Anal Bioanal Chem (2008) 391:759–771
DOI 10.1007/s00216-008-1890-6
ORIGINAL PAPER
Lichens biomonitoring as feasible methodology to assess
air pollution in natural ecosystems: Combined study
of quantitative PAHs analyses and lichen biodiversity
in the Pyrenees Mountains
María Blasco & Celia Domeño & Cristina Nerín
Received: 30 October 2007 / Revised: 10 January 2008 / Accepted: 17 January 2008 / Published online: 12 March 2008
# Springer-Verlag 2008
Abstract The air quality in the Aragón valley, in the
central Pyrenees, has been assessed by evaluation of lichen
biodiversity and mapped by elaboration of the Index of Air
Purity (IAP) based on observations of the presence and
abundance of eight kinds of lichen with different sensitivity
to air pollution. The IAP values obtained have been
compared with quantitative analytical measures of 16 PAHs
in the lichen Evernia prunastri, because this species was
associated with a wide range of traffic exposure and levels
of urbanization. Analyses of PAHs were carried out by the
DSASE method followed by an SPE clean-up step and
GC–MS analysis. The concentration of total PAHs found in
lichen samples from the Aragón valley ranged from 692 to
6420 ng g−1 and the PAHs profile showed predominance of
compounds with three aromatic rings. The influence of the
road traffic in the area has been shown because values over
the median concentration of PAHs (>1092 ng g−1),
percentage of combustion PAHs (>50%), and equivalent
toxicity (>169) were found in lichens collected at places
exposed to the influence of traffic. The combination of both
methods suggests IAP as a general method for evaluating
the air pollution referenced to PAHs because it can be
correlated with the content of combustion PAHs and poor
lichen biodiversity can be partly explained by the air
pollution caused by specific PAHs.
Keywords Lichens . Polycyclic aromatic hydrocarbons
(PAH) . Biomonitoring . Analysis . Principal component
analysis (PCA)
M. Blasco : C. Domeño : C. Nerín (*)
Grupo GUIA-Instituto de Investigación de Ingeniería en Aragón
(I3A). Centro Superior de Ingenieros (CPS),
María de Luna 3,
50018 Zaragoza, Spain
e-mail: cnerin@unizar.es
Introduction
Assessment of air quality in a survey area is a very complex
problem because field analysis which includes sampling
and development of appropriate analytical methodology is
required as well as identification and monitoring of possible
sources of pollution and critical emissions. In addition,
economic aspects have to be integrated in the monitoring
program [1–3]. The use of pollution biomonitors is an
advantageous air pollution-assessment technique comple-
mentary to conventional environmental air analysis which
provides additional and useful information about the area
[4–6].
Biomonitors are organisms that respond to a certain level
of pollution by changing their natural behaviour or by
accumulating the particular pollutant in their tissues [4–7].
The use of pollution biomonitors enables easier sampling,
even in remote areas and areas with difficult access, because
technology for sampling is not necessary. Furthermore, the
sample treatment and analysis steps in the laboratory are
facilitated, making possible the simultaneous determination
of several pollutants in the same matrix. In the other hand, a
long-term sampling with a large number of sampling sites is
required [1]. For air pollution assessment, lichens, mosses,
and pine needles [1, 5, 8–17] may be considered as the most
commonly applied organisms. Lichens are the most studied
biomonitors of air quality, since in 1866 a study was
published on use of epiphytic lichens as bioindicators [18].
They have been defined as “permanent control systems” for
air pollution assessment [4]. Because of their high sensitivity
toward specific pollutants and ability to store contaminants
in their biological tissues, lichens are defined as bioindicators
and/or bioaccumulators, respectively.
Lichens are regarded as the result of symbiotic associ-
ation of a fungus and an alga. In this association, the alga is
760
occupied with the formation of nutrients, since it contains
chlorophyll, while the fungus supplies the alga with water
and minerals [4]. These organisms are perennial and
maintain uniform morphology over time. They grow
slowly, have large-scale dependence on the environment
for their nutrition. They do not have roots, leaves, or
flowers so they take water and nutrients directly from the
atmosphere and other substances also from air, for example
atmospheric pollutants, so those compounds which are not
degraded are accumulated in their tissues [1]. In contrast
with vascular plants, they do not shed parts during growth.
Moreover, their lack of cuticle or stoma means that the
different pollutants are absorbed over the entire surface of
the organism as a biomonitoring effect [19].
During the last 30 years, many studies have shown the
possibility of their use as bioindicators of air quality since
Hawksworth and Rose in 1970 related SO2 concentration to
the presence or absence of lichens [20]. Several methods
have been proposed for assessing environmental quality
based on lichen data [4, 21–27]. More recently, lichens have
been used as bioaccumulators of heavy metals, phenols,
nitrogen oxides, ozone, and, more rarely, PAHs [1, 5–10, 28].
In this work lichens are used as biomonitors of polycyclic
aromatic hydrocarbons (PAHs) in two different ways:
1. by the standardized lichen mapping method, which
studies the distribution of the total lichens species, and
2. by individual sampling of lichens species and quanti-
tative analysis of PAHs accumulated in the tallus.
The combination of all these values from chemical
analysis, biological observations, and environmental varia-
bles also requires use of chemometric procedures, which
even increase the value of all these data.
The most commonly used method in lichen studies
consists in mapping the distribution of the total epiphytic
lichen flora at different distances from a pollution source. It
has been widely used to detect changes in air quality and is
mainly based on specific sensitivity of many lichen species
to gaseous pollutants, because pollution effects on lichens
have been shown to reduce the number of different types of
lichen because of the break-up of symbiotic association [8,
21, 28]. This method has been widely used in air pollution
investigations [21–25] and, more recently, has been adopted
in the International Co-operative Programme on Assessment
and Monitoring of Air Pollution Effects on Forests (ICP
Forests) [19, 29]. In this paper, a lichen distribution map was
produced by means of an Index of Atmospheric Purity (IAP)
calculation, based on the frequency of the lichen species.
The second way, quantitative determination of PAHs by
analytical procedures has been studied in depth in different
matrices. PAHs are emitted to the atmosphere in combus-
tion processes, road traffic exhaust, and tobacco smoke and
also generated by industrial sector and home heating. They
Anal Bioanal Chem (2008) 391:759–771
can be present in a wide variety of forms persisting largely
in air, water, aerosols, soils, sediments, and biota [14, 17,
30–35] and have harmful effects for human health and the
environment because of their carcinogenic effect. However
the PAHs extraction process still remains a critical step in
