Environmental and Sustainability Indicators 16 (2022) 100211

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Environmental and Sustainability Indicators

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Use of lichen species for air pollution biomonitoring: Case of Dar-Chichou

forest (Cap-Bon, North-East Tunisia)
Sonia Dhaouadi a, b, *, Noureddine Khalloufi a, Khaoula Ayati a, Nesrine Ayeb a,
Mustapha Béjaoui a, **
a
University of Carthage, Faculty of Sciences of Bizerte, LR01ES14 Environmental Biomonitoring Laboratory, 7021, Jarzouna, Tunisia
b
University of Carthage, Higher Institute of Sciences and Technologies of the Environment, Borj-Cédria, Tunisia

A R T I C L E I N F O A B S T R A C T

Keywords: In the present work, we investigated the bio-uptake of 4 MTE (Ni, Cd, Pb, Zn) in the thalli of some species of
Air quality lichens near a road crossing the Dar-Chichou forest (NE- Tunisia). In the absence of previous studies on the
Biomonitoring lichenic heritage of this forest, the objectives of this work are.
MTE
first of all to identify for the first time the lichen species present in the forest, to determine the impact of this
Biomarkers
road pollution on certain physiological parameters of these pioneer species, such as the chlorophyll a and b
Lichen species
levels, the content of carotenoids, theperoxidation (H2O2) and the ipoperoxidation (MDA) and on the other
hand, to show the bioindicatory and bioaccumulative importance of these lichens in front of certain trace metals
(Pb, Cd, Zn and Ni). In this study, 23 lichen species with different thallus was detected. The amounts of MTE in
thalli of several lichen species were determined using the flame atomic absorption spectrometry. Significant
positive correlations between the production of H2O2 and the MDA content and on the MTE content (Ni, Pb, Zn)
indicating a defense against oxidative stress. The presence of Evernia prunastri, Xanthoria parietina, Physcia ads­
cendens and Cladonia stellaris, made it possible to classify the air quality of the Dar-Chichou Forest. P. adscendens
present the weakest reaction against oxidative stress (H2O2) and seemed the most resistant compared to the two
other species. This study was conducted as part of a comprehensive approach combining chemical and biological
parameters to assess the effect of road traffic on air quality. It provides the first database in Tunisia referring to
MTE contamination and their probable biological effects. This study fills a necessary gap in the literature.

1. Introduction absence of waxy cuticle, stomata and conductive vessels, presence of a

cortex rich in mucilages and often porous structure) and to their phys­
Air pollution by Metallic Trace Elements (MTE) has now become a iological characteristics. They are therefore sensitive to the fallout of
public health problem that mobilizes the scientific community around pollutants present in the atmosphere (Bieri, 2017).
the issue of remediation. MTEs are given special attention not only The response of lichens to air pollution has been studied for decades,
because of the risks they may pose to human health but also because of by using their diversity, presence or absence in an ecosystem (Conti
the dangers associated with their persistence on ecosystems. This form et al., 2016), but also by using their physiological responses through
of air pollution can be studied by several methods, pertinent among measurable parameters such as the production of photosynthetic pig­
which is the bioindication by lichens. ments, H2O2 content, MDA and other parameters (Conti and Cecchetti,
Indeed, lichens have long been defined as a permanent monitoring 2001). For this, the researchers adopted one of two methods. the first
system for assessing air pollution (Conti and Cecchetti, 2001; Pignata uses indigenous species, naturally present in the study area (Augusto
et al., 2007; Conti et al., 2016). They are particularly well-suited or­ et al., 2007; Blasco et al., 2008). In this case, certain species such as
ganisms for the study of gaseous and particulate air pollutants. They owe Xanthoria parietina accurately reflect the chemical composition of the air
this efficiency to their anatomical particularity (vegetative structure in (Scerbo et al., 1999, 2002; Cuny et al., 2004). the second method uses
the form of a thallus resulting in a very high surface/volume ratio, transplanted species (Frati et al., 2007; Bergamaschi et al., 2007;

* Corresponding author. University of Carthage, Faculty of Sciences of Bizerte, LR01ES14 Environmental Biomonitoring Laboratory, 7021, Jarzouna, Tunisia.
** Corresponding author.
E-mail addresses: [email protected] (S. Dhaouadi), [email protected] (M. Béjaoui).

https://doi.org/10.1016/j.indic.2022.100211
Received 11 July 2022; Received in revised form 6 October 2022; Accepted 27 October 2022
Available online 28 October 2022
2665-9727/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Dhaouadi et al. Environmental and Sustainability Indicators 16 (2022) 100211

