Science of the Total Environment 372 (2007) 717 – 727

www.elsevier.com/locate/scitotenv

Factors to consider for trace element deposition biomonitoring

surveys with lichen transplants
Sophie Ayrault a,⁎, Roberto Clochiatti b , Francine Carrot b ,
Laurent Daudin b , James P. Bennett c
a
Laboratoire des Sciences du Climat et de l'Environnement, CEA-CNRS-UVSQ, F-91198 Gif-sur-Yvette, France
b
Laboratoire Pierre Süe, CEA-CNRS, CEA Saclay, F-91191 Gif-sur-Yvette, France
c
U. S. Geological Survey and Institute for Environmental Studies, University of Wisconsin, Madison, Wisconsin 53706, USA
Received 26 June 2006; received in revised form 27 September 2006; accepted 18 October 2006
Available online 8 December 2006

Abstract

A trace element deposition biomonitoring experiment with transplants of the fruticose lichen Evernia prunastri was developed,
aimed at monitoring the effects of different exposure parameters (exposure orientation and direct rain) and to the elements Ti, V, Cr,
Co, Cu, Zn, Rb, Cd, Sb and Pb. Accumulations were observed for most of the elements, confirming the ability of Evernia
transplants for atmospheric metal deposition monitoring. The accumulation trends were mainly affected by the exposure orientation
and slightly less so by the protection from rain. The zonation of the trace elements inside the thallus was also studied. It was
concluded that trace element concentrations were not homogeneous in Evernia, thus imposing some cautions on the sampling
approach. A nuclear microprobe analysis of an E. prunastri transplanted thallus in thin cross-sections concluded that the trace
elements were mainly concentrated on the cortex of the thallus, except Zn, Ca and K which were also present in the internal layers.
The size of the particles deposited or entrapped on the cortex surface averaged 7 μm. A list of key parameters to ensure the
comparability of surveys aiming at observing temporal or spatial deposition variation is presented.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Air pollution; Biomonitoring; Lichen; Evernia prunastri; Transplants; Trace elements