the analysis procedure, especially with environmental
samples. Lichens are a complex matrix that contains
different kinds of organic compound such as fatty acids,
chlorophylls, alkanes, phenols, esters, polycyclic aromatic
compounds, etc. Due to the complexity of this matrix and
low concentration levels of PAHs in lichens, enrichment
and clean-up procedures are usually required prior to the
final chromatographic analysis. For this reason, conven-
tional PAHs extraction techniques like Soxhlet [11] and
sonication extraction [7], have been substituted by low-
solvent-consumption extraction methods, for example the
dynamic sonication-assisted solvent-extraction method
(DSASE) [7]. Quantitative determination of 16 EPA PAHs
that are accumulated in the biological tissues of lichens has
been performed by the DSASE method followed by a SPE
clean-up step [36]. This sample handling procedure is
presented as a good alternative for achieving greater
efficiency and sensitivity of the global analytical procedure,
since the final extract is concentrated, and clean of
interferences, and sample handling is substantially reduced.
For this reason, it will be applied in this paper.
In order to evaluate atmospheric PAHs concentration
levels in the Aragón valley, in the Pyrenees Mountains, and
to monitor air pollution in this area since the opening of
Somport tunnel in 2003 which connects France and Spain,
a survey was performed in 2004 monitoring eight lichens
species: Parmelia sulcata, Evernia prunastri, Ramalina
farinacea, Pseudovernia, Usnea sp, Lobaria pulmonaria,
Xanthoria parietina and Hypogymnia physodes. The IAP
values were then calculated and from these values, Evernia
prunastri was selected as the best indicator of air pollution
related to traffic. Environmental variables such as the
climate, situation of sampling points, analytical data of
PAHs, and biological data from the lichen species have
been combined and studied by a chemometric procedure.
The power of both the experimental data together with the
chemometry is without doubt one of the main values of
environmental monitoring, in which the importance of the
set of variables which influence the data is critical.
The main purposes of this work were:
1. to evaluate the influence of the road traffic in a
mountain valley crossed by a national road by two
different criteria—the index of air purity (IAP) and the
quantitative analysis of 16 PAHs in lichens;
2. to combine both air quality evaluation methods, to find
out which kind of information can give us the IAP
Anal Bioanal Chem (2008) 391:759–771
value related to PAHs and to study the impact of PAHs
on lichen biodiversity;
3. to expand knowledge on the use of lichens as bio-
monitors of the air pollution in natural ecosystems; and
4. to obtain an added value of all the data through the
application of chemometric procedure.
Experimental
Study area
The study was conducted in the Aragon valley (Spain)
situated in the central Pyrenees Mountains that constitute a
natural border between France and Spain. This valley is
located in the North of Spain where the climate is
characterized by an average annual rainfall of 1763 mm
per year, which is concentrated in spring and autumn while
summer is fairly dry; an average temperature of 15 °C in
summer and 4 °C in winter and two types of wind—winds
moving along the valley (north–south and south–north) and
mountain and valley breezes. The valley was divided into
low valley and high valley according to the elevation, which
varies from 840 m in the low valley to 1640 m in the high
valley. The high valley has a craggy orography and a
characteristic V profile while the low valley has a smoother
relief and a U profile.
The Aragón valley is crossed by the national road N330
which passes through the villages of Jaca, Castiello de Jaca,
Villanúa, Canfranc, Canfranc Station, and Candanchu up to
Somport port where it links with the Aspe valley in France.
In 2003 the international Somport tunnel connecting both
valleys was inaugurated and an increase in the traffic
density in the area has been observed since then.
This is a natural area, without industrial activity, where
people usually goes to spend holidays and free time so the
most important anthropogenic activities are associated with
mountain sports, such as ski and trekking, residential
holidays, and tourism. Therefore, the road traffic along the
valley represents the main pollution source of the area,
especially in Summer, due to the tourism, and in Winter,
because two ski resorts are situated in the Spanish valley.
Domestic heating emissions also contribute to atmospheric
pollution in the area.
Determination of the index of atmospheric purity (IAP)
Elaboration of the IAP has made it possible to map out the
air quality in a determined area. This index gives an
evaluation of the level of atmospheric pollution, which is
based on the number, frequency, and tolerance of the
lichens present in an area.
761
The study area was divided in 27 plots 3 km in latitude ×
2 km in length grid based on 1:25000 scale map. The plots
were distributed along the N-330 road, at different distances
from the road in order to evaluate the effect of the traffic in
lichens and PAHs and near the towns and other places of
interest, such as the ski resorts, the tourist places, etc. There
are several ways in which the IAP can be calculated. In this
paper IAP was based on the frequency of the lichen species,
because it is the easiest way to quantify the presence and
coverage of the lichen flora. IAP was calculated in every
plot by use of the formula:
X
IAP ¼ Fi
where F is the frequency (1 ≤ F ≤ 10) of every species
observed in a determined plot and i is the number of species
observed, eight species in this case: Parmelia sulcata,
Evernia prunastri, Ramalina farinacea, Pseudovernia,
Usnea sp., Lobaria pulmonaria, Xanthoria parietina and
Hypogymnia physodes. Observation of lichens to determine
the frequency of every kind was carried out according to
the ICP-forest [4, 19, 29]. One or two observation sites
were chosen per plot, depending on the abundance of
lichens. The IAP values obtained were plotted in order to
create an air-quality map.
Lichens sampling and sample preparation
From Spring to Autumn of 2004 Evernia prunastri lichens
were collected around survey area in plots where this kind
of lichen was observed. Lichens were collected from trees
approximately 1m from the ground. In the laboratory,
lichens were separated from the rest of the bark and from
other materials like dust, dried at 35 °C for 3–4 days,
ground in order to furnish homogenized samples, and kept
at 5 °C until analysis.
Identification and quantification of PAHs in lichens
16 EPA priority PAHs were identified and quantified in
lichen samples by applying dynamic sonication-assisted
solvent extraction (DSASE) followed by SPE clean up.
Three replicates of every lichen sample were analysed.
Chemicals and standards
The polycyclic aromatic hydrocarbons: naphthalene (Np),
acenaphthylene (Ac), acenaphthene (Ace), fluorene (F),
anthracene (An), phenanthrene (Ph), dibenzofuran (Db),
chrysene (Ch), pyrene (Py), benz[a]pyrene (BaP), fluoran-
thene (Fl), benz[b]fluoranthene (BbF), benz[k]fluoranthene
(BkF), benz[a]anthracene (BaA), dibenz[a,h]anthracene
(dBahA), and benz[g,h,i]perylene (BghiPe), were supplied
762 Anal Bioanal Chem (2008) 391:759–771

