Anal. Chem. 2002, 74, 91-95
Determination of the Carbon Content of Airborne Fungal Spores Heidi Bauer,† Anne Kasper-Giebl,*,† Franziska Zibuschka,‡ Regina Hitzenberger,§ Gu 1 nther F. Kraus,⊥ and Hans Puxbaum†
Institute for Analytical Chemistry, Vienna University of Technology, Getreidemarkt 9/151, A-1060 Vienna, Austria, Institute for Water Provision, Water Ecology, and Waste Management, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria, Institute of Experimental Physics, University of Vienna, Boltzmanngasse 5, A-1090 Vienna, Austria, and Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
Airborne fungal spores contribute potentially to the organic carbon of the atmospheric aerosol, mainly in the “coarse aerosol” size range 2.5-10 µm aerodynamic equivalent diameter (aed). Here, we report about a procedure to determine the organic carbon content of fungal spores frequently observed in the atmosphere. Furthermore, we apply a new (carbon/individual) factor to quantify the amount of fungal-spores-derived organic carbon in aerosol collected at a mountain site in Austria. Spores of representatives of Cladosporium sp., Aspergillus sp., Penicillium sp., and Alternaria sp., the four predominant airborne genera, were analyzed for their carbon content using two different analytical procedures. The result was an average carbon content of 13 pg C/spore (RSD, 46%), or expressed as a carbon-pervolume ratio, 0.38 pg C/µm3 (RSD, 30%). These values are comparable to conversion factors for bacteria and some representatives of the zooplankton. Because biopolymers are suspected of interfering with elemental carbon determination by thermal methods, the amount of “fungal carbon” that might be erroneously mistaken for soot carbon was determined using the “two-step combustion” method of Cachier et al.1 and termed as “apparent elemental carbon” (AEC). This fraction amounted to up to 46% of the initial fungal carbon content. Although the aerosol samples were collected in March under wintry conditions, the organic carbon from fungal spores amounted to 2.9-5.4% of organic carbon in the “coarse mode” size fraction. The carbon content of atmospheric aerosol samples can be classified into organic carbon, elemental carbon, and carbonates. Only a small part of the organic aerosol material is fully characterized.2,3 In recent studies, single contributors, such as cellulose,4 * Corresponding author. Phone: +43-1-58801-15130. Fax: +43-1-58801-15199. E-mail:
[email protected]. † Vienna University of Technology. ‡ Institute for Water Provision, Water Ecology, and Waste Management, University of Agricultural Sciences. § University of Vienna. ⊥ Institute of Applied Microbiology, University of Agricultural Sciences. (1) Cachier, H.; Bremond, M. P.; Buat-Me´nard, P. Tellus 1989, 41B, 379-390. (2) Rogge, W. F.; Mazurek, M. A.; Hildemann, L. M.; Cass, G. R.; Simoneit, B. R. T. Atmos. Environ. 1993, 27A, 1309-1330. 10.1021/ac010331+ CCC: $22.00 Published on Web 11/30/2001
© 2002 American Chemical Society
humic-like substances (HULIS),5,6 and bacteria7 were quantified. Single-particle analysis was used to determine the number of biological particles in aerosols.8,9 At present, no information about the contribution of airborne fungal spores to the carbon content of atmospheric aerosol samples is available. Airborne fungal spores with sizes ranging from 2 µm to more than 20 µm have been observed not only in continental air masses but also above the ocean10 and even in the stratosphere.11 Reports focus on their toxicity, and on allergies or general medical implications of fungi sampled indoors,12 in public places,13,14 or near pollution sources.15 The composition of the airborne fungal flora is very heterogeneous, with representatives of molds, yeasts, and ligniscolous and lichenized