1. Introduction Easy to identify, this fruticose pendant lichen is often

Lichens are widely used for monitoring air pollution,
either as bioindicators of air quality or as bioaccumu-
lators of atmospheric deposition (Nash and Wirth, 1988;
Nimis et al., 1993; Bargagli, 1998; Asta et al., 2002;
Bennett and Wetmore, 1999). Among all the lichen
species commonly used for biomonitoring purpose,
Evernia prunastri (L.) Ach. is one of the most used.
⁎ Corresponding author. Fax: +33 1 69 82 35 68.
E-mail address: sophie.ayrault@lsce.cnrs-gif.fr (S. Ayrault).
0048-9697/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2006.10.032
abundant on neutral to acidic tree barks.
In heavy metal and trace element deposition studies,
the use of lichen transplants has gained in importance
owing to the advantages of being a substratum-free
surface during a defined period, and the possibility of
exposing lichens in sites where they are naturally absent
(Bennett et al., 1996; Pilegaard, 1979; Garty et al.,
1996). The lichen E. prunastri can tolerate open,
windswept places. Easy to collect intact, the contami-
nation of the thallus by parts of the substrate is reduced.
This lichen is a good candidate for transplantation
718 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727
(Cercasov et al., 2002; Conti et al., 2004; Godinho et al.,
2004; Frati et al., 2005).
The exposure of lichen transplants has raised several
questions in the literature: the vitality of the transplants
during the experiment, the effect of the exposure
orientation toward wind, and the size of the transplants.
The vitality of E. prunastri transplants has been assessed
in a site affected by an industrial complex (Godinho
et al., 2004). The vitality of the fruticose lichen
transplants (Ramalina lacera) was also positively tested
(Garty et al., 2000). The only limitation to the vitality of
lichen transplants would be to start an experiment
during a dry and hot period (Freitas and Pacheco, 2004).
The orientation towards wind direction appeared to have
no significant influence on the element levels in the
transplants for a large range of elements except for Zn
and Cr (Marques et al., 2004). Element content also
increases with age (Senhou et al., 2002), and the use of
thalli at a similar stage of development (same size) was
preferred (Conti et al., 2004). The ability of E. prunastri
transplants to accumulate metals has been assessed by
several studies but the observed accumulation rates
differed from one study to another. Furthermore,
decreasing concentrations during exposures were ob-
served (Conti et al., 2004; Frati et al., 2005).
In this study, E. prunastri transplants were exposed to
traffic-related pollution to answer the following questions:
- What are the limitations on the use of E. prunastri
transplants in biomonitoring surveys?
- How do the transplantation conditions during
exposure (exposure orientation and direct rain effect)
influence the accumulation of trace elements?
To answer these questions, we tested two hypotheses:
1. Lichens exposed horizontally will accumulate more
deposition elements than lichens exposed vertically, and
2. Lichens exposed to precipitation will accumulate
more deposition elements than lichens not exposed to
precipitation.
2. Materials and methods
2.1. Lichen transplant exposure
The lichens were collected in 2004 in a forest (Tête
Ronde, Gif-sur-Yvette, France) far from any polluting
sources. The lichens were collected on Quercus
pedunculata oak bark together with a small piece of
the substrate, at least at 1.5 m above soil. Twenty-four
specimens were used to determine average levels of
trace elements. The transplants were immediately
transported to the exposure site, having the same
geologic and climatic characteristics as the collection
site, 5 km away. The exposure site is an area devoted to
air monitoring, free from canopy cover, 20 km south of
Paris. The main pollution source is a motorway (100 m
away from the study site), with an average of 67,560
vehicles/day, including 5% heavy duty vehicles. Several
other sources are weaker and further away, (urban,
incinerators). The 10 × 10 m2 study site is protected from
vandalism. The piece of substrate at the thallus base was
attached with glue to plastic tubes 1.5 m above the soil
surface. One group of 24 specimens was exposed to the
rain and the other was protected from rain by a
translucent plastic roof (2 × 1 m2), 2.5 m above soil
(Fig. 1). In each group, three positions were used:
horizontal, vertical and oblique (three specimens in each
sub-group). These transplants were exposed for two
months. After collection of the exposed lichens, a new
collection was done in the forest. Two groups of 10
specimens were exposed vertically, one group open to
the atmosphere, the other protected by the roof.
Transplant samples were taken approximately every
six weeks during 9 months. The thalli were positioned
with the base pointing up, and the thallus pointing down.
2.2. Elemental analysis
2.2.1. Trace element total content
After collection, the lichens were air-dried to
constant weight in the laboratory and carefully inspected
under a binocular microscope to remove extraneous
material with nylon tweezers. The analytical procedure
used for bulk content analysis is described in Grasso
et al. (1999). The lichens were ground in an agate
mortar. The solutions resulting from the complete
digestion step (HNO3, HF, H2O2) were analyzed by
inductively coupled plasma — mass spectrometry ICP-
MS (Series X7, Thermo Electron, Courtaboeuf, France)
for ten elements: Cd, Co, Cr, Cu, Pb, Rb, Sb, Ti, V and
Fig. 1. Equipment to expose lichen transplants in horizontal (H),
oblique (O) and vertical (V) positions. The roof protected one set of
lichens from direct rain.
 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727 719

Zn. The detection limits can be found in Ayrault (2005).
Quality control was estimated by analyses of the IAEA-
336 lichen standard reference material, which is E.
prunastri from a pristine area of Portugal. The obtained
values, presented in Table 1, were in good agreement
with the certified and information values.
In order to examine zonation of elements in thalli, a
vertically-exposed Evernia thallus was flattened and cut
into five portions. The thallus was cut every 5 mm from
the extremity to its base. The first portion represented
the first 5 mm at the outermost part of the thallus. The
last part constituted the contact zone with the support.
Each portion was then analysed for its trace elements
content.
2.2.2. Nuclear microprobe
To prepare thin sections of fragile materials for
nuclear microprobe analysis, the most common protocol
used is embedding the samples in a resin (Mesjasz-
Przybylowicz and Przybylowicz, 2002). This invasive
procedure (involving solvents and resin) was not
applied, due to possible losses and contamination, and
because of the expected redistribution of ions. Therefore
a simpler procedure was used. The air-dried transplanted
lichen was hand-sectioned with a steel blade, in the axis
of the thallus radius, within the last 10 mm of the
extremity of the thalli. Thin-sections were transferred
onto 22 × 22 × 0.2 mm3 ultra-pure quartz slides (Suprasil
grade, Heraeus, Germany) using a very small drop of
glue. Micro-PIXE measurements were performed using
the Laboratory Pierre Süe Nuclear microprobe, France
(Khodja et al., 2001). The technical details can be found
in Ayrault et al. (in press). Elemental maps and local
spectrum were performed with RISMIN software
(Daudin et al., 2003). Elemental imaging was performed
for the following elements: S, Cl, K, Ca, Mn, Fe, Ni, Cu,
Zn, and Pb. All maps (representing X-rays counts in a
given area) were generated from K X-ray lines, except
for Pb, which were obtained using the L X-ray lines. The
relative elemental concentrations were calculated with
Gupix-2000 software developed by Campbell et al.
(2000).
Prior to microprobe analysis, a morphological
examination was conducted. E. prunastri cross-sections
demonstrated the usual layered structure of lichens. The
thallus is bordered by the lower and upper cortex made
of dense fungal hyphae. At the center, the medulla (less
dense fungi hyphae) appears in white. The algal layer
appears in green on the light microscope image of the
Evernia cross-section just below the upper cortex. The
algal layer is a chain of round green gonidial cells that
give their color to the upper surface of the lichen. The
lower surface exhibits a grey color. To discriminate the
contents of the different layers, a micrometer-scale
lateral resolution is needed. In practice, a 10 μm lateral
resolution was obtained for the total thallus (Fig. 4). A
resolution of 3 μm was obtained for the more detailed
maps (Fig. 5). In these operating conditions, the
detection limits were low (100 mg kg − 1 ). This
supported the choice of this specimen.
2.2.3. Electron microprobe
The Scanning Electron Microprobe (SEM) imaging
was performed by O. Boudouma (University Paris VI,