Pacheco et al., 2008; Paoli et al., 2013; Demiray et al., 2012; Guttova 65% and 2.5 ml of hydrochloric acid at 37%. Mineralization is done
et al., 2011; Oztetik and Cicek, 2011a). In this case, changes in the using a digester (Kjel Digester K-446/K-449) for 20 min at 180 ◦ C. It is
physiology and chemical composition of these lichens provide infor­ finished when the solution becomes clear.
mation on the concentration of inorganic and organic contaminants in After decantation, the filtration is carried out using ash filter paper in
the air (Demiray et al., 2012; Guttova et al., 2011; Oztetik and Cicek, sterile vials to obtain clearer and more transparent solutions. Concen­
2011a; Giordano et al., 2005). trations of specific elements in lichen samples were determined by the
In both cases, significant negative correlations were recorded be­ flame atomic absorption spectrometry using the Varian 280 Rapid
tween MTE concentrations and certain physiological parameters (Nan­ Sequential Atomic Absorption Spectrometer (Varian, Australia) for Zn
noni et al., 2015). In our present work, the forest of Dar-Chichou in the and the Varian Zeeman 280 Atomic Absorption Spectrometer using the
Cap-Bon, a Tunisian place under scrutiny in this study, is an artificial Graphite Tube 120 Atomizer (Varian, Australia) for the Pb, Cd and Ni.
forest created in 1972 (Brun, 2007), subject to intense road traffic
through the C26 road that crosses it. Indeed, the daily flow of vehicles is 2.2.2. Photosynthetic pigments (Chl a, Chl b and carotenoids) measurement
6472 (Anonymous, 2021). The extraction of photosynthetic pigments is done through the
In the absence of previous studies on the lichenic heritage of this presence of 80% acetone according to the method of Lichtenthaler and
forest, the objectives of this work are to identify for the first time the Welburn (1983). The amount of 300 mg of fresh material (sorted under a
lichen species present in the forest, to determine the impact of this road binocular magnifying glass) is cut into thin strips in the presence of 5 ml
pollution on certain physiological parameters of these pioneer species, of 80% acetone. The mixture is maintained for 72 h in total darkness and
and to show the bioindicatory and bioaccumulative importance of these at 4 ◦ C. The extract obtained is used for the determination of chloro­
lichens in front of certain trace metals (Pb, Cd, Zn and Ni). phylls a and b as well as carotenoids. The determination of photosyn­
This study would also make it possible to determine the quality of the thetic pigment content in thalli is done from the simultaneous
forest atmosphere via bioindication. Indeed, it has been shown that the measurement of the optical density at three different wave lengths: 470
concentrations of bioaccumulated trace elements MTE in the thalli are nm, 646.8 nm and 663.2 nm. The pigment contents of the leaves are then
directly correlated with those of the environment [ (Anderson et al., determined by reference to the following formulas (Junglee et al., 2014):
1978; Herzig and Urech, 1991; Sloof and Wolterbeek, 1991; Joyce et al.,
1991; Herzig and Market, 1993; Bari et al., 1998; SenIsil Ezgi Eryilmaz Chl a (μg ml− 1) = (12.7 * DO663.2) – (2.69 * D O646.8)
and Ozakca, 2014); (Nannoni et al., 2015)]. Chl b (μg− 1ml) = (22.9 * DO646.8) – (4.68 * D O663.2)
− 1
2. Materials and methods Carotenoids (μg ml) = (5 * DO470) + (2846 * DO663.2) – (14,876 *
DO646.8)
2.1. Study area and sampling sites

The forest of Dar-Chichou is an artificial forest that spreads over an

2.2.3. Hydrogen peroxide (H2O2) measurement
area of 5000 ha. Located in the north-east of Tunisia, it covers the
The H2O2 amount was determined using the method described by
eastern part of the peninsula of Cap-Bon from the coast of Sidi Daoued to
Junglee et al. (2014). The procedure is to grind 0.15 g of fresh material
Oued el Gsab near Hammam Laghzaz. It is bounded to the northwest by
in the presence of 2 ml of trichloroacetic acid (TCA) (0.1%). The ho­
the Mediterranean and to the east by the Sidi Abderrahmen Mountain
mogenate was centrifuged at 12,000 rpm for 30 min at 4 ◦ C. Then 0.5 ml
range.
of supernatant is mixed with 0.5 ml of phosphate buffer 10 mM (PH 7)
The southern boundary is formed by Jebel Korbous and Jebel Sidi
and 1 ml of KI (1M). The determination of H2O2 is based on measure­
Abid to the north. The climate is a Mediterranean one belonging to the
ment by spectrophotometer at 390 nm. The concentration of H2O2 is
sub-humid bioclimatic stage with mild winter (Abazar, 2015). At the
calculated using the extraction coefficient at 390 nm (ε = 10 mM− 1.
time of its establishment, the main species that make up afforestation
Cm− 1) and the contents are finally expressed in μMol g− 1 FW (Draper
were in the following proportions: 29% Pinus pinea L, 9% Pinus halipensis
and Hadley, 1990).
mill, 9% Resinous mixture, 8% Eucalyptus sp, 15% Acacia cyanophylla
Lindl and 30% miscellaneous. The forest is crossed by the C26 road
2.2.4. Malondialdehyde MDA dosage
which generates a fairly intense road traffic. The study sites (Fig. S1)
The MDA dosing reaction is based on the formation in an acidic and
were chosen on the basis of their distribution on both sides of the C26
hot medium between the MDA and two TBA molecules Draper and
road that crosses them.
Hadley method (Van Haluwyn and Lerond, 1986). MDA was extracted
Samples were all taken on the same day (April 21, 2019). Lichen
from fresh plant material (0.15g) crushed in the presence of 2 ml of TCA
species are harvested with a stainless steel knife and stored in plastic
0.1% (w/v). The homogenate obtained was centrifuged at 12,000 rpm
vials previously labelled and then brought back to the laboratory. They
for 30 min. To 1 ml of supernatant is added 1 ml of 0.5% thiobarbituric
are then identified by observation with a binocular magnifying glass by
acid (TBA), prepared in 20% TCA. The mixture was incubated at 90 ◦ C
referring to guides and determination keys based on morphological
for 30 min, then cooled in ice for 10 min and centrifugated at 12,000
criteria (Clauzade and Roux, 1985; Tiévant, 2001; Van Haluwyn and
rpm for 10 min at 4 ◦ C. Absorbance was read at 532 nm and MDA
Lerond, 1993; Van Haluwyn and Asta, 2009; Van Haluwyn et al., 2012)
content was calculated using the extinction coefficient 155 mM-1.
and on the study of colored reactions (Gavériaux, 2003).
Cm-1). Lipid peroxidation was expressed in nMol MDA g− 1 FW.