as certified standards by the Environmental Protection
Agency (EPA), USA. Acenaphthene d10 from Sigma–
Aldrich was used as internal standard in all the analysis.
Hexane and dichloromethane of analytical grade were
supplied from Scharlab (Barcelona, Spain). Standard
solutions of PAHs and internal standard solution of
acenaphthene d10 were both prepared in hexane.
Silane treated glass wool was from Supelco (USA). The
NH2-SPE cartridges used were 500 mg of solid bed from
Analisis Vinicos (Spain). Florisil, 100–200 mesh from Aldrich
(Germany) and sodium sulfate anhydrous from Merck
(Darmstadt, Germany).
DSASE extraction method
The DSASE procedure used was previously optimized and
validated for the determination of PAHs in lichen samples
[7]. A sample of 0.2 g lichen was inserted with silanized
glass wool in a 1-mL volume stainless steel extraction cell
(Suprex, Pittsburgh, PA, USA) using the sandwich tech-
nique. The extraction cell was placed in an Ultrasonic LC
130H bath (Elma, Singer, Germany) and 0.2 mL min−1
flow of hexane was continuously pumped through the
sample under sonication for an extraction time of 10 min. A
volume of 2 mL of raw extract containing PAHs and
interferences, such as the plant pigments, was collected in
glass vials and purified by SPE clean up. The analytical
characteristics of the DSASE method can be obtained from
Ref. [7].
SPE Clean-up
The SPE procedure was also previously optimized to purify
the raw lichen extract and obtain the PAH-enriched final
extract [36]. The clean-up mini columns were prepared by
adding approximately 0.05 g sodium sulfate anhydrous and
0.05 g Florisil to the top of the commercial 500 mg NH2-
SPE cartridges. The cartridges were placed on a Waters
(Milford, USA) manifold and 6 mL dichloromethane and
3 mL hexane were passed through the bed in order to clean
and condition the solid phase, respectively. After condi-
tioning the solid bed, the 2 mL raw extract in hexane was
loaded on the top of the cartridge. When all the raw extract
reached the sorbent bed, the column was rinsed with 0.5 mL
hexane to remove many of the interfering compounds
retained on the column and the PAHs were then quantita-
tively eluted with 2 mL hexane–dichloromethane 65:35. The
final extract containing PAHs was collected in a glass vial
and concentrated to 0.5 mL under a gentle nitrogen stream
before analysis by GC–MS. Figure 1 illustrates all the
operations and steps carried out in the global analytical
procedure.