Table 1
Initial element contents (mg kg− 1 dry weight, before exposure) of the lichen Evernia prunastri
Reference material IAEA-336 Present study a Cercasov et al. Conti et al. Frati et al.
(2002) b (2004) c (2005) d
Certified S.D. Obtained e S.D. Mean S.D. S.D. S.D.
Cd 0.117 0.100–0.134 0.118 0.021 0.29 0.06 0.080 0.010 0.12–0.10
Co 0.287 0.248–0.326 0.304 0.075 0.63 0.18 0.18 0.04 0.19–0.24
Cr (1.03) 0.80–1.17 0.98 0.21 0.53 0.12 1.1 0.5 1.69 0.40 1.3–2.1
Cu 3.55 3.09–4.01 3.25 0.31 5.1 1.3 4.6 2 3.35 0.77 8.9–10.3
Pb (5.0) 4.4–5.5 4.9 0.2 6.7 1.2 13.9 7.2 2.24 0.66 3.8–4.6
Rb 1.72 1.52–1.92 1.63 0.10 4.5 0.4 2.5–3.4
Sb 0.073 0.063–0.083 0.074 0.005 0.35 0.08 0.28 0.04
Ti 41 3 18.3 4.1
V (1.5) 1.25–1.69 1.1 0.3 0.25 0.03 1.4 0.14 1.4–1.3
Zn 31.6 28.1–35.1 33.7 2.8 16.1 3.9 26.7 2.6 31.3 8.2 24.5–22.5
Information values for the reference material IAEA–336 are shown in parentheses.
a
Average of 24 specimens collected in oak trees, Gif-sur-Yvette France.
b
Average of 10 specimens collected on bark of larch in Croce d'Aune, 1200 m a.s.l., Italy. Washed with water prior to analysis.
c
Average of 22 specimens, collected in the National Park of Abruzzo, 1500 m a.s.l., Italy.
d
Average of 5 specimens, collected in the Chianti region, Tuscany, central Italy. First figure: sampling in 2002, second figure: sampling in 2003.
e
Average of 7 determinations.
720 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727

UFR 928) with a Geol JSM 840 A at 20 keV and a width Table 2
of 25 mm. Element concentrations (mg kg− 1 dry weight) in Evernia prunastri in
three positions and two precipitation exposures

