International Journal of PIXE, Vol. 21, Nos.

3 & 4 (2011) 133−144

 World Scientific Publishing Company
DOI:10.1142/S0129083511002252

ELEMENTAL ACCUMULATION PATTERNS OF THE LICHEN SPECIES

PHYSCIA TRIBACOIDES NYL., HETERODERMIA DISSECTA AND BACIDIA
BECKHAUSII KÖRBER FROM THE WALAYAR RF REGION,
TAMIL NADU, INDIA

BHARATH PRITHIVIRAJ*,†, ¶,║, E. MANIKANDAN‡,§,¶,** , G. N. HARIHARAN* and K. G. M. NAIR‡

*
Lichen Ecology & Bioprospecting Laboratory, M. S. Swaminathan Research Foundation (MSSRF),
Taramani, Chennai 600 113, Tamil Nadu, India
†
Department of Chemistry & Biochemistry, University of Colorado, Boulder, CO 80302, USA
‡
Particle Irradiation Facility Section, Material Science Division,
Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam 603102, Tamil Nadu, India
§
DST/CSIR, Nanotechnology Innovation Centre, Council for Scientific and Industrial Research (CSIR),
Pretoria-0001, South Africa
║
bharath.prithiviraj@gmail.com
**
manicsir@gmail.com
Received 18 May 2009
Revised 13 December 2011

ABSTRACT
Lichens have been used extensively as biomonitors of air quality (Richardson 1992; Seaward 1995,
2005). Lichen thalli lack a protective layer and hence accumulate airborne pollutants and particulate
matter from the environment by wet and dry deposition (Nash 1996). The elements accumulated on
lichen thalli can be quantitatively analysed using techniques such as Proton Induced X-ray Emission
(PIXE) and Electron probe microanalysis. These techniques yield two-dimensional, quantitative
element information with micron-level spatial resolution. This study used Scanning Electron
Microscopy-Energy Dispersive X-ray (SEM-EDX) and PIXE to quantify elements accumulated on
lichen thalli of selected species collected from polluted and unpolluted areas of the Madukkarai −
Walayar forests. Thalli of selected lichen species from the study site (polluted and unpolluted) were
irradiated using 1.7 MV tandem accelerator with Proton beam energy of 2 MeV. The ion induced X-
rays were detected by Si (Li) semiconductor detector. The morphological analysis and localization
of elements accumulated on these lichen samples were also carried out with the use of a SEM-EDX
microanalysis using environmental scanning electron microscopy (ESEM) as supporting evidence.
PIXE spectral elemental output revealed the presence of elements such as As, Ba, Br, Ca, Cu, Fe, K,
Mn, Zn, Cl, Ti, Cr and Pd with the incidence of increased Calcium levels. IAEA 336 lichen
reference material was used for standardization.

Keywords: Lichens; cement dust pollution; SEM-EDX; proton induced X-ray emission; elemental
analysis Western Ghats.

1. Introduction
The use of lichens (symbiotic fungi associated with algae or cyanobacteria) in the study
of environmental quality is widely accepted in many countries. The use of moss and

†
Current address.
¶
Corresponding authors.

133
134 B. Prithiviraj et al.

lichens to asses trace metal deposition is inexpensive, independent of power supply and
can provide information on the continuing biological effects of atmospheric pollutants
(Loppi et al. 2007). Pollution monitoring associated with limestone quarries and their
dust dispersal effects within forest areas can be assessed using epiphytic lichens by
studying the community level changes and accumulation of elemental content within
lichen thalli along with data on photobiont chlorophyll degradation. The chemical
analysis of native lichen thalli will indicate metal accumulation over the lichen’s lifespan
(Purvis et al. 2007). Beeby, 2001 suggests that the use of lichens as bioindicators is
advantageous over other air pollution assessment tools because it can replace expensive
and sophisticated equipment and that intensive lichen monitoring is a pre-requisite for
effective long-term observation of air pollution.
Farmer et al. 2002 indicate that particulate sources known to affect vegetation are
calcareous in origin. Limestone quarries release large amounts of Calcium oxide that is
highly reactive with water causing extensive damage to plants exposed to the chemical.
Cement kiln dust has a pH of 12.0 according to investigations by Darley et al. 1966. The
objective of this paper is to assess the morphological and ultra structural changes,
accumulation of elements and heavy metals within lichen thalli in select lichen species
exposed to cement kiln dust. The present study was designed to analyze the effect of
particulate dust released from limestone quarries and cement kiln processing on the
morphology, ultra structure and membrane integrity of lichens Bacidia beckhausii
Körber, Heterodermia dissecta and Physcia tribacoides. These lichens colonize polluted
substrates within the reserve forest areas close to Malabar cements factory, Kerala and
the blasting sites of ACC limestone mines, Walayar, Tamil Nadu.