Fig. 1 Diagram illustrating all

operations and steps of the ana- 0.2 g. of homogenized
lytical procedure DSASE lichens samples

Conditions:
- FLOW: 0.2 mL/min of hexane
Extraction - EXTRACTION TIME: 10 minutes
- Room temperature
2 mL of raw extract containing
PAHs plus interferences
SPE clean up (chlorophylls, lipids, etc.)

Conditions:
- PREPARATION:
PREPARATIO
Florisil
Clean up
SPE NH2

0.5m Hexane
- RINSING: 0.5mL
- ELUTION: 2mL Hexane-DCM (65-35)

Purified extract containing PAHs

Evaporation under gentle N2

Concentration
stream until 0,5 µL

Analysis
GC-MS
Anal Bioanal Chem (2008) 391:759–771 763

Gas chromatography–mass spectrometry Results and discussion

The analytes were identified and quantified using a Hewlett
Packard HP 6890 Series gas chromatograph coupled to a
5973 mass spectrometer. Separations were carried out on a
Factorfour VF5-ms 60 m×250 μm×0.25 μm column.
Helium was used as carrier gas at 1 mL min−1. Injection
into the GC system was performed in splitless mode, with
the purge valve open at 0.9 min; the injector temperature
was 300 °C. The GC oven temperature was held 50 °C
for 1 min, increased to 180 °C by a temperature ramp of
20°min−1 then ramped at 10°min−1 to 300 °C and held for
10 min. The MS operating conditions were: EM 1900 eV,
transfer line temperature 280 °C, and operation in SIM
(selected ion monitoring) mode, using the following
characteristic m/z: naphthalene, 128; acenaphthylene and
acenaphthene, 152, 153; dibenzofuran, 168; fluorene, 166;
phenanthrene and anthracene, 178; fluoranthene and pyr-
ene, 202; benz[a]anthracene and chrysene, 228; benz[b]
fluoranthene, benz[k]fluoranthene and benz[a]pyrene, 252;
dibenz[a,h]anthracene, 278 and benz[g,h,i]perylene, 276.
Acenaphthene d10 with a characteristic mass of 164, was
added to all the standards and sample extracts before
injection, as internal standard. Quantitative results for PAHs
were based on peak area relative to that of the internal
standard, and comparison with PAH standards.
Multivariate statistical method
Principal component analysis (PCA) was used to analyse
the experimental data obtained. PCA is a multivariate
statistical technique widely used in environmental sciences
[37]. It is based on data reduction and is quite useful when
a great number of variables and samples must be studied
because it allows extraction of a small number of latent
factors (principal components, PCs). In this work PCA was
carried out by The Unscrambler (CAMO) software for
Windows.
Index of air purity (IAP)
Evaluation of the IAP was carried out at 18 plots out of a
grid of 27. Places where the studied lichen species were not
present or access was not possible, often in the high valley
due to the steep slopes, were not evaluated. The target
lichen species grow preferentially on the bark of oaks and
beeches, however pine forests are predominant in many
parts of the survey area where none or very few species
were found. The eight kinds of lichen were observed only
in two plots and the other places showed different
combinations of the studied species. Every frequency value
observed was associated with a percentage of coverage
according to the Braun–Blanquet scale as follows: 1–2 (1–
5% coverage, median 3%), 3–4 (6–25% coverage, median
15%), 5–6 (26–50% coverage, median 37.5%), 7–8 (51–
75% coverage, median 62.5%), and 9–10 (76–100%
coverage, median 87.5%) [38]. Table 1 shows the number
of sites where each kind of lichen was observed and the
average coverage (%) in order to evaluate the abundance of
every species in the Aragón valley. Xanthoria parietina and
Hypogymnia physodes were abundantly present in the
whole area, followed by Parmelia sulcata and Evernia
prunastri, whereas Pseudevernia furfuracea, Ramalina
farinacea, Usnea s.p., and Lobaria pulmonaria were
occasionally found. The last, Lobaria pulmonaria, was
not observed at any plot crossed by the road. According to
the criterion of presence or absence of lichens, preliminary
remarks about the sensitivity of every kind of lichen to the
air pollution associated with the road traffic can be obtained
from the lichen observations. The most frequent species,
Xanthoria p., Hypogymnia p., Parmelia s. and Evernia p.,
apparently showed higher resistance to pollution than the
less frequent ones. That agrees in some cases with the
suggestions of other authors about the sensitivity of each
species to the air pollution, as can be seen in Table 1, where

Table 1 Number of observation sites, mean coverage (%), and estimation of the tolerance to air pollution of every kind of lichen

Lichen species Sites (no.) Average coverage (%) Sensitivity to air pollution according to different authors