2.2.4. Statistical analysis Thallus position Cr Cu Pb Rb Ti V Zn

Statistical analyses were performed using Microsoft Protected from precipitation
Excel Analysis ToolPak and Systat. The significance of Horizontal 1.8 12.5 278.9 6.7 39.3 1.2 76.9
Oblique 0.7 7.3 54.8 2.6 17.9 0.3 24.3
the difference between exposure conditions (protected
Vertical 2.4 19.8 13.7 3.0 40.2 2.0 30.4
and unprotected from rain) was evaluated using t-tests. Average 1.6 13.2 115.8 4.1 32.5 1.1 43.9
The difference was considered to be significant when
p b 0.05. The effects of thallus position and exposure Exposed to precipitation
to precipitation were tested statistically with nested Horizontal 0.7 8.6 392.6 1.7 24.7 0.8 66.4
Oblique 0.7 6.7 69.7 2.5 19.8 0.2 20.6
analyses of variance, with exposure to precipitation as
Vertical 1.2 12.7 20.3 4.5 32.3 1.3 34.5
the main factor, and thallus position nested within it. It Average 0.9 9.3 160.9 2.9 25.6 0.7 40.5
was not possible to test for an interaction between the Precipitation 0 0 0 0.002 0 0 0.13
two because of the nested design. With three replicates F probability
for each treatment, there were 2 precipitation treat- Position F probability 0 0 0 0 0 0 0
ments × 3 thallus positions × 3 replicates = 18 data points
for each element. The relationship between element
concentration and time was tested with a correlation origin (i.e. Pb). Several explanations could be given for
analysis and the correlations compared with critical the higher accumulation in the part nearest to the tree
values of correlation coefficients (Zar, 1974). trunk. First, this area could be the oldest part where the
elements have been accumulating since the beginning of
3. Results and discussion lichen growth. Second, there might have been contam-
ination of this part due to water flowing on the trunk
3.1. Zonation when raining and/or the ability of this part of the thallus,
densely branched, to retain particles. This hypothesis is
The first question was the choice of the part of the supported by the mobility of the elements on the bark
lichen for analysis. Bargagli et al. (1987) and Godinho surface (Ayrault et al., in press). Another explanation
et al. (2004) demonstrated that the metals were more could be the translocation of the elements from
concentrated in the inner zone of the thallus of the foliose biologically active zones, the thallus extremities, to
lichen Parmelia. Such a non-uniform distribution of less active parts. To avoid any artefact (the translocation
trace elements in the thallus of Evernia was hypothe- may differ from one element to another, or the dust
sized. The results for Cu, Ti and Pb are shown in Fig. 2. accumulation may depend on particle diameters), the
The trace element concentrations were rather stable in external 2-cm of the thalli were used in further
the first portions of the thallus and increased in the base experiments.
portions. This result was similar for the studied elements,
included those from soil origin (i.e. Ti) or pollution 3.2. Initial content

The initial content of the lichens used for transplan-

Fig. 2. Ti (divided by 5), Pb and Cu content (mg kg− 1) of portions of an
Evernia prunastri thallus. Cut from extremity of the thallus: A: − 5 mm;
B: 5–10 mm. C: 10–15 mm. D: 15–20 mm. E: zone of the thallus
attached to the tree bark.
tation are presented in Table 1. Initial contents of
Evernia used by other authors in transplantation studies
are listed for comparison (Cercasov et al., 2002; Conti
et al., 2004; Frati et al., 2005). The initial contents of the
thalli used in this work were in the same order of
magnitude as the published data, and significantly
higher than the element concentrations of the reference
material lichen (IAEA-336) that is considered a
pollution free lichen sample. In all the studies, the
initial content was the average of the measurements of at
least five specimens. The uncertainty associated with the
initial concentrations revealed the inter-individual
variability. In this study, the maximum inter-individual
 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727
variability was 30%. The initial concentrations were low
enough to provide support for the choice of the
collection area used in this study.
3.3. Influences of orientation and exposure on deposition
Exposure conditions may affect lichen accumulation
processes. We have studied the influences of two
factors: orientation towards the atmosphere and the
protection from direct precipitation (Table 2). The
effects of thallus position and exposure to precipitation
depended on element, and may possibly be interdepen-
dent. The effects of exposure to precipitation were
significant ( p b 0.001) for six of the seven elements, Zn
being the exception with no significant difference. The
effects of thallus position were significantly different for
all elements. The patterns of differences, however,
varied among elements.
Averaged across thallus positions, the concentrations
of Cr, Cu, Rb, Ti, V and Zn were significantly higher in
lichens that were protected from precipitation, but that
of Pb was significantly lower.
The concentrations of Pb and Zn were consistently
higher in lichens in the horizontal position regardless of
exposure to precipitation. Rb was higher in lichens
protected from precipitation in the horizontal position,
but the opposite was true in those exposed to
precipitation, suggesting an interaction between the
two factors. In addition, other elements were consistently
higher in the vertical position, regardless of exposure to
precipitation: Cr, Cu and V. For Ti there were very small
differences between the horizontal and vertical position
concentrations regardless of exposure to precipitation.
Except for Pb, all other elements exposed in the oblique,
45° position had the lowest concentrations. The
concentration of Pb was midway between the horizontal
and vertical position concentrations.
The concentrations of Pb varied the most among
these treatments, by one order of magnitude. The other
elements, while being statistically significantly differ-
ent, varied much less on a concentration basis, which is
what is meaningful biologically. It appears, therefore,
that the positioning of transplanted lichens and their
exposure to precipitation can influence the uptake of
elements in an ambiguous way; except for Pb. Lead
concentrations in the horizontally positioned lichens
were 20 times higher than those in the vertical position.
This is probably due to Pb being more abundant in the
exposure site, and the greater exposure the horizontal
position confers on the lichen.
The vertical position is the position most similar to
natural growth. The horizontal position is the less
721
Fig. 3. Lead concentration (mg kg− 1) in lichen Evernia prunastri
exposed under rain in horizontal (H), oblique (O) and vertical (V)
position. Specimens sampled after for 2 months of exposure with an
average of 3 determinations.
probable growth orientation for this epiphytic lichen.
The lichens exposed in the horizontal position exhibited
the highest rate of trace elements accumulation in both
groups (exposed to rain or not). The position effect was
very pronounced for lead. After two months exposure,
the lead concentrations reached 392 mg kg− 1 for the
horizontal transplants under rain (Fig. 3) and 279 mg
kg− 1 for the horizontal transplants protected from direct
rain. Such high lead concentrations were never again
observed at this site. They could be attributed to a
discrete event which occurred within a short period prior
to the sampling date.
This comparison showed that the natural, vertical
position of Evernia was less advantageous for accumu-
lation. This should be taken in consideration in
interspecies studies when foliose lichens, naturally
growing on horizontal supports, are compared with
Evernia or other fruticose lichens. St Clair et al. (2002)
suggested that foliose lichens more efficiently accumu-
lated airborne copper than fruticose lichens. If trans-
plants are used for spatial trends studies or repeated
biomonitoring surveys, they will have to be carefully
positioned, preferably in the vertical position, to ensure
the accurate comparison of data. The vertical exposure
is less advantageous for accumulation but reduces
exposure to perturbing events, and is the closest position
to the natural growth position of Evernia.
3.4. Accumulation trends
The accumulations of 10 elements (Ti, V, Cr, Co, Cu,
Zn, Rb, Cd, Sb and Pb) were measured during 9 months
(February 2004–November 2004) on Evernia trans-
plants exposed vertically. One set was exposed to rain,
with the other set protected by the roof. One specimen in
each set was collected every two months. The
concentrations (mg kg− 1) are presented in Table 3. For
each element, the probability that the protection from
722 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727