2. Study Area
Madukkarai − Walayar reserve forest is located within the Nilgiri Biosphere reserve area.
These forest sites are contiguous, bordering north of Palghat gap, in the Kerala-Tamil
Nadu state border, India between E 76° 45’30” and N 10° 49’50” to E 76° 54’10” and
N 10° 54’10”. The study site (Figs. 1 and 2) is located at a distance of 27 km to West of
Coimbatore city, Tamil Nadu and 19 km to the East of Palakkad town, Kerala and south
of the Bolampatti II Reserve Forests. The extent of forest cover within the study site is
197.77 km2. The altitude of this region ranges between 290m Msl at Pudupathi 55th
Reserve Forest Corner, Tamil Nadu to 1,039m Msl at Parappatti village in the Tamil
Nadu − Kerala Border. According to Wilson (1967), the Walayar valley is swept by
strong winds during the monsoons as well as in months of January and February. The
Walayar river forms a natural boundary delimiting the forests of the range in the Walayar
valley to the Kerala state on the west and Tamil Nadu on the east.
The current study centers around the cement industry – Malabar Cements Pvt. Ltd.,
Kerala located within the reserve forest area. The mines are located inside the forest areas
and cause extensive damage (Vagholikar and Moghe, 2003). The forests in Coimbatore
district of Tamil Nadu bordering Walayar, Kerala are affected by extensive limestone
mining.
 Elemental Accumulation Patterns of the Lichen Species 135

Study site

Fig. 1. Map showing India, the Western Ghats with study site Madukkarai – Walayar region.

Fig. 2. Malabar cement factory.

136 B. Prithiviraj et al.

3. Materials and Methods

Analysis was conducted on the following lichen species Bacidia beckhausii Körber,
Heterodermia dissecta and Physcia tribacoides were collected from the macroplot
sampling sites within the Walayar reserve forest close to the cement factory and
limestone quarry for experimental studies between the periods February 2003 to February
2006. Samples were collected from the polluted and unpolluted sites of the Walayar
Reserve forest at about 0.5 m to 1 m height from the ground level. Lichen species
B. beckhausii, H. dissecta and P. tribacoides were selected on the basis of their availability
in both polluted and unpolluted sites on the same host tree species. Sampling from the
same host species was carried out at a height of 1.3 mts from the ground. Sample
preparation was carried out according to the protocol described by Maenhaut et al.
(1984). To avoid contamination of elements such as Fe, Cu and Na from equipment or
hand contact lichen thalli were carefully removed from the substrate ensuring that the
chisel was not in direct contact with the lichen samples and the detached portion of the
bark with the thalli was wrapped immediately in tissue paper to be air dried under shade.
Samples were stored at 4˚C in the laboratory prior to PIXE analysis. 200 mg of fresh
lichen sample was shade dried at 37˚C for 48 hrs; ground to a powder in a ceramic mortar
and pestle. 100 µl of an internal standard reference, Palladium chloride (PdCl2) at
8.138 mg/ml was also added to the powdered tissue sample to monitor the beam current.
200 mg of graphite powder (99.99% purity) was added as binder material to the ground
lichen thalli and this mixture was compressed under hydraulic pressure into pellets.
Elemental profile analysis: The target pellets were subjected to a 2 MeV proton beam
energy source generated from a 1.7 MV tandem accelerator with a collimated proton
beam incident on the sample material. The proton beam size incident was 1.5 mm in
diameter and proton beam current was 10 nA. The material used was Beryllium (Be) with
a window thickness of 25 µm and detector window area of 30 mm². The semiconductor
Si (Li) detector material and thickness of exit foil was 100 µm. The resulting ions
induced by X-rays from the sample were detected by the Si (Li).
The active portion of the detector is a 2−5 mm thick intrinsic region produced by
drifting Li into a P-type Silicon crystal. The detector was maintained by liquid nitrogen
(LN2) temperature to prevent diffusion of Li from the intrinsic region to the Li free
region. When the emitted X-rays strike into the detector, photoelectrons are generated
within the body of the Si surface. As this photoelectron travels through the Si, it
generates electron-hole pairs. These electrons and holes are attracted to opposite ends of
the detector with the aid of a strong electric field. The size of current pulse thus generated
depends on the number of electron-hole pairs created, which in turn depend on the energy
of incoming X-rays. From this information elemental composition of the materials under
examination can be obtained from the X-ray spectrum. The PIXE spectra (Figs. 3(a) and
3(b)) was analyzed using GUPIX computer software package (Campbell, J.L. 2000) that
converts the X-ray intensities into elemental concentrations using a standardization
technique that involves fundamental parameters, predetermined instrument constants and
input parameters such as solid angle and charge collected. Analysis of PIXE data was
 Elemental Accumulation Patterns of the Lichen Species 137