This paper TOa Calatayud et al.b

Evernia prunastri EP 10 38 Resistant 5 Intermediate

Hypogynmia physodes HP 18 59 Resistant 8 Intermediate–tolerant
Lobaria pulmonaria LP 2 10 Sensitive – Sensitive
Parmelia sulcata PS 18 32 Resistant 8 Intermediate
Pseudevernia furfuracea PS 10 18 Sensitive 6 Intermediate
Ramalina Farinacea RF 6 10 Sensitive 5 Intermediate
Usnea s.p. Us 6 8 Sensitive 3 Sensitive
Xanthoria parietina XP 18 45 Resistant 7 Intermediate–tolerant
a
Air pollution tolerance index (TO), from 1 (very sensitive species) to 9 (resistant species) according to Kirschbaum and Wirth [39]
b
Sensitivity to pollution of every lichen species according to Calatayud et al. [19]
764
the different sensitivity values proposed for every kind of
lichen are shown. As the sensitivity is based on the
abundance of every species in the selected environment,
there is not total coincidence, because other environmental
variables also influence lichen diversity.
The IAP values evaluated in the Aragon valley range
from 19 to 43 and are grouped in four pollution levels
(high: 0–20, moderate: 21–40, low: 41–60, and very low:
61–80). The moderate level was split in two additional
categories (moderate/high: 21–30 and low/moderate: 31–
40) because this pollution level was most often found in the
area. The map of the survey area and the pollution level
corresponding to every plot are shown in Fig. 2. The survey
area shows a prevailing low/moderate pollution level (55%
of the observation plots), nevertheless, high and moderate/
high pollution levels were found at plots directly exposed to
the influence of road traffic (28%), such as plots crossed by
the road or near the towns, and low pollution level at
natural sites situated further from the road where diversity
of 6 or 8 kinds of lichen was observed (17%). This fact
confirms the clear influence of the road in the distribution
of the lichen species and the IAP values. However, the
Fig. 2 Map of the Aragon valley showing the 18 evaluated plots
where the IAP value was calculated and the respective pollution level,
and the sites where Evernia prunastri was collected: 1, Near Villanúa;
2, Fuente del Paco; 3, Lierde; 4, Canfranc; 5, Ip; 6, Canfranc station I;
7, Canfranc hydroelectric; 8, Canfranc station II; 9, Canfranc station
III; and 10, Canfranc station IV. At plots where two observations were
made the average IAP was calculated
Anal Bioanal Chem (2008) 391:759–771
limitations of the IAP method have been shown up before
by some authors, because in environmental work it is
difficult to separate the effect of other climatic and
environmental variables that also influence the lichen flora
[1, 19, 22, 24, 28, 40, 41]. Variables such as the rate of
urbanization of a place and the traffic influence determined
the load of atmospheric pollutants in the air and the type of
microclimate, which condition the humidity, vegetation,
and illumination in an area and, furthermore, the amount of
nutrients and pH requirements for the growing of specific
lichen species [27]. Therefore, the influence on lichen
biodiversity of these variables was also studied.
Principal component analysis
To confirm the influence of other variables on lichen
diversity (IAP), principal component analysis (PCA) was
carried out considering the IAP values, the traffic influence,
the urbanization rate, the type of microclimate, the cover
percentage of every kind of lichen, and the distance to the
road. Table 2 shows the evaluation of the influence of
traffic, rate of urbanization, and the type of microclimate in
every plot. The loadings and scores of the first, second, and
third components can be seen in Figs. 3 and 4.
As Fig. 3a shows, the first component indicates that high
values of IAP are associated with plentiful presence of
Parmelia s., Usnea, s.p., Pseudevernia f. and Lobaria p., so
these species contribute most to lichen biodiversity.
Parmelia s. is a kind of lichen resistant to air pollution
and widely distributed around the survey area. Although
abundant, it was more often found in places where large
numbers of other, more sensitive, species, such as Usnea s.
p. and Pseudevernia f., were present. As has been estimated
before, Lobaria p. was related to remote places without
direct influence of traffic. On the other hand, Xanthoria p.
was uniquely abundant at sites where the microclimate was
relatively dry and sunny, because of the scarce vegetation
present, and consequently where the climatic conditions
Table 2 Evaluation of the traffic influence, urbanization rate, and the
type of microclimate at three different levels
Score 1 2 3
Traffic Walking Mountain Road
influence place track (non-
asphalted)
Urbanization Natural Vicinity of Urban place
rate place urban place
Microclimate Wet and shady Forest Relatively
forest with lush dry and sunny,
vegetation alternating
trees and
bushes
Anal Bioanal Chem (2008) 391:759–771 765

were not suitable for growth of the other lichen species.
This lichen has been classified as a nitrophytic species
which helps to fix atmospheric nitrogen [19] and Gombert
et al. associated monospecific lichen communities of
nitrophytic flora with artificial environments [20, 40]. In
general, Xanthoria p. was rarely found in the low part of
the Aragón valley.
The second component positively correlated the frequen-
cy of Evernia p. with the rate of urbanization and the
influence of traffic; this kind of lichen is, therefore, very
interesting for biomonitoring of air pollution associated
with the road traffic. As we can see in Fig. 3b, where sites
with presence of Evernia p. are highlighted, this species is
widely distributed, covering a wide range of lichen
biodiversity conditions, because it can be found either at
places with a high variety of lichens or accompanied by few
other species, or even alone, although this kind was always
observed at forest sites with leafy vegetation and high
humidity. For this reason, Evernia prunastri was selected
for quantitative analysis of PAHs in order to combine the
concentration of the target compounds found in this kind of
lichen with the IAP values obtained in the Aragon valley.
Figure 4 shows the scores from the PC3 versus PC1 plot.
It can be divided in three groups. Group I includes sites
where Evernia p. was present and sampled for later
analysis. The two other groups are sites where this lichen
was not found, because the microclimate was not very
suitable for growth of the lichens studied (group II) or
where the prevailing species were Xhantoria p. and
Hypogynmia p. whose plentiful presence involves the
absence of the other species studied (group III). Both kinds
of lichen are regarded as tolerant to pollution, as is shown
in Table 1.
PAHs in lichen samples
The 16 EPA PAHs were determined in Evernia prunastri
lichen samples collected at ten sampling points from the

Fig. 3 Principal component

analysis (PCA). IAP, index of air
purity; T, traffic influence; U,
urbanization rate; M, microcli-
mate; %PS, frequency of
Parmelia sulcata; %EP,
frequency of Evernia prunastri;
%RF, frequency of Ramalina
farinacea; %Pse, frequency of
Pseudevernia furfuracea; %Us,
frequency of Usnea s.p.; %LP,
frequency of Lobaria
pulmonaria, and distance, the
distance from the sampling point
to the road. (a) Loadings of PC2
versus PC1; (b) scores of PC2
versus PC1, highlighting sites
where Evernia prunastri
was found
766 Anal Bioanal Chem (2008) 391:759–771

Fig. 4 Principal component

analysis (PCA). IAP, index of air
purity; T, traffic influence; U,
urbanization rate; M, microcli-
mate; %PS, frequency of
Parmelia sulcata; %EP,
frequency of Evernia prunastri;
%RF, frequency of Ramalina
farinacea; %Pse, frequency of
Pseudevernia furfuracea; %Us,
frequency of Usnea s.p.; %LP,
frequency of Lobaria
pulmonaria, and distance, the
distance from the sampling point
to the road. (a) Loadings of PC3
versus PC1; and (b) Scores of
PC3 versus PC1. Group I, sites
where Evernia prunastri was
found. Group II, sites where
Evernia prunastri was not found
because the microclimate was
not suitable. Group III, sites
where Xhantoria p. and
Hypogynmia p. were the
prevailing species