basidiomycetes, etc. Methods commonly applied for the determination of airborne fungi yield predominantly cultivable molds and yeasts. Among those four genera of molds, those predominantly found are Cladosporium sp., Penicillium sp., Aspergillus sp., and Alternaria sp.11,14,16-18 Principally, the different species can be identified by cultivation, but only a part (0.3 to 38%) of the total number, which has to be determined by microscopic enumeration, can be cultivated.19 Cultivation depends mainly on three factors: whether a fungus grows on an artificial medium, the type of medium and the (3) Saxena, P.; Hildemann, L. J. Atmos. Chem. 1996, 24, 57-109. (4) Kunit, M.; Puxbaum, H. Atmos. Environ. 1996, 30, 1233-1236. (5) Havers, N.; Burba, P.; Lambert, J.; Klockow, D. J. Atmos. Chem. 1998, 29, 45-54. (6) Decesari, S.; Facchini, M. C.; Fuzzi, S.; Tagliavini, E. J. Geophys. Res. [Atmos.] 2000, 105, 1481-1489. (7) Bauer, H.; Kasper-Giebl, A.; Lo ¨flund, M.; Giebl, H.; Hitzenberger, R.; Zibuschka, F.; Puxbaum, H. Atmos. Res., submitted. (8) Matthias-Maser, S. In Atmospheric Particles; Harrison, R. M., Van Grieken, R., Eds.; John Wiley & Sons Ltd.: New York, 1998; Chapter 10. (9) Matthias-Maser S.; Brinkmann J.; Schneider W. Atmos. Environ. 1999, 33, 3569-3575. (10) Gregory, P. H. The Microbiology of the Atmosphere, 2nd ed.; Leonard Hill Books: Aylesbury, Bucks, U.K., 1973; pp 146-169. (11) Bruch, C. W. Airborne Microbes, Seventeenth Symposium of the Society for General Microbiology, held at the Imperial College, London, 1967. (12) Pastuszka, J. S.; Kyaw Tha Paw, U.; Lis, D. O.; Wlazlo, A.; Ulfig, K. Atmos. Environ. 2000, 34, 3833-3842. (13) Jones, B. L.; Cookson, J. T. Appl. Environ. Microbiol. 1983, 45, 919-934. (14) Picco, A. M.; Rodolfi, M. Int. Biodeterior. Biodegrad. 2000, 45, 43-47. (15) Reinthaler, F. F.; Marth, E.; Eibel, U.; Enayat, U.; Feenstra, O.; Friedl, H.; Ko ¨ck, M.; Pichler-Semmelrock, F. P.; Pridnig, G.; Schlacher, R. Aerobiologia 1997, 13, 167-175. (16) Al-Suwaine, A. S.; Hasnain, S. M.; Bahkali, A. H. Aerobiologia 1999, 15, 121-130 (17) Takahashi, T. Mycopathologia 1997, 139, 23-33
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conditions of incubation, and the vitality of the single spores. When the numerical concentration of fungal spores is available, their contribution to organic aerosol carbon can be estimated using a conversion factor. In the case of bacteria, conversion factors have been determined20-22 and have already been applied to atmospheric samples.7 Nothing comparable exists for fungal spores. Here, we present two procedures to determine the carbon content of the spores of representatives of the predominant airborne genera. Both analytical procedures are routinely used for the determination of carbon in aerosol samples, but they have not yet been used for the analyses of fungal spores. Furthermore, we calculate conversion factors to relate numerical concentrations of fungal spores to their carbon content. On the basis of bulk- and size-classified aerosol samples collected at a mountain site in Austria (Mt. Rax, 1644 m above sea level), the contribution of fungal spores to the carbon content of a continental background aerosol was derived. Because the sampling was carried out in winter and the area was entirely covered with snow, little biogenic activity could be expected; therefore, the numerical concentrations of fungal spores reported from Mt. Rax can be regarded as a lower limit for the continental lower troposphere. EXPERIMENTAL SECTION Preparation of the Spore Suspensions. The first set of samples comprised the spores of two test strains of molds commonly found in environmental samples, Aspergillus niger van Tieghem (ATCC16404) and Penicillium chrysogenum Thom (VIAM269). They were cultivated on trypticase