Bot. Bull. Acad. Sin. (2005) 46: 53-59

PETERNEL et al. — Airborne pollen spectrum in Croatia

Airborne pollen spectra at three sites in inland Croatia, 2003

Renata PETERNEL1, Josip CULIG1, Bozena MITI ´C2, Ivana HRGA1, and Ivan VUKUŠI ´C1

1Research Department, Zagreb Institute of Public Health, Mirogojska 16, HR-1000 Zagreb, Croatia, Europe

2Department of Botany, School of Science, University of Zagreb, Zagreb, Croatia

(Received January 5, 2004; Accepted August 13, 2004)

Abstract. The aim of this study was to determine whether there were major differences in the seasonal incidence and abundance of pollen grains and pollen types in pollen fall between sites of different land use. The material was collected from 5 January until 20 December 2003 at three sites in central Croatia. The sampling sites were located in an average urban setting (Zagreb), a rural setting (Ivani´c Grad), and a small town surrounded by a large woodland with partially thermophilic vegetation (Samobor). Using the volumetric method of pollen collection, pollen grains of 35-37 taxa were identified, depending on the sampling site, eight of them producing the greatest amount of pollen (Alnus sp., Ambrosia sp., Betula sp., Carpinus sp., Poaceae, Quercus sp., Taxus/Juniperus, and Urticaceae). Differences among the sampling sites were recorded according to seasonal pollen concentration, total monthly pollen count, and total annual pollen count. The proportion of particular plant classes (tree, grass and weed) was quite comparable among the three sampling sites because of their relative geographic proximity (i.e. within the same climatic region). A difference was observed in the air pollen concentration. All three monitoring sites revealed the total annual airborne pollen concentration in inland Croatia to be dominated by highly allergenic pollen (Zagreb 54%, Samobor 58% and Ivani´c Grad 82%) of the following taxa: Alnus sp., Ambrosia sp., Betula sp., Carpinus sp., Corylus sp., Poaceae, Urticaceae and Artemisia sp. Accordingly, there were no distinct phenologically induced differences in pollen species recorded at the three monitoring sites. However, substantial differences were observed in seasonal pollen grain count and in the percentage proportion of some allergenic pollen grains (Ambrosia sp., Betula sp., Carpinus sp., Poaceae), providing valuable information to individuals suffering from pollen allergy.

Keywords: Aerobiology; Central Croatia; Pollen grain; Volumetric method.




At present, pollen is definitely one of the most potent known allergens. The number of individuals allergic to plant pollen has been steadily increasing, especially in large cities and industrial areas (Hofman and Michalik, 1998; Nilson and Persson, 1981; Obtulowicz et al., 1996). Allergenic plants are characteristic of particular areas, depending on geographic and climatic region and vegetation. The occurrence of airborne pollen is temporally limited and specific for each plant species. The principal aim of aeropalinological studies is to assess the seasonal appearance of pollen grains in the air, and to make quantitative and qualitative analyses of pollen fall or aeroplankton. Atmospheric pollen has been measured for many years at various locations in Europe. Pollen calendars for particular countries have been published in a number of national or regional pollen atlases (Nilsson et al., 1977; Horak and Jäger, 1979; Stix, 1981; D'Amato, 1984; Driessen et al., 1988). Prediction of the initial occurrence of airborne pollen allows individuals allergic to a particular plant pollen to be informed and to adjust their daily activities so as to avoid contact with the allergens, the most efficient preventive measure.

Papers published to date on spatial variation in the incidence of pollen refer to distant sites from different climatic regions (Hyde, 1959). Only rarely have aerobiological investigations been carried out at several sites within a small area of diverse land use (Emberlin and Norris-Hill, 1991).

The first research into allergenic pollen distribution in Croatia was launched in 1959 at four locations: Zagreb, Hvar, Crikvenica, and Dubrovnik (Volari´c-Mrši´c, 1972). Using a gravimetric method, data were collected weeky throughout the year. Since 1973, allergenic pollen has been continuously studied in Zagreb (Lovašen-Eberhardt, 1979). Modern aeropalinological investigations using daily volumetric monitoring of atmospheric pollen began in Croatia in 2002 (Peternel et al., 2003).