2.2. Lichen analysis

2.3. Data interpretation and statistical analysis
2.2.1. Metals uptake in the lichen’s thallus
The lichens were carefully washed in deionized water (Leblond, In order to determine the state of air quality, we used the floristic
2004). They were sorted under a binocular magnifying glass to remove approach that uses lichen in its entirety. We have chosen, among the
all traces of foreign matter (dust, leaves, soil, wood chips) (Vitali et al., floristic methods, the qualitative one that is based on lichen associations
2019). Samples taken underwent dehydration at 105 ◦ C for 72 h, then (Van Haluwyn and Lerond, 1986).
grinding using a ball mill for 15 min at a frequency of 25 Hz until The MTE measurements were compared with the Nimis and Bargagli
obtaining a homogeneous and very fine powder. After sieving, we add scale (Nimis et al., 1999). It is composed of 7 classes, ranging from a
300 mg of dry matter of the grind, a volume of 7.5 ml of nitric acid at natural environment ("naturalness") to a polluted environment

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S. Dhaouadi et al. Environmental and Sustainability Indicators 16 (2022) 100211

("alteration") (Table S1) This scale is based on data obtained over several In the Nd2 site, A.cu, is characterized by low accumulation for the 4
years and on different taxa of cortical lichens including X. parietina. metals. In Nd3, Cladonia foliacea endiviifolia (C.Fo.en) is the most
Results are expressed of box plots as mean ± 2 standard error (2SE) accumulating species of Ni, Pb and Zn (respectively contents: 198.33,
and statistical analyses were performed using Statistica 8 for Windows. 158.33 and 242.9 μg g− 1 DW) while Cladonia firma (C.fim) and Cladonia
Structure of the data was first tested in order to assess normality (Sha­ ramulosa (C.ram) are the poorest. In Sd2, Evernia prunastri E.pr has a very
piro-Wilk test) and equality of variance (Bartlett test). Variation of MTE low accumulation power for all MTEs. Finally, in the site Sd3 Cladonia
contents and biomarkers reactions for all samples were tested using one ciliata (C.ci) has a considerable accumulation power for Pb.
way ANOVA. Whenever the ANOVA detected significant differences, The species X.pa is one of the 12 recommended lichen species in the
post-hoc comparisons were made using Tukey’s HSD test. A probability NF X43-904 standard as frequently used in bioaccumulation studies of
level of less than 0.05 was considered significant (95% confidence MTEs. These species can thus be used to identify the threshold of
interval). naturalness of the ecosystem of Dar-Chichou according to the scale of
Principal Component Analysis (PCA): an ordination method for Nimis and Bargagli (Nimis et al., 1999). According to this scale, the state
analyzing any table of statistical data representing n individuals of naturalness or deterioration in this forest differs according to the
described by p quantitative variables (Nimis et al., 1999; Celeux et al., accumulated metal. Indeed, concerning the Cd, all the recorded values
1989). PCA was performed on the mean values of all the measurements (0.33–0.89 μg g− 1 DW) are below the threshold of naturalness (below
taken (H2O2, MDA), the levels of bioaccumulated heavy metals and the the limit of class 4 “low naturality/alteration” indicated by a green line
23 lichenic species analyzed (Table 1). Data processing was performed in Fig. 2). For the Pb, all the values are between the threshold of natu­
using XLSTAT software 2020 version; after y = log (x + 1) ralness class 3 ′′ middle naturality" and the threshold of alteration (class 6
transformation. ′′
high alteration" indicated by a red line in Fig. 1, with the exception of the
maximum value (111.283 μg g− 1 DW) which slightly exceeds the
3. Results alteration threshold. Concerning Zn, the median value is below the
threshold of naturalness, but some values far exceed the threshold of
Lichen surveys carried out at the six sites in the study area show an alteration. Finally, all values of Ni are above the level of alteration and
overall diversity of 23 species (appendix). Indeed, depending on the type fit into the class 7, indicating a "very high alteration" by this metal.
of thallus, the species identified can be divided into 4 categories: 5
crustose species, 5 fruticose, 2 foliose and 11 squamous species. 3.2. Principal components analysis (PCA)
The specific wealth recorded in the sites located north of the C26
road (Nd1, Nd2 and Nd3) is greater than that of the sites located in the To better understand the relationships that exist between the levels
south (Sd1, Sd2 and Sd3) regardless of the distance from this road. There of MTE in the different lichen species studied and their biochemical
are 9 species in Nd1 against 3 in Sd1; 12 in Nd2 against 6 in Sd2 and 12 responses (photosynthetic activities and responses to stress), a principal
in Nd3 against 8 in Sd3 (Table S2). component analysis has been necessary. In this PCA, the first two main
The most frequently encountered species are Xanthoria parietina, axes PC1 and PC2, having an eigen value greater than 2 and extracting
Physcia adscendens foliaceous species often present in highly anthropized 62.15% of the global variance, are then suitable to explain the distri­
and disturbed environments, and Cladonia stellaris (in all study sites bution of the various parameters and sites studied (Table 1) (Fig. 2).
except Sd1). The PC1 axis, which extracts 39.28% of the variance, makes it
possible to identify on the positive side the three metals bioaccumulated
3.1. Metals bio-uptake (Ni, Pb, Zn) by the different species as well as the marker of oxidative
stress (H2O2) and the marker of lipid peroxidation (MDA). This grouping
The spatial distribution of the specific accumulation of MTEs (Pb, Ni, on the same side of the axis is justified by positive and significant cor­
Cd and Zn) (Fig. S2) has made it possible to note that the species X.pa, P. relations of each of these metal pollution parameters (Ni, Pb, Zn) and
ad and C.st are almost present in all the sites prospected and are the most biomarkers (H2O2, MDA). The PC2 axis which extracts 22.87% of the
accumulative. However, Ramalina canariensis (R.ca) shows high levels of total variance makes it possible to identify on its positive pole an
Ni in Sd1 (83.13 μg g− 1 DW), Sd2 (82.9 μg g− 1 DW) and Nd2 (31.1 μg g− 1 increasing gradient of the chlorophyll parameters (Chl a and Chl b).
DW); in Pb in Sd1 (32.3 μg g− 1 DW) and Sd3 (26.5 μg g− 1 DW) and in Zn This factorial plane, PC1-PC2, also makes it possible to identify the
in Sd1 (55.8 μg g− 1 DW) and Nd2 (31.2 μg g− 1 DW) (Fig. S2) as for characteristic species of the different sites in relation to their bio­
species with restricted distribution, the accumulation of MTEs is accumulative properties and their biochemical reactions against oxida­
different depending on the species and the site where it is recorded. tive stress and lipid peroxidation. Indeed, the PC1 axis makes it possible
Indeed, for the species Pertusaria leioplaca (P.le), Amandinea punctata to distinguish 09 species among the 23 species analyzed and to divide
(Am.pu), Cladonia foliacea (C.fol) harvested only in the Nd1 site, the them into two opposite groups, the first grouping together 03 species (P.
accumulation of metals is very low. While for Trapelia glebulosa (Tr.gle), ad, C.st, C.fo.en), located on the positive side of the axis and the second,
Zn (98.32 μg g− 1 DW) and Pb (98.36 μg g− 1 DW) reach high levels. containing 06 species (P.le, C.gr, Am.pu, C.ch (Cladonia chlorophaea), C.
ca (Cladonia carneola), C.ce (Cladonia cervicornis) on the negative side of
Table 1 the axis. In relation to their distributions and the relative contributions
Codes of parameters used on PCA’s. of the sites, the first group is associated with an increasing gradient of
Parameter PCA Code bioaccumulated Ni, Pb and Zn and a strong antioxidant (H2O2) and
lipoperoxidase (MDA) reaction (Table 2), then that the second is rather
Hydrogen peroxide H2O2
malondialdehyde MDA
associated with a lower bioaccumulation of metals and less efficient
Chlorophyl a Chl a antioxidant and lipoperoxidase reactions. As for the axis PC2, it makes it
Chlorophyl Chl b possible to identify two groups; the first, on the positive side, contains 05
Carotenoids Carot species; (R.ca, E.pr) from the southern sites and R.po (Ramalina polli­
Nickel Ni
naria), A.cu (Alyxoria culmigena), R.la (Ramalina lacera) from the
Cadmium Cd
Plomb Pb northern sites characterized by a more advanced photosynthetic ca­
Zinc Zn pacity materialized by Chl a rate and higher Chl b. The second group, on
Nd1, Nd2, Nd3 North-Sites of the road the negative side, with 05 species (Xpa, Sp.cr, Tr.gle, C.ran (Cladonia
Sd1, Sd2, Sd3 South-Sites of the road rangiformis), C.ci) exhibiting the weakest photosynthetic capacities
Colored marks and abreviation Species names
(Table 2).