soy agar (Oxoid CM131) at 25 °C for 20 days. For analysis, the mature spores of A. niger and P. chrysogenum were suspended in a sterile, particle-free NaCl solution (9 g NaCl in 1 L demineralized water). To get a more representative data set, a second set of samples was analyzed, which consisted of more test strains and fungal spores cultivated from ambient aerosol and rainwater samples. The test strains were Penicillium aurantiogriseum Dierckx (VIAM1455), Cladosporium cladosporioides (Fres.) de Vries (VIAM 1448), and Cladosporium herbarum (Pers.) Link (VIAM1449), cultivated on 2% malt extract agar (Oxoid L39 with a supplement of 0.01 g/L FeSO4‚7H2O, 0.01 g/L ZnSO4‚7H2O, and 0.005 g/L CuSO4‚5H2O) at 25 °C for 14 days, as well as Alternaria alternata (Fr.) Keissler (VIAM1450), cultivated on corn meal agar (Difco 238620) at 25 °C for 14 days. Thus, representatives of all four genera predominantly found in indoor and outdoor aerosol samples11,14,16,17 were analyzed. The aerosol and rainwater samples were collected on Mount Rax in March 2000 during an intensive measurement campaign of aerosol and cloud properties. A detailed description of the sampling site is given elsewhere.23 Rainwater was collected in a heat-sterilized glass bottle equipped with a flame-sterilized steel funnel on top. (18) Introduction to Food- and Air-Borne Fungi, 6th ed.; Samson, R. A., Hoekstra, E. S., Frisvad, J. C., Filtenborg, O., Eds.; Centraalbureau voor Schimmelcultures: Utrecht, The Netherlands, 2000. (19) Lappalainen, S.; Nikulin, M.; Berg, S.; Parikka, P.; Hintikka, E.-L.; Pasanen, A.-L. Atmos. Environ. 1996, 30, 3059-3065. (20) Loferer-Kro ¨ssbacher, M.; Klima, J.; Psenner, R. Appl. Environ. Microbiol. 1998, 64, 688-694. (21) Pelegrı´, S. P.; Dolan, J.; Rassoulzadegan, F. Aquat. Microb. Ecol. 1999, 16, 273-280. (22) Sattler, B.; Puxbaum, H.; Psenner, R. Geophys. Res. Lett. 2001, 28, 239242. (23) Hitzenberger, R.; Berner, A.; Giebl, H.; Drobesch, K.; Kasper-Giebl, A.; Lo ¨flund, M.; Urban, H.; Puxbaum, H. Atmos. Environ. 2001, 35, 51355141.
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Aerosol samples were collected on open-face cellulose nitrate filters (Sartorius; 47 mm Ø; pore width, 0.8 µm). After cultivation of the samples on dichloran-glycerol agar (DG-18, Oxoid CM729 with 0.1 g/L chloramphenicol supplement) at 25 °C for 20 days, four different fungi were selected for analysis. One of them was identified as Aspergillus versicolor (Vuill.) Tiraboschi; the others were unidentified species belonging to the genera Aspergillus, Penicillium, and Cladosporium (P11, P12, P13). The mature spores of the second set of samples (airborne fungi as well as the test strains) had to be suspended in “dilution water” (0.008% Tween 80 detergent in sterile, particle-free distilled water) because of their hydrophobic properties. During the preparation of the spore suspensions, special precautions had to be taken to avoid a contamination by the agar. Therefore, the Petri dishes were turned upside down, and the mature spores were transferred into the lid by slightly shaking the Petri dishes. In this manner they could be separated from the rest of the mycelium without being contaminated by the agar. After homogenizing them using a vortex for 3 min (Autovortex SA6, Stuart Scientific), the concentrations of the spore suspensions were determined by microscopic enumeration using a Thoma chamber (depth, 0.100 mm; 0.0025 mm2; microscope, Olympus CH2; magnification, 400). At least 64 fields were counted. Under these conditions, the relative standard deviations of the numerical concentrations were, on average, 37%. Only in the case of A. alternata were higher values obtained because of the small numerical concentration. The diameter or the length and width of the spores were measured