Table 3
Composition (mg kg− 1 dry weight) of Evernia lichen transplants as a function of time and exposure conditions (exposed to direct rain or protected
from it)
Date Cd Co Cr Cu Pb Rb Sb Ti V Zn
Exposed
3-17-2004 0.5 0.6 7.6 21.4 28.1 6.6 3.4 124.3 5.8 95.4
5-10-2004 0.5 0.5 2.3 11.8 22.0 4.1 1.8 36.2 2.5 38.5
6-9-2004 0.3 0.5 2.9 14.2 24.7 7.8 2.1 49.7 2.0 45.6
7-16-2004 0.3 0.3 2.2 10.1 16.4 7.7 1.4 30.6 1.2 52.0
9-9-2004 0.5 0.4 1.9 13.5 15.9 4.4 2.4 47.0 2.2 76.2
10-20-2004 0.6 0.3 1.4 11.2 15.2 6.9 1.7 38.7 1.6 66.0
11-19-2004 0.5 0.2 0.8 9.8 23.4 6.0 1.1 34.4 1.1 102.5
Correlation with date - r 0.315 − 0.912 −0.812 − 0.716 − 0.588 − 0.031 −0.686 −0.644 − 0.754 0.309
Average 0.46 0.40 2.73 13.14 20.81 6.21 1.99 51.56 2.34 68.03

Protected
3-17-2004 0.2 0.4 4.5 10.3 11.9 12.8 1.4 49.1 2.1 56.6
5-10-2004 0.5 0.5 2.8 14.1 11.6 7.8 2.3 44.9 2.0 46.6
6-9-2004 0.4 0.5 3.1 16.9 13.2 8.4 2.6 78.4 2.7 63.4
7-16-2004 0.3 0.4 2.1 12.1 9.2 6.9 1.8 32.0 1.6 57.6
9-9-2004 1.1 0.4 1.0 14.9 10.2 6.3 2.2 38.6 1.2 40.9
10-20-2004 0.9 0.7 2.6 21.3 19.6 7.6 3.1 51.5 2.3 61.8
11-19-2004 0.5 0.5 2.3 23.2 12.8 8.2 3.7 60.5 2.3 64.8
Correlation with date - r 0.619 0.462 − 0.680 0.842 0.382 − 0.617 0.809 0.025 − 0.063 0.232
Average 0.56 0.49 2.63 16.11 12.64 8.29 2.44 50.71 2.03 55.96
t probability 0.386 0.308 0.870 0.350 0.016 0.055 0.424 0.952 0.637 0.239

