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372 S. Orecchio et al. / Environmental Research 107 (2008) 371–379

Table 1
Polycyclic aromatic hydrocarbons analysed and classification regarding their carcinogenic potentials (Toxic equivalence factors (TEF)) hydrocarbons (mean of three analysis)
in Pine Bark samples
TEF (a) Larsen and TEF (b) Schneider TEF (a) Larsen and TEF (b) Schneider TEF (a) Larsen and TEF (b) Schneider
Larsen, (1998) et al., (2002) Larsen, (1998) et al., (2002) Larsen, (1998) et al., (2002)
Acenaphythylene 0.001 Fluoranthene 0.05 0.001 Penzo (a) 1 1
pyrene
Acenaphythene 0.001 Pyrene 0.005 0.001 Perylene
Fluorene 0.001 1 Methyl Indeno (1,2,3- 0.1 0.1
pyrene cd)pyrene
Phenanthrene 0.0005 0.001 Benzo (a) 0.005 0.1 Dibenzo (a,h) 1.1
anthracene nthracene
Anthracene 0.0005 0.01 Chrysene 0.03 0.01 Benzo (g,h,i0 0.02 0.01
perylene
2 Methyl Benzo (b) 0.1 0.1
anthracene fluoranthene
9 Methyl Benzo (k) 0.05 0.1
anthracene fluoranthene

Animal and vegetal organisms including lichens have been recommended by the USA Environmental Protection Agency, in each sample 19
PAHs were analysed, with particular attention to 16 recognized as priority
widely used for environmental biomonitoring purposes (Ru ¨ hling
pollutants (Table 1).
and Tyler, 1968; Walkenhorst et al., 1993; Raffaelli, 1996; Baumard
et al., 1998; Catsky et al., 2001; Corte ´s, 2004; Madejon et al.,
2006). Some studies, carried out by our research group using 2.1. Chemicals
lichens and leaves as bioaccumulators (Giovenco et al, 1996;
All chemicals used were of analytical grade with high purity. In particular,
Alaimo et al., 2000; Macaluso et al., 2000; Lombardo et al., 2001;
organic solvents (dichloromethane, cyclohexane, pentane), supplied by Sigma
Culotta et al., 2002, 2005) showed a general increasing of heavy Aldrich (Milano, Italy), were of 99.8% purity. Standard reference PAHs mixture (EPA
metals and/or PAHs concentration in the urban areas under 610 PAHs mixture) and perdeuterated internal standards (fortification solution B)
investigation owing to human activities. were from Supelco (Milano, Italy).
Tree bark has also been used as a bioaccumulator of both heavy
metals and organic toxicants in a study comparing the bioaccu- 2.2. Sampling and sites
mulation capacity of moss and pine bark samples towards heavy
metals in an industrialized area of Turkey (Tu ¨rkan et al., 1995). Palermo is a densely populated town (more than 1 million habitants) with a
Schultz et al. (1999) used pine barks to characterize temporal heavy load of vehicular traffic and major industrial activities are located within the
variations and spatial patterns of some inorganic and organic urban area. Pinus sylvestris L., a vegetal species common in the entire
Mediterranean region, is widespread both in the city and in the outskirts. Pine
substances and clearly showed the suitability of this biomonitor-
bark samples were collected, between April 2004 and February 2005, from 50- to
ing tool. Some authors (Bo ¨hm et al., 1998) used oak tree bark for 60-year-old pine trees, free of mosses and lichens. When it was possible three trees
determining the distribution of air pollution by heavy metals and for every sampling station were chosen. Sampling sites are shown in the maps
other elements in Bohemia. Schulz et al. (1999) analysed the reported in Figs. 1 and 2.
In order to compare analytical results obtained in sites having different
concentrations and natural isotope ratios of both N and individual
meteorological conditions and vehicular traffic, some pine barks samples were also
compounds in bark samples from various locations to obtain collected in sites far from the city. Detailed information about the characteristics of
information on the origin of atmospheric N depositions (Schulz sampling sites are reported in Table 2.
et al., 2001). Using a hard steel knife, the external surface of the bark, not exceeding 2 mm
in depth, was removed from different areas of the whole circumference of each
Due to its very porous surface, tree bark is well recognized to
tree, at 1–1.5 m height, obtained by combining the sub-samples of 5–10 g. For
be an excellent biosorbent material of airborne pollutants. In this
analysis we used the laboratory sample obtained by combining the sub-samples of
work, we optimize an analytical method for the bark of pine trees, the same tree into one mixed sample. Barks were collected using rubber gloves and
which are widespread in Palermo town, as well as throughout the were immediately refrigerated (4 1C), stored avoiding exposure to light, and then
Mediterranean area, and use it as a bioaccumulator of PAHs, with rapidly carried to the laboratory where they were frozen before analysis.
Two samples of bark for each site were dried to a constant weight in an oven at
the aim of evaluating the air quality of Palermo.
a temperature of 70 1C to report PAH values on a dry weight basis.
2. Material and methods 2.3. Sample treatment
Among the various steps of the procedure for the detection of PAHs in The dried samples were pulverised to uniform size with a laboratory mill. The
whatever environmental sample, the sampling, the extraction and the quantitative mill was scrupulously cleaned and dried after each grinding to avoid contamina-
recovery are by far most important. First, the repeatability of the sampling was tion. About 5 g of bark was added to pre-cleaned (Soxhlet extracted with
checked by analysing for PAHs in four different samples of bark collected from dichloromethane for 24 h) anhydrous Na 2 SO 4 (Carlo Erba, Milano). A solution of
different points of the same tree, giving a relative standard deviation of about 7%, a perdeuterated surrogate standard (benzo(a)anthracene-d 12 ) was added to the
while analysing four bark samples taken from different trees of the same site gave sample of bark.
a relative standard deviation of about 8%. Therefore, we can consider the standard
deviation from sampling to be negligible relative to that of the analytical process.
2.4. Extraction of PAHs
For the subsequent analyses, we collected about 25–50 g bark from each station
and homogenized it before extraction in the laboratory.
Particular attention was paid in this study to the extraction method in order to To find the best extraction method to obtain the maximum recovery
obtain the best recovery of PAHs from barks under investigation. The PAHs percentage of PAHs under investigation, different recovery tests were carried out
concentration in the various bark samples and their distribution in the different on tree bark samples. These were preventively extracted for 48 h, using a Soxhlet
sampling sites were related to hypothesize the possible origin of PAHs in the urban apparatus and successively spiked with a known quantity of PAHs using different
area of Palermo. Qualitative and quantitative analysis of PAHs was carried out by methods. The average recoveries calculated for all the compounds and the relative
the GC–MS technique using reference standards and spectra libraries. As standard deviations are reported in Table 3.