at a magnification of 1600 at an accuracy of (0.2 µm (Axioplan 2, Zeiss). For the calculation of the spore volumes, a spherical (A. niger, P. aurantiogriseum, P. chrysogenum, P11 and P12) or ellipsoidal (A. alternata, A. versicolor, Cladosporium ssp. and P13) form was assumed. For the calculation of the volume of the spores of Cladosporium sp. and A. alternata, which are ellipsoidal or obclavate to ellipsoidal and irregular in size (C. cladosporioides, 3-7 µm in length and 2-3.5 µm in width; C. herbarum, 5-11 µm in length and 3-5 µm in width; P13, 3-6 µm in length and 3.5-5 µm in width; A. alternata, 12-22 µm in length and 9-11 µm in width), the arithmetic mean (n ) 7) of length and width was taken. Determination of the Total Carbon Content. To determine the carbon content of the fungal spores, two different analytical procedures were used. Procedure I. A standard method for the determination of total carbon and total organic carbon in liquid samples was modified for the analysis of the first set of samples. It is based on a commercial instrument (GOTOC 100, Gro¨ger & Obst) that operates on continuous flow (flow rate, 0.5 mL/min). The liquid samples are combusted in a catalytic combustion furnace operating at 850 °C and are purged by precleaned (sodalime, activated carbon) ambient air (air flow, 0.4 L/min). CO2 resulting from the combustion is determined using a nondispersive infrared (NDIR) detector. Originally, the instrument was set up to evaluate steadystate concentrations. To allow the analysis of smaller sample volumes, which is often necessary for environmental samples, the instrument was modified using a 6-port injection valve and, thus, was converted to a flow injection system. To eliminate the influence of dissolved CO2, the samples were acidified with nitric acid (100 µL 1 N HNO3/mL), stripped with pure N2, and then
Table 1. Carbon Content and AEC of the Different Spore Suspensions
a
fungus
spore concn (spores/mL)
na
A. alternata A. niger C. cladosporioides C. herbarum P. aurantiogriseum P. chrysogenum
1.1 × 105 2.5 × 107 8.4 × 106 2.0 × 106 9.7 × 106 6.4 × 107
3 6 2 4 3 6
test strains 32 360 80 160 88 870
A. versicolor P11 P12 P13
9.2 × 106 1.8 × 107 6.0 × 106 3.5 × 107
8 9 8 8
airborne fungi 67 152 84 769
C content (µg C/mL)
RSD (%)
na
15 24 4.2 5.3 0.19 17
2
19 24 29 31
2 4 2
3 5 5 5
AEC (µg C/mL) 15 ndb bdlc 17 28 nd 7 14 bdl 33
RSD (%) 11 4.6 18
3 22 16
Number of replicates. b Not determined. c Below detection limit.
injected into the sample loop (300 µL). Standard solutions for calibration were prepared from phthalic acid (p.a., Merck). The limit of quantification (6 s of the multiple (n ) 9) injection of demineralized water) was 0.4 µg C/mL, corresponding to an absolute limit of quantification of 0.12 µg C. This method was used for A. niger and P. chrysogenum, that is, the first set of samples. The NaCl solution was used as a blank. Procedure II. A combustion method based on the system described by Puxbaum and Rendl24 was used for the analysis of the second set of samples. A 30-µL aliquot of the sample was pipetted onto small pieces of aluminum foil that had been precleaned by heating at 450 °C for 10 hours. Then the liquid samples were evaporated in a drying oven at 70 °C for 40 min. For analysis, the dried samples were combusted at 1050 °C in a pure oxygen flow (0.8 L/min). The resulting CO2 was detected by a standard NDIR analyzer for continuous emission monitoring (MAIHAK Unor 6N) using a nominal detection range of 0.0-0.2% (vol) CO2. The calibration was performed using standard solutions prepared from phthalic acid (p.a. Merck). The absolute limit of quantification (6 s of multiple (n ) 9) analysis of demineralized water) was 0.4 µg C. The carbon content of the dilution water was used as a blank value. Determination