carried out and different elements were identified and their concentrations were estimated
after plotting them using Origin 7.5 (Demo Version). A reference point was generated by
comparing the concentration of Palladium (Pd) solution obtained from the internal
standard used with the known concentration. IAEA 336 was used as a lichen standard.

a b
c d

Fig. 3. Micrographs of Physcia tribacoides thallus and EDX spectrum.

Morphological observations on lichen thalli and reproductive structures: Lichen

thalli and characters corresponding to the texture of thalli were observed with a dissection
microscope. The presence of metal rich particulates on thallus surface and in intercellular
spaces of the medulla was demonstrated by SEM and Electron microprobes. Particle
trapping by lichen thalli also resulted in an increased ash and metal content in lichens
growing near regions of high metal exposure. Composition of accumulated elements,
distribution of trace elements within lichen layers, quantitative elemental deposition per
unit area and ultra structural details of target lichens from polluted and unpolluted sites
were investigated through Scanning Electron Microscopy, associated with energy
dispersive X-ray microanalysis (EDX).
138 B. Prithiviraj et al.

4. Results and Discussion

The scanning electron microscopy (SEM) observation of thalli surfaces morphology and
semi-quantitative measurement of element counts (Table 1) were carried out by means of
the energy dispersed X-ray (EDX) system integrated with the SEM. Observations were
made on thalli collected from both polluted and unpolluted sites. The micrographs of
P. tribacoides samples from unpolluted macroplots reveal prominent formation of pruina
at 2,275× magnification and elemental concentrations at Ca 5.16% by weight, Si 3.39%
by weight (Table 1, Fig. 3). The thallus sample from polluted macroplot showed
perforations instead of pruina at 1,138× magnification (Fig. 3(b)). The EDX spectra
(Figs. 3(c) and 3(d)) of the upper thallus surface indicated the presence of Ca at 1,138×
magnification and showed concentration of Ca 37.68% by weight, Al at 9.57 % by
weight, Fe at 10.88 % by weight (Table 1). H. dissecta from polluted sites indicated
soredial clumps along the margin of the thalli at a magnification of 28×. At a
magnification of 117×, the soredial clumps showed Potassium incidence equivalent to Ca
accumulation (Fig. 4(a)) as EDX spectral output. The deposition of Ca flakes is
visualized in the medulla at 2000× magnification (Fig. 4(b)). Elemental data of
H. dissecta thallus from polluted areas revealed deposition of Ca at 40.11%, Si at 10.84,
Al 10.21% and Fe at 5.59% by weight (Table 1). H. dissecta from unpolluted areas
showed Ca at 23.31% by weight and Si at 28.94% by weight. The soredia of H. dissecta
of unpolluted macroplots showed lesser accumulation of Ca 4.77%, Si 25.66% (Table 1,
Fig. 4(d)). In samples from polluted sites soredia showed the following accumulation
pattern, Ca 18.83 %, Si 25.66 %, and Fe 10.71 % by weight (Table 1).

a
b
d

c d

Fig. 4. Ultra-structural SEM micrographs with EDX data on Heterodermia dissecta.

 Elemental Accumulation Patterns of the Lichen Species 139

Table 1. SEM-EDX spectral data of elements localized within select lichen species.