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S. Dhaouadi et al. Environmental and Sustainability Indicators 16 (2022) 100211

Fig. 1. Boxplot of MTE concentration in thallus of Xanthoria parietina in all sites prospected. (Red line: max; Green line: min). (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Principal Component Analysis of biomarkers

(H2O2, MDA), levels of chlorophylliens pigments (Chl
a, Chl b, Carotenoids) and levels of Ni, Cd, Pb, Zn
accumulated in the various lichenic species collected
in Dar Chichou Forest around the C26 road at Avril
2019. X.pa: Xanthoria parietina, P.le: Pertusaria leio­
placa, (C.gr) Cladonia grayi, (Am.pu) Amandinea
punctata, (C.gr) Cladonia grayi (C.fol) Cladonia folia­
cea, (Sp.cr), Sporodophoron cretaceum, (P.ad) Physcia
adscendens, (C.st) Cladonia stellaris, (R.la) Ramalina
lacera (A.cu) Alyxoria culmigena, (C.fim) Cladonia
firma, (C.ch) Cladonia chlorophaea, (R.po) Ramalina
pollinaria, (R.ca) Ramalina canariensis, (C.ran) Clado­
nia rangiformis, (C.ce) Cladonia cervicornis, (C.fir)
Cladonia fimbriata, (C.fo.en) Cladonia foliacea endivii­
folia, (C.ram) Cladonia ramulosa, (C.ca) Cladonia car­
neola, (E.pr) Evernia prunastri and (C.ci) Cladonia
ciliata.