Aragon valley (Fig. 2). The concentration (ng g−1) of every
compound is shown in Table 3 as well as the Σ16 PAH
concentrations, called hereafter PAHs. The concentrations
were calculated as averages of three replicates for every
sampling point.
The total PAH content of Evernia prunastri varied from
696 to 6240 ng g−1 with a median value of 1092 ng g−1.
Although studies of PAHs in native lichens are very scarce,
a previous study carried out in the same survey area
reported concentration range of PAHs in Parmelia sulcata
at a similar order of magnitude, from 352 to 1652 ng g−1
[36], and a study performed in a forest ecosystem in which
PAHs were determined in Hypogynmia physodes samples
reported concentrations from 1184 to 2253 ng g−1. In that
work, PAHs were also determined in other vegetable
matrices used as biomonitors for the air pollution, such as
mosses, pine needles, or tree bark. The concentration range
found in mosses was slightly lower than that found in
lichens, 587–622 ng g−1, while PAHs in the other matrices
were found at lower concentrations and the range of PAHs
was smaller [11]. Other PAHs biomonitoring studies in
natural areas reported concentrations of these compounds
from 183 to 1629 ng g−1 in two kinds of moss from different
forest communities in Poland [12] and from 300–500 ng g−1
in Quecus ilex leaves in a control site in Naples [42].
In this work, concentration values over the median value
(>1092 ng g−1) were found in five samples collected at sites
near the road or the towns, as in the IAP evaluation,
although in this case the highest values were located in the
high Aragon valley. The highest concentrations of PAHs
were obtained in lichens from sites around the first 500 m
from the road. Samples collected at greater distance showed
very similar concentration of PAHs, for example lichens
from sampling points 1, ,3 and 2 situated 483, 1542, and
2525 m, respectively, from the road in which concentrations
were 932±25, 976±90 and 1008±6 ng g−1, respectively.
The distribution profile of accumulated PAHs in Evernia
prunastri according to the percentage of 2, 3, 4, 5, and 6-
Table 3 Concentration (ng g−1) of each PAH and Σ16 PAHs (PAHs), percentage of combustion PAHs (PAHscomb), and equivalent toxicity (TEQ) found in Evernia prunastri at every sampling
point

1 2 3 4 5 6 7 8 9 10

Naphthalene 86 ±3 414 ±16 127 ±21 231 ±23 21 ±4 107 ±7 102 ±17 198 ±42 73 ±2 95 ±7
Acenaphthylene 17 ±2 44 ±1 37 ±4 74 ±14 85 ±15 36 ±3 22 ±5 69 ±5 14 ±2 24 ±1
Acenaphthene 56 ±6 178 ±1 178 ±30 192 ±2 14 ±2 90 ±3 69 ±8 116 ±27 53 ±10 55 ±2
Dibenzofuran 45 ±5 35 ±0.5 60 ±8 434 ±16 169 ±31 69 ±1 36 ±3 74 ±12 32 ±2 42 ±1
Fluorene 51 ±5 41 ±2 47 ±4 509 ±24 159 ±29 62 ±4 29 ±2 76 ±17 29 ±4 45 ±2
Phenanthrene 217 ±6 95 ±14 117 ±6 2659 ±301 234 ±42 236 ±7 121 ±25 501 ±31 112 ±27 230 ±13
Anthracene 19 ±6 9 ±2 27 ±3 140 ±30 15 ±3 17 ±3 5 ±2 57 ±3 8 ±1 19 ±2
Fluoranthene 122 ±1 33 ±3 80 ±23 1170 ±96 114 ±21 227 ±16 57 ±4 670 ±67 92 ±22 338 ±19
Anal Bioanal Chem (2008) 391:759–771

Pyrene 80 ±4 16 ±0.1 41 ±8 276 ±32 116 ±21 102 ±11 55 ±5 256 ±36 76 ±13 146 ±10
Benz[a]anthracene 73 ±1 17 ±4 53 ±7 166 ±13 86 ±16 122 ±7 111 ±4 344 ±14 74 ±5 124 ±4
Chrysene 42 ±9 7 ±0.2 35 ±5 79 ±22 21 ±4 72 ±21 50 ±4 95 ±3 23 ±2 73 ±4
Benz[b]fluoranthene 27 ±0.1 29 ±4 47 ±7 123 ±47 55 ±10 37 ±7 26 ±5 96 ±16 31 ±5 43 ±7
Benz[k]fluoranthene 28 ±2 31 ±0.2 42 ±12 81 ±35 38 ±7 38 ±6 47 ±9 103 ±10 35 ±2 33 ±1
Benz[a]pyrene 31 ±8 19 ±4 46 ±3 43 ±13 15 ±3 34 ±12 13 ±1 43 ±5 15 ±3 13 ±2
Dibenz[a,h]anthracene 16 ±1 16 ±0.2 18 ±5 24 ±8 21 ±4 18 ±1 30 ±5 31 ±5 15 ±4 55 ±11
Benzo[g,h,i]perylene 23 ±4 24 ±4 20 ±4 37 ±14 15 ±3 21 ±2 21 ±5 47 ±7 14 ±2 40 ±1
PAHs 932 ±25 1008 ±6 976 ±90 6240 ±356 1177 ±213 1288 ±103 794 ±55 2777 ±258 696 ±69 1376 ±55
%PAHscomb 47 ±1 19 ±0.5 39 ±1 32 ±2 41 ±3 52 ±2 52 ±3 61 ±1 54 ±0.3 63 ±1
TEQ 127 ±5 108 ±2 155 ±27 214 ±64 181 ±13 147 ±19 181 ±24 257 ±18 105 ±23 311 ±55

