e., too cool) to bedetected within the 1 km2 MODIS footprint (Justice et al., 2002).Furthermore, the whole Sichuan Basin experienced stagnant meteo-rological conditions with low wind speeds on May 17 (Fig. 5b),resulting in the air masses being trapped in the Sichuan Basin foralmost 3 days (figure not shown), with the consequence of strongaccumulation of biomass smoke particles. Both conditions resultedin elevated concentrations of aerosol and smoke tracers (levoglucosanand K+) in particular, as well as fungal tracers in the basin. With thesecond highest concentrations of arabitol (52.3 ng m−3) and mannitol(72.5 ng m−3) onMay 13, a similar biomass burning episode character-ized by enhanced biomass burning activities and relatively stagnantmeteorological conditions occurred in this region on that day and theday before (Fig. 5a). It is worthwhile to note that compared with May

Fig. 4. The normalized concentrations (differences between episode (or non-episode)and overall average concentration against the overall average concentrations) of allmeasured species.

Fig. 5. Wind vectors at 08:00 (UTC) on May 13 (a) and May 17 (b) based on WRF modeling. The scale of the vertical bar indicates the wind speed, with the colors denoting regionswith certain wind speeds. Fires for the past 48-h period and the location of Chengdu were indicated by red squares and stars, respectively.

75Y. Yang et al. / Science of the Total Environment 431 (2012) 68–77

17, wind speeds onMay 13were slightly higher, resulting inmore effec-tive pollutant dispersion and thus smaller accumulation of aerosol com-pared to May 17.

4. Conclusions

In this paper, we present the measurement results of fungal molec-ular tracers (arabitol and mannitol) in fine particle samples collected inChengdu city in the Sichuan Basin during the biomass burning season inspring 2009. Biomass burning tracers (levoglucosan and K+), active firecount maps and air flow patterns were used to identify the predomi-nant sources of fungal tracers. Arabitol and mannitol measured inChengdu city showed the highest concentrations among any previousreports in the literature. The fungal tracers were significantly correlatedwith simultaneously measured levoglucosan and K+, suggesting thatthe elevated fungal tracers were mainly associated with biomass burn-ing activities, constituting an important source of airborne particles. Theresults from this study thus may imply the important role of biomassburning activities as sources of fungal material. However, in order todraw a solid conclusion, many uncertainties remain to be solved. Forinstance, how do the concentrations of these fungal tracers actually re-late to the number of spores? What kinds of spores do these fungaltracers represent?How large are the relative contributions of other pos-sible sources?More careful work, where we check the molecular tracertechnique againstmore robustmolecular biology techniques,may bringus closer to solve these issues, as discussed by Lang-Yona et al. (2012).

Acknowledgements

We thank Dr. Yinon Rudich for critical comments and valuable dis-cussions of the manuscript. We also thank three additional anonymousreviewers for valuable and helpful suggestions and comments on themanuscript. This study was financially supported by the Special Scien-tific Research Funds for Environment Protection Commonwealth Sec-tion (No. 200809143 and No. 201009001), Natural Science Fund ofChina (Project No 40875075), the Fundamental Research Funds forthe Central Universities (2010380003161542), and the Tans‐Century

Training Programme Foundation for the Talents offered by theMinistryof Education of China.

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