of the Apparent Elemental Carbon (AEC). Regarding the determination of organic carbon (OC) and elemental carbon (EC) in aerosol samples, a variety of methods have been described in the literature, starting with thermal methods in the mid-1970s.25-27 However, thermal methods have difficulties in separating polymeric organic substances, for example, biological material, such as plant debris or lignin, from EC. Consequentely, a siginificant amount of OC might be erroneously attributed to EC because of charring of organic substances during pyrolysis.28,29 Here we use the term “apparent elemental carbon” to describe (24) Puxbaum, H.; Rendl, J. Microchim. Acta 1983, 1, 263-272. (25) Ga`l, S.; Paulik, F.; Pell, E.; Puxbaum, H. Fresenius’ Z. Anal. Chem. 1976, 282, 291-295. (26) Malissa, H.; Puxbaum, H.; Pell, E. Fresenius’ Z. Anal. Chem. 1976, 282, 109-113. (27) Novakov, T. In Proceedings of the International Microchemical Symposium; Malissa, H., Grasserbauer, M., Belcher, R., Eds.; Springer: Vienna, Austria, 1981; pp 141-165. (28) Puxbaum, H. Fresenius’ Z. Anal. Chem. 1979, 298, 250-259. (29) Cadle, S. H.; Groblicki, P. J. In Particulate Carbon: Atmospheric Life Cycle; Wolff, G. T., Klimisch, R. L., Eds.; Plenum Press: New York, 1982; pp 89109.
this fraction of OC. Optical correction procedures to overcome the interference have been developed and successfully applied.30 As an alternative to optical correction procedures, a two-step combustion procedure was described1 that compares well with the thermal-optical methods.31,32 However, testing of the twostep combustion method for analytical artifacts showed that for natural products, such as pollen or plant debris, charring still can be a problem.1 This led us to investigate a potential AEC contribution of fungal spores. Sample preparation and analysis is the same as described for the determination of the carbon content with procedure II, but as in the two-step combustion method,1 a preheating step is interposed after the evaporation of the samples. During this step, the samples are kept at 340 °C in a pure oxygen flow (1.3 L/min) for 2 h in a muffle-oven. The intention of this treatment is to oxidize organic compounds to CO2, leaving EC on the substrate. In the subsequent combustion step at 1050 °C, only elemental carbon should be detected. AEC was analyzed from the spores of the second set of samples only. Determination of the Dry Weight. The dry weight of the fungal spores was determined by drying 1 mL of sample of a known numerical concentration at 104 °C until constant weight. The gravimetric determinations were carried out at 20 °C and 50% RH using a microbalance. RESULTS AND DISCUSSION Carbon Content of Fungal Spores. Table 1 presents the spore concentrations, the carbon concentrations, and the apparent elemental carbon concentrations of the investigated suspensions of 6 test strains and 4 strains isolated from airborne samples. Comparing the carbon content and dry mass of the suspensions of the airborne spores yields carbon contents from 42 to 66% (average, 51%) of the dry mass of the spores. (30) Delumyea, R. G.; Chu, L. C.; Macias, E. S. Atmos. Environ. 1980, 14, 647652. (31) Guillemin, M.; Cachier, H.; Chini, C.; Dabill, D.; Dahmann, D.; Diebold, F.; Fischer, A.; Fricke, H. H.; Groves, J. A.; Hebisch, R.; Houpillard, M.; Israel, G.; Mattenklott, M.; Moldenhauer, W.; Sandino, J. P.; Schlums, C.; Sutter, E.; Tucek, E. Int. Arch. Occup. Health 1997, 70, 161-172. (32) Schmid, H.; Laskus, L.; Abraham, H. J.; Baltensperger, U.; Lavanchy, V.; Bizjak, M.; Burba, P.; Cachier, H.; Crow, D.; Chow, J.; Gnauk, T.; Even, A.; ten Brink, H. M.; Giesen, K. P.; Hitzenberger, R.; Hueglin, C.; Maenhaut, W.; Pio, C.; Carvalho, A.; Putaud, J. P.; Toom-Sauntry, D.; Puxbaum, H. Atmos. Environ. 2001, 35, 2111-2121.