Table 2
Coefficients in the linear combinations of variables making up PC’s axes. (Hydrogene Peroxyde H2O2, Malondialdehyde: MDA, Chl a: chlorophylle a, Chl b: chlor­
ophylle b and Carotenoid: Carot).
Chl a Chl b Carot H2O2 MDA Ni Cd Pb Zn

PC1 0,057 0000 0,246 0563 0,733 0755 0,000 0634 0,548
PC2 0,776 0799 0,020 0152 0,039 0039 0,090 0130 0,014

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The study of the correlations between the various parameters 4. Discussion

measured for the entire lichen biocenosis of Dar-Chichou, shows sig­
nificant positive correlations between, on the one hand, the production Several studies have shown that lichen richness and diversity are
of H2O2 and the MDA content and on the other hand the Ni, Pb and Zn influenced by several factors. The higher specific richness recorded in
contents. The production of H2O2 is correlated with the production of the sites located to the north of the C26 road (Nd1, Nd2 and Nd3) than
MAD. For photosynthetic pigments, only carotenoids are positively that of the sites located to the south (Sd1, Sd2 and Sd3) could be related
correlated with the increase in Ni, Pb and Zn contents and it seems that to the influence of the wind. Indeed, according to the wind rose of the
Ni only among the 4 metals studied influences the synthesis of Chl a. region studied, the prevailing wind throughout the year is from the
WNW to NW sector, especially during the spring (Abazar, 2015). Wind
direction and proximity to the sea are factors that influence the distri­
3.3. Choice of lichen species for biomonitoring bution of lichen species (Brahimi et al., 2007). Other factors can influ­
ence this distribution, namely, tree essence, circumference, recovery
The ascending hierarchical classification of lichen species, according and orientation of the sampling site (Paoli et al., 2006). Indeed, the four
to their presence/absence in different sites, allowed us to gather 3 lichen groups (crustaceans, leaves, fruticose and scaly) show a disparity
groups of species according to the Euclidean distance (see Fig. 3). This according to the studied sites which can be the result of all these factors.
distance is 1.25 between groups 1 and 2, 1.21 between groups 1 and 3 This lichen diversity can, on its own, constitute a method for evaluating
and 1.19 between groups 2 and 3. the first group contains the species P. (the air quality of a given ecosystem. The most used method is that of the
le, C.fir, C.fo.en, R.ca, C.gr, Am.pu, Sp.cr, Tr.gle and R.la, the second Haluwyn and Lerond scale (Van Haluwyn and Lerond, 1986) which is
group contains E.pr, A.cu, C.ch, Xpa, R.po, P.ad, C.st and a third group based on lichenic association, and which makes it possible to distinguish
contains C.ran, C.ce, C.fim, C.ram, C.ca, C.ci and C.fol. 7 atmospheric quality zones ranging from A to G. In the current study,
The presence of the species X.pa, P.ad and C.st in all sites and their the presence of lichen association of Evernia prunastri, Xanthoria parie­
high bioaccumulations of metals compared to the other species (Fig. 1 tina, Physcia adscendens and Cladonia stellaris, allows to classify the air
additional) justify the choice of them for the remainder of the study. C.st quality of the forest of Dar-Chichou in zone E representing the average
shows the highest bioaccumulation potential for Ni, Pb and Zn while X. pollution level (Van Haluwyn and Lerond, 1986).
pa and P.ad show comparable spectra, slightly lower than that of C.st. We always base ourselves on the specific richness, we can advance
For Cd, the 3 species have almost identical spectra and weakly accu­ that the sites of the south (Sd1, Sd2 and Sd3) are more polluted than the
mulate this metal (Fig. 4). Tukey’s HSD test reveals non-significant sites of north (Nd1, Nd2 and Nd3). Indeed, and according to several
differences between these three species for the levels of the 4 bio­ works (Paoli et al., 2006; Larsen et al., 2007; Calvelo et al., 2009), the
accumulated MTEs. diversity of lichens constantly decreases with the increase in stress
The P.ad species, which shows the weakest reaction against oxidative linked to atmospheric pollution (Sujetoviene and Sliumpaite, 2013). Our
stress (H2O2), appears to be the most resistant compared to the other two results confirm those of the file as for the crustacean species known for
species. This finding explains, at least in part, the lower damage to their weak exchanges compared to the foliaceous and fruticose species.
biological membranes materialized by a relatively lower level of MDA The crustacean species such as P.le, Am.pu, sp.cr, A.cu accumulate less
(Fig. 5). However, there are not significant differences between these air pollutants that the leafy and fruticose species X.pa, P.ad, C.st
three species with respect to these two reactions (p » 0.5). It is therefore (Fig. S2).
concluded that a similar bioaccumulation of MTEs detected between In the case of rapid thallus growth, the MTE contents in the lichens
these three species triggers similar physiological responses. may express temporary contamination and in the case of slow growth
Regarding the synthesis of chlorophyll pigments (Fig. 6) there are such as the case of X.pa, they may indicate chronic contamination
highly significant differences between the three species (p = 0.00018) (Daillant, 2003). Indeed, according to the scale of Nimis and Bargagli
for chl a and chl b.

Fig. 3. Dendrogram showing groupings of lichen

species according to Euclidean distance clustering. (X.
pa) Xanthoria parietina, (P.le) Pertusaria leioplaca, (C.
gr) Cladonia grayi, (Am.pu) Amandinea punctata, (C.
gr) Cladonia grayi (C.fol) Cladonia foliacea, (Sp.cr),
Sporodophoron cretaceum, (P.ad) Physcia adscendens,
(C.st) Cladonia stellaris, (R.la) Ramalina lacera (A.cu)
Alyxoria culmigena, (C.fim) Cladonia firma, (C.ch)
Cladonia chlorophaea, (R.po) Ramalina pollinaria, (R.
ca) Ramalina canariensis, (C.ran) Cladonia rangiformis,
(C.ce) Cladonia cervicornis, (C.fir) Cladonia fimbriata,
(C.fo.en) Cladonia foliacea endiviifolia, (C.ram) Cla­
donia ramulosa, (C.ca) Cladonia carneola, (E.pr) Ever­
nia prunastri and (C.ci) Cladonia ciliata.