The data presented here were obtained during a one-year study conducted at three sites in inland Croatia. Our aim was to determine whether there were any major differences in seasonal occurrence or in abundance of pollen grains and pollen types in pollen fall among the sites of different land use.

Materials and Methods

Material was collected at three sites in central Croatia from 5 January to 20 December 2003. Pollen sampling and floristic research were both conducted. Pollen sampling

*Corresponding author. Phone: ++385(0)1-4696-108; Fax: ++385(0)1-4678-002; E-mail:

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

was done by use of 7-day Hirst volumetric pollen and spore traps. The samplers were placed in Zagreb (45°49' N and 15°59' E, 157 m above sea level and 19.7 m above ground level), Ivani´c Grad (45°43' N and 16°24' E, 101 m above sea level and 18.5 m above ground level), and Samobor (45° 48' N and 15°43' E, 168 m above sea level and 17.3 m above ground level). The distance between measuring sites was about 30 km (Figure 1). The sampler absorbs 10 L air per min, allowing for determination of pollen concentration at 2-h intervals. It is supplied with a timer which moves adhesive tape (2 mm/h) to which pollen grains adhere. The tape was removed twice weekly, cut to a length corresponding to 24-h pollen sampling, applied onto a glass slide, and embedded in the following medium: 70 g polyvinyl alcohol (Gelvatol) and 4 g phenol C6H6O, dissolved in 200 mL of distilled water. After resting overnight, 100 mL glycerol C3H8O3 was added and warmed up in water bath until the solution turned liquid and clear. Then, 4 drops of alcohol solution of basic fuchsin C20H20CIN3 per 100 mL were added. Samples were examined under a light microscope, magnification x400, to determine pollen type and count per 1 m3 air per day. Pollen concentration was expressed as pollen grain count/m3. Preliminary floristic research included plant collection at all three sites, in the surroundings of pollen samplers. This part of the investigation included standard field studies of autochthonous and cultivated plants (plant collection, botanizing, and data entry in floristic lists), and laboratory plant determination.

Criteria for definition of pollen allerginicity (HAP-highly allergenic pollen, AP-allergenic pollen) are based on results of skin-test positivity (percentage positive reaction in patients with pollinosis) in Europe (Bousquet et al., 1984; D'Amato and Lobefalo, 1989; Spieksma, 1986).

Results and Discussion

Zagreb is situated at the base of Medvednica Mountain, and the composition of airborne pollen there is strongly influenced by the forest vegetation to the north, which consists of the following species: Acer sp., Ulmus glabra Hudson, Fraxinus excelsior L., Carpinus betulus L., Ostrya carpinifolia L., Corylus avellana L., Fagus silvatica, Quercus petraea (Mattuschka) Liebl., Taxus

baccata L., Abies alba L. (at higher altitudes), Castanea sativa L., various species of the genus Rubus, Sambucus nigra L., Robinia pseudoaccacia L., and others. Grassy and weedy grounds prevail in rural areas east and south of the city. In these areas, the vegetation consists of a number of ruderal and weedy, wild-growing and adventive plants, primarily from the families Poaceae, Chenopodiaceae, Compositae, Brassicaceae, Urticaceae, Lamiaceae, and Fabaceae. Among the adventive plants, the allergenic species Ambrosia artemisiifolia L. and Artemisia vulgaris L. are the most widespread. In the urban area along the Sava river, the species Alnus glutinosa (L.) Gaertn. and various species of the genera Salix and Populus prevail. In addition to this more or less natural vegetation, the city has numerous parks containing the following species: Betula pendula L., various species of the family Cupressaceae and of the genera Pinus and Picea, Aesculus hippocastanum L., Platanus hybridus, Populus sp., Juglans regia L., and Tilia sp.

Ivani´c Grad is most rural of the three monitoring locations, with a lot of weedy areas. The respective airborne pollen also showed some elements of typical inland forest vegetation. A great impact of ruderal and weedy vegetation was recorded, with numerous species of the families Brassicaceae, Asteraceae, Amarantaceae, Apiaceae, Plantaginaceae, Poaceae, and others. The cultivation also contained some taxa from the family Cupressaceae, then Betula pendula, Taxus baccata, Pinus, Picea and Abies sp., and Tilia sp.