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Table 2. Carbon Content of Spores and Carbon to Volume (C/V) Ratio and Percentage of AEC on the Carbon Content of Different Molds fungus
spore size diam (µm)
A. alternata A. niger C. cladosporioides C. herbarum P. aurantiogriseum P. chrysogenum
la ) 12-22, wb ) 9-11 4 l ) 3-7, w ) 2-3.5 l ) 5-11, w ) 3-5 3.2 3.5-4
A. versicolor P11 P12 P13
l ) 3.5, w ) 2.5-3 4 5 l ) 3-6, w ) 3.5-5
vol (µm3) test strains 890c 34 14 63 17 25 airborne fungi 14 34 65 53
av
34
C/spore (pg C/spore)
C/V ratio (pg C/µm3)
AEC (%)
292c,d 10e 5.2d 23d 9.1d 14e
0.33 0.31 0.36 0.37 0.53 0.53
46 nd f bdl g 30 32 nd
7.3d 8.3d 14d 22d
0.52 0.25 0.21 0.42
12 10 bdl 3.5
13
0.38
a Length. b Width. c Not included in the average. d Determined with procedure II. e Determined with procedure I. f Not determined. g Below detection limit.
Figure 1. Relationship between C/V ratio and volume of spores of 10 different fungal species. The error bars include relative standard deviations of the counting statistics of microscopic enumeration of the spore suspensions, of the size, and of total carbon determination.
To obtain a more universally applicable result of the analysis, the carbon content per single spore and the carbon per spore volume were calculated. These parameters are conversion factors commonly used for biomass estimations in limnology and are given in Table 2, together with the dimensions of the spores. Disregarding A. alternata, the carbon content per spore ranges from 5.2 to 23 pg C/spore. No difference was obtained between the test strains (5.2-23 pg C/spore) and the airborne fungi (7.322 pg C/spore). A. alternata showed a markedly higher carbon content per spore. Generally, A. alternata will not be present in samples of ambient aerosols, because it is too large to be collected under standard sampling protocols (PM 10, PM 2.5). Consequently, A. alternata was excluded for the calculation of the average carbon content. This average value amounts to 13 pg C/spore (RSD, 46%). 94
Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
In limnology, volume-based conversion factors are used more often than factors based on the carbon content of individual spores. The C/V ratio of fungal spores (in this case A. alternata can be included) ranges from 0.21 to 0.53 pg C/µm3, with an average of 0.38 pg/µm3 (RSD, 30%). In Figure 1, C/V ratios are plotted versus the spore volume. No dependency of the C/V ratio on the spore size could be observed, although the highest ratio (0.53 pg/µm3) was measured for the smallest spores. Literature values of conversion factors for bacteria vary between 0.05933 and 0.73 pg C/µm3.34 On the other hand, Pelegrı´ et al.21 analyzed the carbon content of two marine flagellates having different volumes (39 and 1590 µm3) and one ciliate with an average volume of 846 µm3, and obtained very similar conversion factors: 0.12 pg C/µm3 for (33) Nagata, T.; Watanabe, Y. Appl. Environ. Microbiol. 1990, 56, 1303-1309. (34) Bjørnsen, P. K. Appl. Environ. Microbiol. 1986, 51, 1199-1204.
Table 3. Contribution of Fungal Carbon to Organic Aerosol Carbon in Different Size Classesa
date
fungi (spores/m3)
C fungi (µg/m3)
OC impactor 0.1-10 µm (µg/m3)
contribution of fungi to OC (%)
OC impactor 2.12-10 µm (µg/m3)
contribution of fungi to OC (%)
Mar 14, 2000 Mar 22, 2000 Mar 25, 2000
322 174 3234
0.004 0.002 0.023
0.92 0.29 2.54
0.4 0.8 0.9
0.14 0.05 0.43
2.9 4.2 5.4
av
1243
0.010
1.25
0.7
0.21
4.2
a
The spores were collected with an impinger with an upper cut diameter of 7 µm.