Region Wt
Lichen species analyzed Ca Na Al Mg Si Fe Cu K S %

Bacidia Thallus 23.23 0.00 14.22 0.00 41.14 21.42 0.00 0.00 0.00 100
beckhausii (P)* Ascomata 94.54 0.00 0.00 0.00 5.46 0.00 0.00 0.00 0.00 100

Bacidia Thallus 8.14 0.00 0.00 3.86 56.92 0.00 0.00 7.44 0.00 100
beckhausii (UP)* Ascomata 43.05 0.00 0.00 2.17 10.23 0.99 0.00 12.72 0.00 100

Heterodermia Thallus 40.11 7.59 10.21 8.00 10.84 5.59 15.65 2.01 0.00 100
dissecta (P)*
Soredia 18.83 3.07 18.09 7.31 25.66 10.71 11.51 4.81 0.00 100

Heterodermia Thallus 23.31 0.00 0.44 4.31 28.94 0.00 0.00 6.25 0.00 100
dissecta (UP)* Soredia 4.77 6.09 0.55 8.05 25.66 0.00 0.00 4.80 0.00 100

Physcia Thallus 37.68 1.64 9.57 3.37 8.5 10.88 3.75 2.07 0.00 100
tribacoides (P)*

Physcia Thallus 5.16 1.47 2.5 2.64 3.39 2.34 0.00 2.58 0.00 100
tribacoides (UP)*
(P)*− Polluted site, (UP)* − Unpolluted site (% of elemental composition of the region analyzed by EDX)

SEM micrographs of B. beckhausii thallus (Fig. 3(a)) showed a smooth upper surface
without undulation that was completely covered by white crystals at 19× magnification.
The Ascomata (Fig. 5(a)) appeared as circular bodies of varying sizes that were covered
by white powdery substances at 87× magnification. The thallus top surface (Fig. 5(a))
was uniformly covered by a crystalline substance of varying sizes at a magnification of
724×. Energy Dispersive X-ray spectral analysis focused on the thallus upper surface
indicated the presence of Ca 23% by weight of thallus. The SEM micrograph of ascomata
showed that the epithecial layer with crystalline deposits and its corresponding EDX
spectral data revealed the presence of Calcium quantified at 94.54% weight at 87×
magnification (Table 1, Fig. 5(b)). Thallus from unpolluted environments showed higher
levels of silicon compared to calcium.
P. tribacoides thalli were found to possess ten elements from both polluted and
unpolluted sites. The spectral data output revealed a maximum concentration 16.8 mg/kg
of Ca in polluted thalli when compared to 13.1 mg/kg in unpolluted thalli (Table 2,
Fig. 6(b)). There is a slight increase of iron (Fe) at 1.51 mg/kg in polluted and 0.77 mg/kg
in non-polluted macroplots. There is a double fold increase of sulphur (S) concentration
within polluted thalli (46.2 mg/kg) in comparison to unpolluted thalli at 21.6 mg/kg.
Thalli from unpolluted sites show a higher potassium (K) concentration at 2,889 mg/kg
than 2,162 mg/kg from polluted sites.
140 B. Prithiviraj et al.

a b

c d

Fig. 5. (a) B. beckhausii EDX of the ascomata. Fig. 5. (b) B. beckhausii EDX of upper cortex.

H. dissecta thalli from the polluted sites were found to possess a total of ten elements
with the maximum concentration of Ca 15.28 mg/kg compared to Ca 4.18 mg/kg in
unpolluted thalli (Table 2). There was Fe concentration of 4.51 mg/kg and sulphur
concentration at 23.6 mg/kg from polluted site in comparison to the unpolluted site where
concentration was lower at Fe 1.31 mg/kg and S 13.4 mg/kg. B. beckhausii from polluted
macroplots was found to possess eleven elements. In B. beckhausii thalli within
unpolluted macroplots other elements excluding bromium (Br) were observed in
comparatively low quantity. The spectral data output revealed a maximum concentration
of Ca in polluted thalli 25.8 mg/kg compared to 9.8 mg/kg in unpolluted thalli (Table 2,
Fig. 6(c)). There is a slight increase of Fe at 1.31 mg/kg and S at 65.6 mg/kg. Thalli from
unpolluted site showed higher amount of “K” than thalli from polluted site.
 Elemental Accumulation Patterns of the Lichen Species 141
M S S 002
5
10 Sc
Ca
K
Ti
4
Co
10 Cl

Mn k (α+β)
S
Cr C u k (α + β )
3 P
10
Zn
Yield

10
2 Hg Pd
Pb
Se
B r R b k (α + β )
1
10
Sr

0
10
5 10 15 20 25
E n e rg y (k e V )
Fig. 6. (a) PIXE spectral output, IAEA336.