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1
Fig. 4. Boxplots of Ni, Cd, Pb and Zn concentrations (μg.g− DW) in thallus of Xanthoria parietina, (XPA), Physcia adscendens, (PAD) and Cladonia stellaris (CST).
Values are given as means ± 2SE.

Fig. 5. Boxplots of Hydrogen Peroxide (H2O2) and malondialdehyde (MDA) Fig. 6. Boxplots of Chl a, Chl b and Carotenoid (Carot) content (μmol.g− 1 FW)
content (μmol.g− 1 FW) in thallus of Xanthoria parietina, (XPA), Physcia ads­ in thallus of Xanthoria parietina (XPA), Physcia adscendens (PAD) and Cladonia
cendens, (PAD) and Cladonia stellaris (CST).. Values are given as means ± 2SE. stellaris (CST). Values are given as means ± 2SE.

(Draper and Hadley, 1990), certain values of the accumulation of Zn far chemical and biochemical parameters studied (Table 2) shows differ­
exceed the threshold of alteration. ences in correlations according to the metal studied and according to the
The impairment of certain physiological functions in lichens in chemical and/or biochemical parameter considered.
connection with the accumulation of certain pollutants makes them Concerning Zn, Pb and Ni, significant positive correlations were
good bioindicators (Nimis et al., 1999; Bouziane, 2006). Indeed, many recorded between each of these metals and the carotenoid contents on
studies establish a direct link between metallic trace elements and the one hand and the H2O2 and MDA contents on the other hand. It sems
oxidative stress in higher plants (Cuny et al., 2002). They have shown a that neither the synthesis of chl a, nor that of chl b are disturbed by the
deleterious effect on chlorophyll pigments but also on membranes by contents of MTE studied exception between chl a and Ni. According to
increasing the concentration of malondialdehyde (MDA) (Nimis et al., some authors, pollutants cause damage to photosynthesis, decrease the
1999). Metal trace elements have many toxic effects. They affect the integrity of cell membranes (Zambrano and Nash, 2000; Piccotto et al.,
various metabolic pathways such as photosynthesis, respiration, and 2011; Paoli et al., 2011), and/or induce oxidative stress (Carreras et al.,
others (Cuny et al., 2002; Garty et al., 1985; Chettri et al., 1998). Ac­ 2009; Oztetik and Cicek, 2011b). Contrary to all expectations, the levels
cording to Brown and Beckett (Brown et al., 1985), membrane damage of chlorophyll compounds are very high in some lichenic species of
by trace elements is the primary cause of their toxicity. polluted sites compared to control lichens (Cuny et al., 2002). Von Arb
The PCA study of the correlation between the MTE contents accu­ and Brunold (Von Arb and Brunold, 1990) and Carreras and Pignata
mulated by the entire lichen biocenosis of Dar-Chichou and the various