the flagellates and 0.14 pg C/µm3 in the case of the ciliate. A dependency of C/V on bacterial cell size was described, but no general relationship was determined.33,35 The reason for the high variation of bacterial C/V ratios might be the difficulty of bacterial size determination and the shrinkage of the cells caused by sample conservation (in the literature, the samples were usually preserved with formalin, which leads to a shrinkage of more than 40%).21 Generally, the C/V ratios for fungal spores are on the same order of magnitude as the values for bacteria and zooplankton given above. For our application in atmospheric chemistry, a conversion factor based on a single individual (i.e., C/spore or C/cell) is preferable from a practical point of view. In this case, no measurements of spore or cell volumes have to be carried out, and if preservatives are used, the shrinkage effects do not influence the results. Apparent Elemental Carbon (AEC) of Fungal Spores. The concentrations of AEC in the spore suspensions are given in Table 1, and the percentage of the initial organic matter that is determined as AEC is listed in Table 2. Quite variable results were obtained, ranging from below the detection limit up to 46% of the initial organic matter. The highest value was obtained for A. alternata, the largest spores. These results again point to the difficulties that can be encountered by separating OC and EC in samples containing a high amount of biological material. Fungal Spores in Atmospheric Aerosol Samples. A first estimate of the contribution of fungi to organic aerosol carbon was performed for aerosol samples collected in Austria on Mt. Rax in March 2000. As mentioned earlier, because of the wintry conditions, low biological activity was expected. Bioaerosols were collected using modified impingers (0.9% NaCl solution). The impingers were equipped with an inlet having an upper cut diameter of 7 µm aed. A detailed description of the experimental setup is given elsewhere.7 The number concentrations of fungal spores collected by the impingers was determined by microscopic enumeration and ranged from 174 spores/m3 to 3234 spores/m3 in aerosol. Using the average conversion factor of 13 pg C/spore, these values could be converted to carbon concentrations ranging from 2 to 23 ng/m3, as given in Table 3. Simultaneously size-classified aerosol samples (size range, 0.110 µm) were collected using seven-stage low-pressure cascade impactors (Berner impactor, LPI 80). Analyses of the size-classified aerosol samples were performed for TC (procedure II) and for
EC (two-step combustion1). Organic carbon was calculated as the difference between TC and EC. Summarizing OC concentrations within the single-size classes, fungal spores contributed 0.4 to 0.9% of organic carbon. Visual confirmation during microscopic enumeration showed that fungal spores can be attributed to the coarse mode of the aerosol. Therefore, the contribution of fungal spores to the carbon content was apportioned only to the size range between 2.12 and 10 µm. For this case, the contribution of spores to organic carbon ranges from 2.9 to 5.4%. The results of three independent samples are given in Table 3. Because the impinger was operated using an upper cut diameter of 7 µm, the average percentage of the fungal contribution of 4% to the OC in the size range of 2.12 to 10 µm can be regarded as a lower limit, because a marked contribution of fungal spores is expected for the size range between 7 and 10 µm.
(35) Kroer, N. FEMS Microbiol. Ecol. 1994, 13, 217-224.
AC010331+
CONCLUSIONS On the basis of the analysis of test strains and airborne samples using two independent combustion procedures, the average carbon content of fungal spores was determined to be 13 pg/ spore (RSD, 46%). The volume-based conversion factor was 0.38 pg C/µm3 (RSD, 30%) on average. Using these conversion factors, the contribution of airborne fungal spores to the organic carbon content of aerosols and precipitation can be estimated by simple microscopic enumeration of the spores. Airborne fungal spores will contribute to PM 10 (because they occur predominantly in the 2-10 µm size range) and will contribute nothing or very little to PM 2.5. First results obtained from samples collected at a background site in Austria during wintry conditions showed that fungal spores represent an average of 4.2% of organic carbon detected in the coarse mode of aerosol samples. ACKNOWLEDGMENT The authors thank the Rax team for assistance in the field campaign. Special thanks go to Barbara Schuster for laboratory work and Maria Lo¨flund for helpful technical assistance. Financial support by the Austrian FWF Project P13143-CHE is acknowledged. We also thank the O ¨ sterreichischer Alpenverein (O ¨ AV) for their support. Received for review March 19, 2001. Accepted October 5, 2001.
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