The concentration values are expressed as the average (n=3) ± 95% confidence interval
767
768
aromatic ring PAHs is shown in Fig. 5 as a box-and-whisker
diagram. As we can see, three-ring PAHs were dominant
followed by four-ring PAHs (68–81.5%), whereas the low-
est amount is of PAHs with six rings (0.4–3%). The same
distribution of PAHs according to the number of aromatic
rings was found in Parmelia sulcata in previous work [36]
while the proportion of four-ring PAHs was higher in at-
mospheric particulate matter [5, 7]. As a rule of thumb,
light molecular weight PAHs (2 and 3 rings) are mainly
present in the gas phase, 4-ring PAHs are present in both
the gas phase and particulate matter, and PAHs of higher
molecular weight (5 and 6 rings) are generally associated
with particulate matter, although this distribution depends
on the ambient temperature, the relative humidity, the
properties and concentration of PAHs, and the transport
mechanisms, among other factors [43]. The exposure of
lichens to gas and particulate-associated PAHs reflected that
these organisms can act as biomonitors either the lighter
PAHs, commonly present in the gas phase and taken up in
their tissues during the nutrition process, or the heaviest
PAHs, characteristic of atmospheric particulate matter
which is adsorbed on the lichen surface [16]
The most abundant PAHs were phenanthrene (95–
501 ng g−1) and fluoranthene (33–1170 ng g−1) in all
Evernia prunastri samples except those collected more
distant from the road, in which the lighter PAHs were
present at the highest concentrations. Lichens from site 3
(1542 m) contained the highest concentrations of both Np
and Ace (127±21 and 178±30 ng g−1, respectively) and
samples from site 1 (2525 m) in which Np was the
dominant compound (414±16 ng g−1). This suggests the
low influence of PAHs associated to particulate matter at
places situated far from the road considered the main
Fig. 5 Box-and-whisker plot showing the profile of 2, 3, 4, 5, and 6-
aromatic ring PAHs, as percentages. The central box represents the
values from the lower and upper quartile and the middle line
represents the median. The vertical line extends from the minimum
to the maximum value, excluding extremes (asterisk)
Anal Bioanal Chem (2008) 391:759–771
emission source of PAHs in the valley. Another special
profile was present in lichens from site 4 where the highest
PAHs concentration (6240±356 ng g−1) was found and dB,
F, Ph, An, Fl, and Py were the compounds contributing
most to the total PAHs concentration, in notably higher
proportions than for the other samples. This kind of profile
can be associated with diesel engine emissions whose PAHs
profile is richer in three-ring compounds and specifically
exhaust from diesel heavy-duty vehicles which contain high
concentrations of Ph, An, Fl, and Py [43]. The high PAHs
concentration and profile are probably because of this
sampling site was much closer to the railway lines of a
diesel train already travelling along the valley.
Besides the concentration of individual and total PAHs,
Table 3 also shows the amount of PAHs with more than
four aromatic rings which are called combustion PAHs
(PAHscomb), because these compounds are formed at
combustion process at very high temperature and are
characteristic of the vehicle exhaust emissions, and the
equivalent toxicity (TEQ) of the mixture of PAHs,
calculated as the sum of the concentration of every
compound as per their corresponding toxicity factor
referred to BaP and tabulated by Nisbet and LaGoy
(1992) [44]. The amount of combustion PAHs ranged from
19 to 63% with a median value of 50% and the TEQ values
from 105 to 311 with a median value of 168. For all the
samples collected less than 300 m from the road the amount
of combustion PAHs was over the median value (>50%)
while the two samples taken at a greater distance had the
lowest values (39 and 19%, respectively). In the case of
toxicity, TEQ values over the median value (>168) were
only found for some lichen samples collected less than
450 m from the road. These results, together with the Total
PAHs, show the relevant impact the road traffic exerts on
the air quality of a natural ecosystem, as the Aragon valley
is. The values of PAHs, combustion PAHs, and TEQ found
in Evernia prunastri were considered for correlation with
the IAP method.
Correlation between IAP method and concentration
of PAHs in lichens
The concentration of total PAHs, the percentage of
combustion PAHs, and the toxicity values found in the
Evernia prunastri samples were directly compared with the
IAP values classified in every pollution level, to explore
possible relationships between both methods. In Fig. 6 it
can be seen that the pattern of combustion PAHs and
toxicity is decreasing as the air quality according to IAP
improves, however the same pattern is not observed in the
case of total PAHs due to the high dispersion presented in
the results for the low/moderate pollution level. That
suggests good agreement between IAP–TEQ and IAP–
Anal Bioanal Chem (2008) 391:759–771
Fig. 6 Concentration of total PAHs, percentage of combustion PAHs
(PAHscomb) and toxicity (TEQ) values found in the Evernia prunastri
samples compared with the IAP values classified at every pollution
level (high, 0–20; moderate, 21–40; low, 41–60; and very low, 61–80)
PAHscomb. A Pearson correlation test was applied to the
four variables to find out significant correlations among
them. Table 4 shows the correlation coefficients matrix
which indicated no significant correlation between IAP–
PAHs and IAP–TEQ whereas there was a negative
significant correlation between IAP–PAHscomb (r=−0.394,
P<0.05). This study indicates the sensitivity of the studied
lichen species to the PAHs characteristic of the combustion
process emissions. Moreover, sites where high diversity of
the studied lichen species was observed reveal good air
769
quality referred to the content of combustion PAHs and
toxicity of these compounds.
Principal component analysis was carried out to study
the influence of individual PAHs on lichen biodiversity
evaluated by the IAP values. Commonly, baP is the most
representative compound in PAHs studies because of its
well-documented carcinogenic activity. Nowadays, the
importance of more volatile PAHs, for example Ph, Fl,
and Py, has increased, because of their plentiful presence in
ambient air, because the exhausts from modern diesel
engines and fuels contain less of the high-molecular-weight
PAHs. Fl can be also considered as a main contributor to
the carcinogenicity of PAHs in ambient air, together with
dBahA which is classified as a more potent carcinogenic
compound than baP. As well as abundance and carcino-
genic activity, other PAHs are important because they
indicate the origins, for example BghiPe is specific
indicator of petrol exhaust. To complete the work, the
study focused on the PAHs recommended as indicators in
monitoring programs according to air quality and biological
considerations [43]. As is shown in Fig. 7a, which
represents the loadings of the first and second components,
the compounds used as indicators of PAHs in ambient air
were opposed to the IAP: Fl, Py, dBahA and BghiPe by the
first component (29%), which were also correlated with the
traffic exposure, and Ph by the second component (24%).
For this reason, the pollution due to these compounds can
be considered as responsible for low IAP values in the
Aragón valley and consequently for the decrease in the
lichen flora because of the phytotoxicity of these pollutants.
Finally, in Fig. 7b it can be observed that the survey area
can be split into three different groups according to the air
pollution assessed by both methods combined (IAP and
quantitative analysis of PAHs in lichens). The most
numerous group is formed by lichen samples from sites
exposed to the influence of road traffic, where intermediate
and low IAP values were obtained and where the amounts
of the heaviest PAHs, such as BaA, Ch, Fl, Py, dBahA and
bghiPe (group I), were high. Another group containing the
two samples collected at the greatest distance from the road
has high IAP values and predominant amounts of Np, Ace
Table 4 Pearson correlation matrix
IAP PAHs TEQ
IAP Correlation coefficient
Significance level
PAHs Correlation coefficient −0.105
Significance level (0.609)
TEQ Correlation coefficient −0.261 0.343
Significance level (0.198) (0.087)
% Correlation coefficient −0.394 −0.221 0.508
PAHscomb Significance level (0.046) (0.278) (0.008)
770 Anal Bioanal Chem (2008) 391:759–771