6
S. Dhaouadi et al. Environmental and Sustainability Indicators 16 (2022) 100211

(Carreras et al., 1998) showed that the concentrations of chlorophylls a products formed during lipid peroxidation, malonedialdehyde (MDA),
and b were high in samples exposed to downtown pollution. This has studied as a marker of lipid peroxidation. MDA, a terminal product of
been attributed to a fertilizing influence of pollutants (Carreras et al., lipid degradation and whose content is closely related to cell membrane
2009). Air pollutants are probably responsible for the degradation and degradation, is an early indicator of toxic aggression and can therefore
increase of pigments at the same time (Carreras et al., 2009; Reed et al., be used as a biomarker of oxidative stress (Ladhar-Chaabouni et al.,
1989). This could explain the positive correlation between the chl a and 2007). Indeed, significant positive correlations were recorded between
the high Ni contents in the samples of this study. the level of H2O2 and the content of MDA in higher plants such as wheat.
The content of a pollutant in a lichen species may also depend on the In lichens, increased MDA concentration has been shown to be an in­
type of the photobiont partner in this symbiotic association. Generally, dicator of oxidative damage (Canas et al., 1997; Gonzalez and Pignata,
the photobiont is more sensitive to heavy metals than the mycobiont. 1994). This early biomarker of oxidative stress is correlated with the
This sensitivity determines that of the lichen (Reed et al., 1989; Van­ contents of some metals (Sujetoviene and Sliumpaite, 2013). Other
kataraman et al., 1992). Brown and Beckett (Brown and Beckett (1983) Work have shown a direct relationship between the levels of S, Al, Pb
found that Zn, Cd and Cu inhibited photosynthesis in lichens containing and the MDA content in the thalli of some lichens [76].
cyanobacterial protobionts at significantly lower concentrations than in According to the PCA and the correlation study, X.pa, C.st and P.ad
lichens containing green algae as photobionts. In this work, it was found are the three species which best characterize the distribution and
that the Zn tolerance was proportional to the Zn content. This could be therefore constitute the essential link in any lichen group that can be
related to some molecules produced by lichens. Indeed, a greater pro­ used in bioindication of Dar-Chichou. The study of these three species,
duction of usnic acid by some lichenic species or by the same species allowed us in the rest of the work to compare the variation of the spatial
under different conditions, is at the origin of a greater accumulation of accumulation of the studied MTE, the variation of their biochemical
Zn (Cuny et al., 2002). This could be the case for the species X.pa in Nd1 responses to the different contents of MTE and to look for the impact of
(408.21 μg g− 1 DW), C.st in Nd3 (561,675,408, μg.g− 1 DW) and P.ad in this metallic pollution on the synthesis of some photosynthetic pig­
Nd3 (276.726 μg g− 1 DW). However, this production of usnic acid is ments. The wide distribution, in all the study sites, of these three species
itself dependent on other factors such as road traffic, the source of some could explain their similarity of accumulation of MTEs and which would
pollutants, itself influenced by topographical and meteorological char­ be related to their types of thalli having a large accumulation surface
acteristics (Carreras et al., 2009). compared to the covered surface. Indeed, the thallus is foliaceous for X.
In addition, the type of substrate carrying the lichen can also be the pa and P.ad while it is fruticose for C.st (Canas et al., 1997).
cause of the difference in the bioaccumulation of MTEs by some lichen In addition, the comparison of the three species concerning on the
species. In fact, the elements transported from the substrate to the one hand the contents of Chl a and chl b, and their reactions to oxidative
thallus, by the particle trapping mechanism, depend above all on the stress (H2O2) and lipoperoxidation (MDA) on the other hand allows to
physicochemical characteristics of the substrate. A study carried out by deduce that the three species have not the same photosynthetic physi­
Weed and Norton (1991) on liquid extracts from a lichen colonizing ology, nor the same biochemical reactions against contamination by
sandstone rock, shows the presence of oxalate-iron structures. On basalts certain MTEs. P.ad seems to be the most sensitive, C.st the most resistant
rocks, lichens accumulate magnesium and iron (Iskandar and Syers, species and X.pa takes an intermediate place between the two. C.st,
1972). The disintegration of granitic rocks, rich in aluminum, shows the having the greatest potential for bioaccumulation of MTE, seems that its
transformation of biotite into hydro-y-aluminum vermiculite (Pietro resistance offers it stronger protection against the degradation of chlo­
et al., 1994). On limestone rocks colonized by Cladonia callopisma, rophyll parameters. This is confirmed by a stronger reaction against
Diploschistes ocellatus, Squamarina oleosa, and Protoblastemia testacea, lipoperoxidation (MDA content almost double that of the other two
calcite has decreased considerably due to the activity of oxalic acid species), which offers it a strong protection against the degradation of
(Ascaso et al., 1982). the chlorophyll parameters. According to Brown (Canas et al., 1997), the
Among the physiological responses of lichens to pollutants is the multicellular and reticular form of many lichens makes it possible to trap
production of reactive oxygen species (ROS), such as hydrogen peroxide insoluble particles containing metals, which will contribute to the
(H2O2). Under normal growth conditions, cell production of ROS is low analysis of total metals but will not have an immediate impact on their
in the chloroplast (240 μM s− 1O -2 and 0.5 μM H2O2) (Polle, 2001). metabolism.
Several stress can disrupt cell homeostasis and stimulate the production
of ROS (240–720 μM s− 1 O−2 and 5–15 μM H2O2) (Calvelo et al., 2009). 5. Conclusions
This corroborates with our results for the whole of the lichen biocenosis
collected. In fact, significant positive correlations between the Pb, Ni This global study combining chemical and biological parameters to
and Zn contents and the H2O2 content were recorded for all the lichenic assess the effect of road traffic on air quality, provided the first database
species of Dar-Chichou Forest. These ROS are capable of oxidizing in Tunisia referring to contamination by MTE and their probable bio­
various cellular compounds and can lead to oxidative destruction of the logical effects. In this work, we studied the bio-uptake of 4 MTE (Ni, Cd,
cell. However, they also act as messengers for the activation of defense Pb, Zn) in the thalli of some species of lichens near a road crossing the
systems. Faced with the increase in ROS, the cell activates these forest of Dar-Chichou (NE-Tunisia). In this study, 23 species of lichens
detoxification mechanisms, and it would be interesting in future work to with different thalli were detected and the amounts of MTE in the thalli
research the contents of certain antioxidants produced in the event of of several species were determined.
metal stress (Example: catalase, …). This would make it possible to test The spatial distribution of the specific accumulation of MTEs (Pb, Ni,
the differential competence of different lichen species to protect them­ Cd and Zn) showed that the species X.pa, P.ad and C.st are the most
selves against this type of stress. The significant positive correlation accumulating. However, Cladonia firma (C.fim), Cladonia ramulosa (C.
recorded between the content of carotenoids and the level of H2O2 in the ram) and Evernia prunastri (E.pr) are the poorest in these ETMs. These
lichenic species studied confirms the advanced conclusions by Adams species can thus be used to identify the naturalness threshold of the Dar-
et al. (1993), which confirm that carotenoids participate in photosyn­ Chichou ecosystem according to the scale of Nimis and Bargagli (Nimis
thetic activity in plants and that they contribute to the protection of et al., 1999). Indeed, according to this scale, all Cd values are below the
chlorophylls by reducing their photo-oxidation. naturalness threshold. For Pb, the values are between class 3 (average
The primary targets of ROS are lipids, especially those present in cell naturalness) and class 6 (high weathering). Concerning Zn, the median
and subcellular membranes. Membranes rich in polyunsaturated fatty value is lower than the threshold of naturalness. Finally, for Ni, all
acids (PUFA) are very sensitive to oxidation due to their high degree of values indicate very strong weathering.
unsaturation (Pamplona et al., 2002; Hulbert, 2005). Among the Principal component analysis (PCA), dealing with the sampling sites