The region of Samobor is surrounded by the Zumberak-Samobor Mountains with large forests around the town and with numerous autochthonous and planted trees also found in it. Airborne pollen is influenced by this forest vegetation, which is dominated by many forest species such as Acer campestre, A. pseudoplatanus, A. platanoides, Carpinus betulus, Corylus avellana, Fagus silvatica, Quercus petraea, Ulmus glabra, Castanea sativa, Taxus baccata. In addition, this vegetation also includes a number of species characteristic of a sub-Mediterranean climate. Such a type of vegetation is mostly found on the south mountain slopes and includes woody plants such as Quercus pubescens, Ostrya carpinifolia (more common than Carpinus betulus), and Fraxinus ornus. A small river runs through the town, favoring the growth of woody plants characteristic of humid habitats, with a predominance of the species Alnus glutinosa and various species of the genera Populus (mostly P. alba in lower areas, and P. tremula in higher areas) and Salix (mostly S. caprea). Ruderal and weedy vegetation is less abundant than in the Zagreb area, and here adventive taxa of the families Asteraceae and Poaceae predominate. The city parks and gardens feature a great variety of different taxa (autochthonous and horticultural) of the genera Pinus, Juniperus/Cupressus, Abies and Picea, then widely planted Betula pendula, Juglans regia, and Tilia sp.

The highest amount of pollen grains in 2003 was counted in Ivani´c Grad (n=53,710), in Zagreb (n=52,521) and lowest in Samobor (n=32,692). The number of identified


Figure 1. Measuring sites in inland Croatia.

PETERNEL et al. — Airborne pollen spectrum in Croatia

pollen taxa was 35 in Zagreb, 36 in Ivani´c Grad, and 37 in Samobor. Eight of these taxa produced the greatest amount of pollen (Alnus sp., Ambrosia sp., Betula sp., Carpinus sp., Poaceae, Quercus sp., Taxus/Juniperus, Urticaceae), and nine occurred sporadically or in a small amount (Aesculus sp., Asteraceae, Forsythia sp., Ligustrum sp., Morus sp., Robinia sp., Rosaceae, Rumex sp., Umbelliferae) (Table 1). Monthly pollen concentrations measured at all three monitoring sites in 2003 showed two peaks (in April and August), with between-site differences. In April, pollen concentrations measured at monitoring sites in Zagreb and Samobor exceeded those recorded in Ivani´c Grad (Zagreb n=17,846; Samobor n=14,697; and Ivanic Grad n= 9,238). In August, an inverse pattern was observed (Zagreb n=7,561; Samobor n=2,847; and Ivanic Grad n=19,781) (Figure 2). The earliest airborne pollen grains recorded in March originated from the following species: Alnus sp.,

Corylus sp., Fraxinus sp. and Taxus/Juniperus. In April, the number of species increased, with the pollen originating from Betula sp. as the most common, accounting for 30.8% in Samobor, 32.6% in Zagreb, and 37.4% in Ivani´c

Figure 2. Monthly variation in airborne pollen total count according to measuring sites in inland Croatia, 2003.

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

Grad. In this month, the pollen from Quercus sp. was also found in abundance. In May, plants of the family Poaceae clearly predominated, accounting for 22.9% to 50.3% of total monthly pollen count. In June, pollination of plants from the family Poaceae continued, but the pollen originating from Castanea sp. prevailed with 22.0% in Ivani´c Grad, 57.9% in Zagreb, and 59.6% in Samobor. In July, the lowest monthly pollen count was recorded at all sampling sites, with a predominance of plants from the family Urticaceae. Ambrosia sp., the most allergenic plant of our climate, was in full bloom in August when it accounted for 70.1% to 91.8% of total monthly airborne pollen. This percentage rose further to 95.1%-99% in September. In 2003, the pollen season of the plants of the continental climate terminated in October (Table 2). Classification of plant species into the tree, grass and weed groups revealed exclusively tree airborne pollen to be found in March and April. Then in May and June the grass and weed pollen occurred while an absolute predomonance of weed pollen was recorded in July, August, September, and October. Dif