Fig. 7 Principal component

analysis (PCA). Considered
variables: IAP, index of air pu-
rity; T, traffic influence; and
amount (%) of the 16 PAHs
(Np, naphthalene; Ac, acenaph-
thylene; Ace, acenaphthene; Db,
dibenzofuran; F, fluorene; Ph,
phenanthrene; An, anthracene;
Fl, fluoranthene; Py, pyrene;
BaA, benz[a]anthracene; Ch,
chrysene; BbF, benz[b]fluoran-
thene; BkF, benz[k]fluoranthene;
BaP, benz[a]pyrene; dB[ah]A,
dibenz[a,h]anthracene; BghiP,
benz[g,h,i]perylene). (a) Load-
ings of PC2 versus PC1 and (b)
Scores of PC2 versus PC1.
Group I, lichen samples from
sites exposed to road traffic
influence with intermediate and
low IAP values and high content
of BaA, Ch, Fl, Py, dBahA and
BghiP. Group II, samples at the
greatest distance from the road
with high IAP values and pre-
dominant content of Np, Ace
and Ac. Group III, samples with
special profile richer in dB, F,
Ph and Ant

and Ac (group II). The last group was that containing the
two samples which had a special profile richer in dB, F, Ph,
and An because of their proximity to the railway lines
(group III).
Concluding remarks
This work provided information about the air quality in the
Aragón valley obtained by two quantitative methods based
on use of lichens as biomonitors of atmospheric pollution—
the IAP method, based on observation of the presence and
coverage of eight kinds of lichen studied and which showed
low/moderate level pollution prevailing in the Aragon
valley, and analytical measurement of 16 PAHs in samples
of Evernia prunastri collected at the survey area. Both
methods demonstrate the influence of the road traffic on air
pollution in the valley because either a decrease in the
lichen flora or content over the median value in PAHs
characteristic of combustion processes (>50%) were found
at sites exposed to the influence of traffic. It can be
emphasized also that principal component analysis, as a
chemometric procedure, applied to the set of data enables
correlation between the analytical values, the environmental
variables, and the biological survival of lichen species.
It can be concluded that the air quality in natural
ecosystems can be assessed by quantitative analysis of
PAHs in lichens which behave as good biomonitors of such
compounds in ambient air, as well as by monitoring lichens
through established indexes such as the IAP. Combination
of both methods reveals that low IAP values and therefore
poor lichen flora can be related to high equivalent toxicity
values and a high content in those compounds recommen-
ded as PAHs indicators in ambient air. However, the
complexity of natural ecosystems means that other varia-
bles also influence the lichen vegetation and only part of
the effects which cause low lichen biodiversity can be
explained by air pollution. Therefore, in environmental
networks, lichen biodiversity should be evaluated in natural
ecosystems at sites with similar microclimatic and environ-
Anal Bioanal Chem (2008) 391:759–771
mental conditions to integrate lichen biomonitoring as a
reliable method for the air pollution assessment.
Finally, the status of the lichen vegetation at the Aragón
valley in 2004 has been established in this work which can
be used as a reference study to monitor further environ-
mental changes and impacts.
Acknowledgements This study was financed by the project PAP
(Pyrenees Air Pollution) from the Pyrenees working Community
(Comunidad de Trabajo de los Pirineos).
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