7
S. Dhaouadi et al. Environmental and Sustainability Indicators 16 (2022) 100211

and the parameters studied, makes it possible to identify on the PC1 axis Bouziane, M., 2006. Etude physico-chimique de l’accumulation des métaux lourds par les
lichens. Impacts sur les voies de biosynthèse et interprétation du mécanisme de
a first group formed by the three bioaccumulated metals (Ni, Pb, Zn) and
tolérance chez Diploschistes muscorum. Thèse pour l’obtention du grade de Docteur
the stress markers oxidative (H2O2) and lipid peroxidation (MDA). On de l’Université des Sciences et Technologies de Lille Discipline : instrumentation et
the side of the positive pole of the PC2 axis, an increasing gradient of Analyses Avancées. Université des Sciences et Technologies de Lille. Ecole Doctorale
chlorophyll parameters (Chl a and Chl b) is individualized. As for the des Sciences pour 1 ’Ingénieur de Lille, 3780, pp. 1–185.
Brahimi, M., Koutitonsky, V., Bejaoui, B., Sammari, Ch, 2007. Bull. Inst. Natn. Scien.
lichen species, this PCA also made it possible to identify the character­ Tech. Mer de Salammbô 34, 157–165.
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Brown, D.H., Beckett, R.P., 1985. Minerals and lichens: acquisition localization and
lipid peroxidation. effects. In: Vicente, C., Brown, D.H., Legaz, M.E. (Eds.), Surface Physiology of
The ascending hierarchical classification of lichen species, according Lichens. Universidad complutense de Madrid, Madrid, pp. 127–149.
to their presence/absence in the different sites, allowed us to distinguish Brun, S., 2007. De l’Erg à la forêt. Dynamique des unités paysagères d’un boisement en
région littorale. Forêt des dunes de Menzel Belgacem, Cap Bon. In: Tunisie. Thèse de
3 groups of species, including the one which contains the species X.pa, P. Doctorat Centre de Biogéographie-Écologie - FRE 2545 CNRS/ENS. Université de
ad and C.st noted in all the sampling sites and having more or less similar Paris IV-Paris-Sorbonne. U.F.R. de Géographie et Aménagement.
accumulation profiles of MTEs. This distinction justifies our choice to Calvelo, S., Baccala, N., Liberatori, S., 2009. Lichens as bioindicators of air quality in
distant areas in Patagonia (Argentina). Environ. Bioindic. 4 (2), 123–135. https://
use these species as bioindicators of possible metallic air pollution from doi.org/10.1080/15555270902963459.
road traffic. Canas, M.S., Orellana, L., Pignata, M.L., 1997. Chemical response of lichens Parmotrema
austrosinense and P. conferendum transplanted to urban and non-urban environments.
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Carreras, H.A., Gudino, G.L., Pignata, M.L., 1998. Comparative biomonitoring of
Declaration of competing interest atmospheric quality in five zones of Cordoba city (Argentina) employing the
transplanted lichen Usnea sp. Environ. Pollut. 103, 317–325.
The authors declare that they have no known competing financial Carreras, H.A., Rodriguez, J.H., Gonzalez, C.M., Wannaz, E.D., Garcia Ferreyra, F.,
Perez, C.A., Pignata, M.L., 2009. Assessment of the relationship between total
interests or personal relationships that could have appeared to influence suspended particles and the response of two biological indicators transplanted to an
the work reported in this paper. urban area in central Argentina. Atmos. Environ. 43, 2944–2949.
Celeux, G., Diday, E., Govaert, G., 1989. Classification automatique des données.
Environnement statistique et informatique. BORDAS, Paris.
Data availability
Chettri, M.K., Cook, C.M., Vardaka, E., Sawidis, T., Lanaras, T., 1998. The effect of Cu, Zn
and Pb on the chlorophyll content of the lichens Cladonia convoluta and Cladonia
No data was used for the research described in the article. rangiformis. Environ. Exp. Bot. 39, 1–10. https://doi.org/10.1016/S0098-8472(97)
00024-5.
Clauzade, G., Roux, C., 1985. Likenoi de Okcidenta Europo. Ilustrita determinlibro.
Acknowledgment Royan, S.B.C.O. Édit. Bull. Soc. bot. Centre-Ouest, n◦ spéc. 7, 893 + 2 pp.
Conti, M.E., Cecchetti, G., 2001. Biological monitoring: lichens as bioindicators of air
pollution assessment–a review. Environ. Pollut. 114, 471–492. https://doi.org/
The authors wish to thank Karim ben Hamed and Aderrezek Smaoui 10.1016/s0269-7491(00)00224-4.
(LPE Borj-cédria) for their help in identifying lichen species and carrying Conti, M.E., Jasan, R., Finoia, M.G., Iavicoli, I., Plá, R., 2016. Trace elements deposition
out certain analyzes, and the anonymous reviewers for their construc­ in the Tierra del Fuego region (south Patagonia) by using lichen transplants after the
Puyehue-Cordón Caulle (north Patagonia) volcanic eruption in 2011. Environ. Sci.
tive comments and the editor for his review of the manuscript. Pollut. Res. 23 (7), 6574–6583. https://doi.org/10.1007/s11356-015-5858-8.
Cuny, D., Pignata, M.L., Kranner, I., Beckett, R., 2002. Biomerkers of pollution-induced
Appendix A. Supplementary data oxidatitve stress and membrane damage in lichens. In: Nimis, P.L., Scheidegger, C.,
Wolseley, P.A. (Eds.), Monitoring with Lichens-Monitoring Lichens, 97-110, vol. 7.
NATO Science Series. IV. Earth and Environ. Sci.
Supplementary data to this article can be found online at https://doi. Cuny, D., Davranche, L., Thomas, P., Kempa, M., Van Haluwyn, C., 2004. Spatial and
org/10.1016/j.indic.2022.100211. temporal variations of trace element contents in Xanthoria parietina thalli collected
in a highly industrialized area in northern France as an element for a future
epidemiological study. J. Atmos. Chem. 49, 391–401.
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