Environmental pollution is increasingly higher due to human activities and results in the accumulation of heavy metals that cause various types of problems to living beings (Rai et al., 2005). Man's daily activities cause pollution of different kinds and degrees; indeed, air, soil and water get contaminated as a result of industrial and agricultural activities, transport, etc.
In big cities, air pollution levels are ground of concern. Monitoring programs and search for metals in environmental samples have become widely established as direct and indirect measurement methods. Biological monitoring constitutes an economic alternative for atmospheric pollution studies (Bellis et al., 2003; Castro & Faggi 2008; De Temmerman et al., 2004; Dion et al., 1993; Jasan et al., 2004; Panichev & McCrindle, 2004; Pignata et al., 2002; Walkenhorst et al., 1993). Plants have been observed to be far more sensitive to pollution than animals and man and are therefore used as indicators (Rani et al., 2006). Studies have recorded changes in plants due to all kinds of environmental pollutants, and most of these works refer to physiological alterations (Kabata-Pendias & Pendias, 2001; Linster, 1991; Ochiai, 1987; Patra & Sharma, 2000). Among passive monitor's assays, the multielement analysis of tree bark from urban trees (Castro et al., 2008; Faggi et al., 2008; Jasan et al., 2004; Perelman et al., 2006; Steubing et al., 2002) has been recently introduced to detect and measure the elements of air pollution retained in tree bark.
The degree of impact in plants depends on pollutants concentration, location of entry into plant and species under consideration. Different species may present varying sensitivity/tolerance levels to different contaminating agents (Srivastava, 1999). Alderdice (1967) set two categories of toxic effects: acute toxicity (due to high pollution levels during short periods of time, usually lethal) and chronic toxicity (due to low pollution levels over long periods of time, lethal or sublethal).
Toxic effects produced by pollutants on soil organisms and plants have usually been studied in laboratories under controlled conditions, but field studies have been scarce (Patra & Sharma, 2000). Stomatal and epidermal cell size, lower frequency, thickening of cell wall, epicuticular wax deposition alterations and chlorosis are among the structural modifications in leaves deriving from pollution (Mhatre & Chaphekar, 1985; Mohapatra et al., 1991; Rao & Dubey 1991; Setia et al., 1994; Srivastava, 1999). In Brazil, Sant'Anna-Santos et al., (2008) worked with Genipa americana L. (Rubiaceae) in order to characterize injuries on leaf structure and micromorphology, and to evaluate its degree of susceptibility to simulated acid rain. They found necrotic interveinal spots on leaf blade, with plasmolized guard cells and cuticle rupture. Their results highlighted the relevance of anatomical data for precocious diagnosis of injury and to determine species sensitivity to acid rain. Working with Celosia cristata samples subjected to gaseous and particulated pollution deriving from heavy traffic in Indian routes, Srivastava (1999) observed that general plant growth was affected with severe distortions in foliar epidermal characters. Pointing out the importance of cuticle and epidermal features in the determination of tolerance/sensitivity of each species to environmental pollutants.
Taking into account these characteristics, some authors consider foliar epidemis as a bioindicator of environmental quality (Masuch et al., 1992; Alves et al., 2008; Balasooriya et al., 2009). A multi-element analysis was carried out in the metropolitan area of Buenos Aires, using Fraxinus pennsylvanica tree bark as a bioindicator of air pollution (Castro et al, 2008; Perelman et al., 2006). Results confirmed the existence of a distribution pattern along an urban-periurban gradient (central, residential, periurban). Simultaneously, basic properties and heavy metal levels were analysed in soil from the basis of the same trees (Lopez et al., 2006). Results were then compared with those from studies performed in the city of Mendoza (Faggi et al., 2008). Later, Fujiwara et al. (2006) correlated the concentration of elements in particulate matter (PM) from street dust with the concentration of pollutants retained in tree bark and with the concentration of pollutants in air in the same sites. They concluded that tree barks are more sensitive as passive bioindicators than street dust.
Our objective was to evaluate the possibility of using epidermis as a pollution indicator in trees from Buenos Aires metropolitan urban and periurban areas, given the ease and low cost of the technique. To recognise areas of different pollution levels and the effect produced by the same level of pollutants over a short time period (deciduous species) and over a long time period (evergreen species).
MATERIALS AND METHODS
Selection of species
A deciduous and an evergreen tree were selected, namely Fraxinus pennsylvanica Marshall, and Ficus benjamina L, respectively. These species were selected given their high frequency along the streets of the metropolitan area of Buenos Aires, and because of the different life span of their leaves. If morphological or epidermal modifications were observed, their life span could allow us to determine whether they responded to a stimulus over a short period of time (stationary) or to the cumulative action of pollutants over long periods of time. Perelman et al. (2006) conducted sampling for tree bark at the same time as that reported for soil by Lopez (2006).
Leaf samples were collected from urban and periurban areas characterized by different contamination levels. Three different sites belong to the urban area: (Ur 1) Constitucion, affected by heavy car traffic and a railway terminal, (Ur 2) Flores, a residential neighbourhood affected by car traffic, and (Ur3) downtown, with dense traffic incidence. A public park affected by the international airport and its access highway (Figure 1) conform the periurban area Ezeiza (Per). Chascomus (Buenos Aires Province), a rural environment (Rur), was selected as the "non-contaminated zone" where leaves from the same species under study were used as "blank samples", to provide a baseline to test foliar anatomic characters.
Collection of Samples
Ten to twenty leaves were taken from the external part of the canopy, nearly 2 m high. Samples were taken from 10 trees per site every season, at the same time as...