5 Physcia tribacoides
10 Ca
Pd L (α+β)

Ca
Co

Ti
4
10
Yield

Cl Mn
V Cu
Fe
S
P
10
3 Cr

Zn

Hg

2
10 1 2 3 4 5 6 7 8 9 10 11
Enegry (keV)
Fig. 6. (b) PIXE spectral output, P. tribacoides.

Bacidia beckhausii
5
10 Ca

K
Cl Ti
4 Co
10
Mn k (α+β)

S
P Cu k(α+β )
3
10
Cr
Zn
Yield

2 Hg
10 Pd
Pb
Se
Br Rb k (α+β )
1
10
Sr

0
10 5 10 15 20 25

Energy (keV)
Fig. 6. (c) PIXE spectral output, B. beckhausii.
142 B. Prithiviraj et al.

Table 2. Elemental analysis values through Proton Induced X-ray Emission spectroscopy.

IAEA 336 B. beckhausii P. tribacoides H. dissecta

control Polluted Unpolluted Polluted Unpolluted Polluted Unpolluted
Element (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)
As 0.62 Bdl* Bdl Bdl Bdl Bdl Bdl
Ba 6.8 Bdl Bdl Bdl Bdl Bdl Bdl
Br 13.2 0.2 Bdl Bdl Bdl Bdl Bdl
Ca 36.4 25.8 9.8 16.8 13.1 15.28 4.18
Cu 3.4 0.49 0.11 0.73 0.14 3.4 0.49
Fe 451 1.31 0.8 1.51 0.77 451 1.31
K 1930 2100 2890 2162 2889 1923.35 2243.52
Mn 65 26 17.3 31 15.7 18 11.3
Zn 28.4 7.4 2.1 4.7 0.765 5.4 2.51
Cl 18.4 200 272.2 70 124.6 192 272.2
Ti 8.6 6.0 0.616 9.2 3.4 2.12 0.765
Cr 1.1 1.24 0.358 1.16 0.4 0.45 0.5
S 23.1 65.6 33.4 46.2 21.6 23.6 13.4
*
Bdl − Below detection levels

5. Conclusion
Elemental accumulation patterns on lichen thalli showed gradients concomitant to
locations from where sampling was carried out. Mean threshold of limestone based
particulate deposition reveal Normal, Struggle and Desert zone within surface of thalli.
Thus for microhabitat level assessment elemental accumulation is represented as the
calcium level per unit thallus area. A total of nine trace elements were identified/
quantified through EDX.
The ascomata of Bacidia beckhausii Körber within the cement dust polluted sites
exhibited high calcium (Ca) accumulation levels of 94.54% and Silicon levels of 5.46%
of element concentration in 20 µm2 analyzed area using SEM-EDX. None of the other
lichen species inhabiting this area showed such a high amount of calcium accumulation
in thallus and reproductive structures. Particulate material within lichen thalli exhibit a
threshold of sub µm to >100 µm trapped directly from the environment. Various
metabolic products including metal compounds and complexes of certain metals
especially Ca, Mg and Cu with organic acids produced by the mycobiont also existed
within the thalli (Bargagli et al. 2002). The compositions of accumulated metals,
elements, distribution of trace elements within photobiont and mycobiont layers of the
thallus and quantitative elemental deposition per unit area with ultra-structural details of
target lichens from polluted and unpolluted sites were investigated through scanning
electron microscopy, associated with energy dispersive X-ray microanalysis and PIXE.
Thus elemental accumulation pattern of lichen species inhabiting this region supports a
trend line to particulate deposition affecting the lichen biodiversity of the study region.
 Elemental Accumulation Patterns of the Lichen Species 143

Acknowledgements
Funding support from Department of Biotechnology, New Delhi. Encouragement from
Prof. M. S. Swaminathan, Chairman and Prof. P. C. Kesavan, Homi-Bhabha Chair, M. S.
Swaminathan Research Foundation, Chennai, India. Special thanks to Dr. P. Balaji,
Dr. Baldev Raj, Director, IGCAR Dr. M. Vijayalakshmi, Dr. Parameswaran, Mr. P.
Magudapathy, IGCAR, Kalpakkam. Kathiravan for assistance, Chockalingam, Senthil
and Radhan for assistance during forest survey.

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