Accumulation factor (AcF) Exposed

3-17-2004 1.5 1.0 15.3 4.2 4.2 1.5 9.8 6.9 23.1 6.0
5-10-2004 1.7 0.8 4.5 2.3 3.3 0.9 5.0 2.0 10.1 2.4
6-9-2004 0.9 0.9 5.8 2.8 3.7 1.7 6.1 2.8 8.1 2.9
7-16-2004 0.9 0.6 4.4 2.0 2.4 1.7 4.1 1.7 4.9 3.2
9-9-2004 1.8 0.7 3.7 2.6 2.4 1.0 6.8 2.6 8.7 4.8
10-20-2004 2.0 0.4 2.8 2.2 2.3 1.5 4.9 2.1 6.5 4.1
11-19-2004 1.8 0.4 1.5 1.9 3.5 1.3 3.1 1.9 4.5 6.4

Protected
3-17-2004 0.6 0.6 9.0 2.0 1.8 2.8 4.1 2.7 8.5 3.5
5-10-2004 1.8 0.8 5.6 2.8 1.7 1.7 6.4 2.5 8.0 2.9
6-9-2004 1.5 0.8 6.3 3.3 2.0 1.9 7.5 4.3 10.9 4.0
7-16-2004 0.9 0.7 4.2 2.4 1.4 1.5 5.0 1.7 6.5 3.6
9-9-2004 3.7 0.6 1.9 2.9 1.5 1.4 6.3 2.1 4.8 2.6
10-20-2004 2.9 1.2 5.3 4.2 2.9 1.7 8.8 2.8 9.3 3.9
11-19-2004 1.6 0.8 4.7 4.6 1.9 1.8 10.5 3.3 9.0 4.0
Correlations significant at p = 0.05 with 5 degrees of freedom are shown in bold font. Means significantly different are shown in bold font.
All the thalli were vertically exposed. The accumulation factor (AcF) is the ratio between concentrations before exposure (see Table 1) and after
exposure.