ferences were observed among the monitoring sites according to the tree to grass to weed ratio. In May, a higher proportion of grass pollen was measured at the monitoring sites in Ivani´c Grad (50.4%) and Samobor (43.7%) than in Zagreb (23.5%) while in June the proportion of grass pollen was considerably higher in Ivanic Grad (52.8%) than at the other two sampling sites. In August, September, and October, weed pollen predominated at all sampling sites, ranging from 98.6% to 100% (Figure 3). Total annual pollen concentration in the air was strongly dominated by highly allergenic pollen grains at all three sampling sites located in inland Croatia, with a percentage proportion of 54% in Zagreb, 58% in Samobor, and 82% in Ivanic Grad, including pollen of the following taxa: Alnus sp., Ambrosia sp., Betula sp., Carpinus sp., Corylus sp., Poaceae, Urticaceae and Artemisia sp. The percentage proportion of ragweed and grass pollen was highest at the Ivani´c Grad sampling site, and that of birch at the Samobor and Zagreb sampling sites (14% and 12%, respectively) (Figure 4).

Figure 3. Monthly variation in percentage of tree, grass, and weed airborne pollen in inland Croatia, 2003. Legend: AP, allergenic pollen; HAP, highly allergenic pollen.

Figure 4. Variation in airborne highly allergenic pollen according to measuring sites in inland Croatia, 2003.

PETERNEL et al. — Airborne pollen spectrum in Croatia

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

Pollen of anemophilous plants prevailed at all monitoring sites while pollen of entomophilous taxa occurred sporadically or in very low amounts. A similar pattern was reported in southeast Poland, however, with a different floral composition due to climatic differences. In Poland, pollen of the allergenic alder tree (Alnus sp.) accounts for most of the pollen count while in central Croatia the most allergenic weed, ragweed (Ambrosia sp.) does (Kasprzyk, 1999). Monthly pollen concentrations showed two peaks, in April and August, at all three sampling sites. These peaks coincided with the flowering of generally anemophilous tree and weed, especially ragweed. Similar results have also been reported from central Italy and south France (Clot, 2001; Emberlin et al., 1993; Spieksma and Frenguelli, 1991; Jäger et al., 1991). Between-site differences were observed: monthly pollen concentrations measured in April at the Zagreb and Samobor sampling sites exceeded those recorded at the Ivani´c Grad sampling site, a fact ascribed to the variation in their respective flora compositions. Ivanic Grad is a typical rural, lowland region abundant in overgrown, weedy ground, with little forest vegetation. Thus only a minor amount of tree pollen is found in the total pollen count in spring. Zagreb, and Samobor in particular are closely surrounded by large forest areas, thus the amount of tree pollen is high in the spring. A reverse pattern was observed in August, when a huge amount of ragweed pollen in the air was recorded in Ivani´c Grad, with the pollen grain count at this monitoring site approaching 20,000 or twofold that measured in Zagreb. Hyde (1959) observed the vegetation patterns to influence the pollen fall, and the highest pollen grain counts were found in samples taken at rural or suburban sites. The proportion of tree, grass and weed groups at the three sampling sites was quite comparable, with a variation recorded only in the amount of pollen in the air because the sampling sites were a relatively small distance apart, in the same climatic region, with a concurrent onset and termination of a particular plant group's pollination. This resulted in the seasonal concurrence of pollen in the entire area's atmosphere. The spectrum of highly allergenic pollen isolated from total pollen count was generally identical to that recorded in inland Europe (Lejoly-Gabriel and Leuschner, 1983; Dalen and Voorhorst, 1981; Eriksson, 1978; Spieksma et al., 1989; Horak et al., 1981; Kasprzyk, 1999), differences being only found in their percentage proportion. Ragweed pollen shows a specific pattern, as the plant has mostly spread over east and Central Europe. In our study, ragweed pollen accounted for a high proportion of total pollen in late summer and early autumn, which is not the case in northern and western European countries.

The phenology and type of pollen species showed no distinct variation among the three sampling sites. However, substantial differences were recorded in pollen grain count and percentage proportion of some highly allergenic plant pollen (Ambrosia sp., Betula sp., Carpinus sp. and Poaceae). The latter is of utmost importance for the individuals suffering from pollen allergy. This was a preliminary study, obviously calling for longterm research to be undertaken in the future.

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