rain influences the accumulation trends was calculated
(t-test), using the 5% probability level (Table 3).
Protected and unprotected transplants were significantly
different only for Rb and Pb.
The correlations between exposure time and concen-
tration values were statistically tested for each element
and whether they were protected or unprotected from
rain. In the exposed lichens, Co and Cr significantly
decreased over time, but none of the other elements were
affected. In the protected lichens, Cu and Sb signifi-
cantly increased over time but none of the other
elements were affected. Obviously Evernia transplants
are suitable for monitoring these elements over time.
The accumulation factors (AcF) were calculated as
the ratio of the concentration after exposure to the
concentration prior to exposure (Table 3). The concen-
trations ‘prior to exposure’ are presented in Table 1.
Frati et al. (2005) presented a 5-class scale to interpret
accumulation factors. An AcF higher than 1.25 would
indicate accumulation, while an AcF less than 1 would
 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727
indicate a loss. Applying this to our data led to the
conclusion that there was an accumulation of all 10
elements except Co, in the protected and exposed
lichens. Our AcF were higher than those measured by
Frati et al. (2005), most of them occurring in the ‘severe
accumulation’ class (AcF N 1.75) as defined by these
authors. The interpretative scale is probably site-
dependent, as the ‘prior to exposure’ values are linked
to the geochemical background of the site. As we
noticed an inter-individual variability equal to 30% and
less, accumulation cannot be stated for an AcF lower
than 1.3. Even higher AcFs would be found if values
from remote sites would be used as denominators
(Bergamaschi et al., 2004). However, using such data
would not allow observing a pollution signal over the
geochemical background of a specific site. To conclude,
the use of endogenous lichens (sampled in an area
presenting the same geochemical background as the
studied site) for transplants and the measurements of the
inter-individual variability allowed the interpretation of
the accumulation trends. Following Frati et al. (2005),
the initial content has to be measured prior to each
exposure experiment, as an inter-annual variability
(Table 1) can be observed, mainly for soil origin
elements. An alternative is to measure the local
geochemical background of the substratum (Grasso
et al., 1999; Varrica et al., 2000). If the background level
is low, local background data are preferable (Reimann
and Garret, 2005).
During the first months of exposure (1–2 months),
the unprotected transplants exhibited higher accumula-
tion concentrations and AcFs than the thalli protected
from direct rain, for all elements except Rb. However,
the element contents of the unprotected transplants
decreased subsequently.
The rapid increase in trace elements of the unpro-
tected lichens could be attributed to the deposition on
their surfaces of coarse particles. These coarse particles
were then washed off by rain, causing a subsequent
decrease of the element contents. The negative effect of
rain on trace elements accumulation by lichens was
measured by Asta and Garrec (1980b). The sampling of
endogenous lichens after heavy rains led to an under-
estimation of the pollution signal. A biological excretion
due to toxic and or saturation effects can also explain
these element losses. These biological mechanisms
could be stimulated by the rapid increase in element
contents. On the other hand, the lichen surfaces
protected from rain were never exposed to precipitation,
so no negative effects of rain washing, and no positive
effects deposition accumulation could occur. Only fine
aerosols due to air turbulence reached the lichens. The
723
increase in trace elements levels observed during this
experiment in protected lichens could be attributed to
the deposition of fine particles on the surface of the
protected lichens. Discontinuous trace elements accu-
mulation trends have been observed in previous Evernia
transplantation experiments (Cercasov et al., 2002;
Conti et al., 2004; Frati et al., 2005). While heavily
polluted sites appeared to exhibit constant accumulation
rates, the sites proximate to diffuse pollution sources
exhibited discontinuous trends that short-term experi-
ments cannot detect. It could be hypothesised that, when
the dust concentrations in air are very high (close to a
continuous huge source), the deposition of particles on
lichen surfaces dominates the mechanisms of trace
elements release (e.g. biological regulation, rain run-off,
blown-off by wind). At the end of the long-term
exposure presented here, the AcFs measured for
protected lichens were equal or higher than the AcFs
for the unprotected lichens, suggesting that fine aerosols
were the main source of atmospheric elements in the
studied site.
The break in the accumulation curves during summer
(sampling date: 16-July-2004) could be due to the cell
damages caused by the oxidative environmental condi-
tions during this period (Conti and Cecchetti, 2001). As
suggested by Gombert et al. (2006), the impact of high
ozone levels on lichen's accumulation ability has to be
validated, taking into account the ecological parameters
occurring during summer days.
In the course of this study, some high levels of metals
were observed. These high concentrations were attrib-
uted to discrete pollution events that occurred a short
time before sampling. The speed of the increase and
decrease of the element content in lichens after a
pollution event was confirmed by R. Clocchiatti
(unpublished data). A fire event occurred in a factory
in an area where endogenous thalli of Parmelia sulcata
Tayl. had been sampled two months previously. Three
samplings were made: A few hours after the fire, 5 and
then 12 days after the fire event. The lichens sampled the
day of the fire had accumulation factors up to 3 for some
elements (Ti, Sn, Bi). Five days after, the thalli contents
were close to the concentration levels registered two
months before the fire. The speed of these phenomena
points out that one single observation would be unable
to reveal the environmental conditions of a site.
The discontinuous trends in the accumulation rates
could be attributed to the release of elements during rain
events. The surface wash-off could be associated with a
biologically-assisted release mechanism. From a prac-
tical point of view, this suggests exposure durations not
shorter than 6 months, and preferably up to 12 months
724 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727

(Bennett et al., 1996). Long term observations, punctu-
ated by monthly or bi-monthly samplings, and the use of
a protecting non-metallic roof allowing the air to
circulate, appear to be important for atmospheric
biomonitoring with lichen transplants.
3.5. Micro-scale elemental distribution in the thallus
Thalli horizontally exposed to rain and exhibiting an
exceptional high lead concentration (Fig. 3) were
selected for further studies on micro-scale localisation
by μPIXE. Several distinctive distribution patterns
emerged from the PIXE maps (Figs. 4 and 5). The
highest S, Cl, K, Ti, Mn, Cr and Fe concentrations were
detected in the cortex. S and Cl were distributed
similarly, with elevated concentrations in the upper
cortex, and no measurable concentrations in the lower
cortex. K and Mn were distributed similarly, with
elevated concentrations in the upper cortex–algal layer
region, and lower concentrations in the lower cortex. It
was not possible to distinguish upper cortex and algal
layer on the K and Mn maps. Ti, Cr, Cu and Fe were
randomly distributed, mainly detected in the upper
cortex and slightly less so in the lower cortex. Lead was
widespread in the cortex.
The highest level of Ca was in the medulla, with very
low concentrations in the upper cortex. The granular
aspect of the calcium distribution in the medulla is
consistent with the existence of calcium oxalate or
sulfate crystals along the medulla hyphae (Edwards
et al., 1997). Zn had a relatively uniform level across the
thallus, with decreasing concentrations in the medulla.
This supports the fact that Zn plays a biological role
(Cuny, 1999). A region void of any significant amount
of any element was detected between the medulla and
the lower cortex.
Studies about the localisation of the metals in the
lichen thallus, with reference to its layered microstructure

Fig. 4. PIXE pictures of transversal cutting (1300 × 190 μm2) on a thallus of Evernia prunastri showing the Cl, Fe, Zn, Pb and Ti distribution. Grey
scales are arbitrary and element-dependent. The grey level represents the total counts per pixel in the region of interest of the X-ray spectrum. The
structure of the thallus is labelled as follows: uc: upper cortex, al: algal layer, m: medulla, lc: lower cortex.
 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727 725

Fig. 5. PIXE pictures of a portion of a transversal cutting (180 × 164 μm2) on a thallus of Evernia prunastri showing the Cl, K, Ca, Mn, Fe and Ti
distribution. Grey-scales are arbitrary and element-dependent. The grey level represents the total counts per pixel in the region of interest of the X-ray
spectrum. The structure of the thallus is labelled as follows: uc: upper cortex, al: algal layer, m: medulla, lc: lower cortex.

exist (Asta and Garrec, 1980a; Clark et al., 2001; another, independent of growth form. This proves
Budka et al., 2002). The element distribution that the distribution of key nutrients in the lichen is
between mycobiont (fungi) and photobiont (algae) due to active translocation mechanisms inside the
of Ca, Mg, K and P was identical from one species to symbiosis.

Fig. 6. Electron microprobe images of particles entrapped on the upper cortex of Evernia prunastri exposed in a rural/traffic site. A: Titanium-rich
magnetite balloon (FeO.Fe2O3–TiO2). B: drop of mullite (3Al2O3.2SiO2) bearing small (less than 2 μm) particles of metallic lead.
726 S. Ayrault et al. / Science of the Total Environment 372 (2007) 717–727
The average diameter of the particles observed in the
cortex was 7 μm. Particles detected in the lower cortex
were smaller than the particles entrapped in the upper
cortex, where particles with diameters up to 15 μm were
found. Due to the beam lateral resolution, no particle
smaller than 3 μm in diameter could be observed. The
binding of the particles (free deposit or incorporated in
the cortex) would have to be studied, as a function of
particle size and composition, to determine the ability of
rain to wash-off these particles.
The electronic microprobe provided us with the
morphology and elemental composition of the particles
entrapped on the lichen upper cortex surface. It showed
titanium-rich magnetite balloons and mullite (3Al2O3.2-
SiO2) particles (Fig. 6). This was consistent with the
PIXE results. On the surface of the mullite particles,
small (b 2 μm in diameter) dots of lead were observed.
We hypothesized that the lichen surface was widespread
with these very small lead particles.
On the basis of the elemental distribution analyses,
we can conclude that E. prunastri is an effective particle
accumulator for particles less than 20 μm in diameter.
This is consistent with the conclusion of the accumu-
lation trends experiments: protected from rain and
coarse particles, the Evernia transplants accumulated
significant amounts of trace elements.
4. Conclusions
This study confirms the ability of E. prunastri lichen
transplants for atmospheric metal deposition monitor-
ing. The superficial location of the metals (except Zn) in
the lichen thallus was investigated in vivo.
Several pitfalls of air biomonitoring using Evernia
transplants were identified in this work. A zonation of
the metals was observed in the lichen, between the
extremities of the thallus and the parts attached to the
substratum. To ensure the comparability of the data
from one survey to another, the method used to sample
the thallus has to be strictly identical between surveys.
The position of the thallus during exposure affected the
accumulation levels. Once again, this position has to be
strictly defined to ensure comparability. The use of a
roof (non-metallic), allowing air to circulate, to protect
the lichen from direct rain appeared desirable. These
recommendations, in addition to not starting an
experiment during a dry and hot period (Freitas and
Pacheco, 2004), and not to collect samples during a very
humid period (Asta and Garrec, 1980b), will hopefully
improve the comparability and the significance of the
air deposition biomonitoring surveys using lichen
transplants.
Element accumulation is the result of two competitive
phenomena: the deposition of particles on the lichen
surface and the metal release by several mechanisms
(biological regulation, rain run-off, blow-off by wind,
and cell leakage under oxidative stress). As such, the
accumulation rates are not continuous and several
months are needed for observation. We propose
experiment durations for up to one year, punctuated by
intermediate monthly or bimonthly samplings.
Acknowledgements
The authors thank Dr J. Asta (Univ. Grenoble,
France), C. Garrec (LSCE, France) and the anonymous
reviewers. This is contribution LSCE no 2107.
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