Ecotoxicology and Environmental Safety 78 (2012) 184194

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Ecotoxicology and Environmental Safety

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Trace metal contents in wild edible mushrooms growing on serpentine and

volcanic soils on the island of Lesvos, Greece
M. Aloupi a,n, G. Koutrotsios a,1, M. Koulousaris a, N. Kalogeropoulos b
a
b

Water and Air Analysis Laboratory, Department of Environment, University of the Aegean, GR-81100 Mytilene, Greece
Laboratory of Chemistry, Biochemistry, Physical Chemistry of Foods, Department of Nutrition and Dietetics, Harokopio University, GR-17671 Athens, Greece

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 June 2011
Received in revised form
7 November 2011
Accepted 16 November 2011
Available online 14 December 2011

The objectives of this survey were (1) to assess for the rst time the Cd, Cu, Cr, Fe, Mn, Ni, Pb and Zn
contents in wild edible mushrooms (Russula delica, Lactarius sanguiuus, Lactarius semisanguiuus,
Lactarius deliciosus, Suillus bellinii) from the island of Lesvos, (2) to investigate the metals variability
among the species, as well as in relation to the chemical composition of the underlying soil, comparing
mushrooms collected from volcanic and serpentine substrates and (3) to estimate metal intake by the
consumption of the mushrooms under consideration. The trace metals in 139 samples were determined
by ame or ameless atomic absorption spectroscopy. The median metal concentrations were as
follows: Cd: 0.14; Cr: 0.10; Cu: 8.51; Fe: 30.3; Mn: 5.26; Ni: 0.34; Pb: 0.093 and Zn: 64.50, all in
mg kg  1 dry weight. The observed concentrations are among the lowest reported for mushrooms from
Europe or Turkey, while Pb and Cd values did not exceed the limits set by the European Union.
Signicant species- and substrate-related differences in the metal contents were found, but the
variability did not follow a uniform pattern for all the metals in all mushroom species. As a general
trend, the mushrooms growing in serpentine sites contained higher Cd, Cr and Ni than those from
volcanic sites. The calculated bioconcentration factors (BCFs) showed that none of the mushrooms can
be regarded as a metal bioaccumulator, although BCF values slightly above unity were found for Zn in
the three Lactarius species, and for Cu in R. delica.
The studied mushrooms could supply considerable amounts of essential metals such as Zn and Cr.
On the other hand, the consumption of R. delica collected from volcanic soils could provide 12% of the
Cd daily tolerable intake and as high as 53% when collected from serpentine soils. Nonetheless, our
results indicate that the regular consumption of wild edible mushrooms from Lesvos is quite safe for
human health.
& 2011 Elsevier Inc. All rights reserved.

Keywords:
Trace metal
Wild edible mushroom
Serpentine soil
Volcanic soil
Greece

1. Introduction
Mushrooms are valuable health foods, both for their texture
and avor as well as for their low energy content, high proportion
of indigestible ber, specic b-glucans and antioxidant constituents (Kalac, 2009). In addition they contain signicant amounts of
vitamins, minerals and trace elements like Fe, Zn, Se, K (Elmastaset al., 2007; Kalac, 2009). The chemical composition of mushrooms is the main cause for their therapeutic properties in
preventing diseases such as hypertension (Talpur et al., 2002),
hypercholesterolemia (Jeong et al., 2010) and several types of
cancer (Lavi et al., 2006; Sullivan et al., 2006). Various wild edible

Corresponding author.
E-mail address: malou@env.aegean.gr (M. Aloupi).
1
Present address: Laboratory of General and Agricultural Microbiology,
Department of Agricultural Biotechnology, Agricultural University of Athens,
GR-11855 Athens, Greece.
0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.11.018

mushroom species exhibit signicant antioxidant activity, and

therefore, can be used as an easily accessible source of natural
antioxidants, as a potential food supplement, or in the pharmaceutical industry (Elmastas- et al., 2007).
The consumption of wild growing mushrooms has been preferred over that of cultivated ones in many countries of central
and eastern Europe, and is considered to be increasing, even in
the developed world (Agrahar-Murugkar and Subbulakshmi,
2005). The collection of mushrooms in Greece, although more
restricted than in some other European countries such as Poland,
France or Italy, is part of the national tradition and they have been
an important ingredient in both traditional cuisine and gastronomy (Keltemidis, 2005).
From an ecological point of view, mushrooms exert a major
inuence on biogeochemical processes involving soil, rock and
mineral surfaces, and the plant rootsoil interface, and play a key
role in the cycling of elements and the transformation of both
organic and inorganic substrates. These processes can affect plant

M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

productivity and the mobility of toxic elements and substances,

with socioeconomic consequences even on human health (Gadd,
2007). Furthermore, ectomycorrhizal fungi may increase plant
tolerance to heavy metals, as for example in plants growing on Ni
and Cr rich serpentine soils (Aggangan et al., 1998).
Compared to green plants, mushrooms can accumulate large
amounts of some toxic heavy metals, such as Pb and Cd (Sesli and
Tuzen, 1999; Demirbas- , 2001; Alonso et al., 2003; Falandysz et al.,
2007; Konuk et al., 2007; Chudzyjski and Falandysz, 2008;
Brzostowski et al., 2011a, 2011b; Falandysz et al., 2011; Garca
et al., 2009; Gucia et al., in press; Jarzynska et al., 2011). A well
documented example is the high accumulative capacity of
Agaricus macrosporus and other Agaricus species for Cd, and of
Coprinus comatus for Pb (Garca et al., 1998 and references
therein; Melgar et al., 1998 and references therein). Many other
metals are essential for fungal growth and metabolism (e.g. Ca,
Cu, Fe, Mg, K, Na and Zn). Both essential and non-essential metals
can cause toxic effects to living organisms, including fungi, when
present above certain threshold concentrations (Gadd, 1993).
Metals exert toxic effects in many ways, e.g. by causing breakdown of cellular and organelle membranes, blocking operational
groups of signicant biological molecules such as enzymes or
interacting with systems, which normally protect against harmful
effects of free radicals generated during normal metabolism
(Gadd, 2007). Nevertheless many fungi have developed a variety
of mechanisms, both active and incidental, which allow them to
survive, grow and ourish on substrates with high metal levels
(Branco, 2010). Serpentine soils are typically inhospitable for
many plants because of their soil chemistry, with high levels of
potentially phytotoxic elements like Ni, Cr, Co and sometimes Mn
and/or Cu (Chiarucci and Baker 2007; Branco, 2010; Kazakou
et al., 2010). These stressful environmental conditions result in
limited ora diversity along with high endemism and ecotypic
specialization (Branco, 2010 and references therein; Kazakou
et al., 2010). However, there is strong evidence that this general
trend is not followed by ectomycorrhizal fungi, which show no
specialization to edaphic chemical composition. On the contrary,
it appears that serpentine soils support higher fungal diversity
than non-serpentine ones (Branco, 2010).
On the island of Lesvos, Greece, about 60 species of mushrooms have been identied up to now in the herbarium of the
Agricultural University of Athens (Dimou and Polemis, personal

185

communication) but no studies on their heavy metal content have

been undertaken. In this study we assess the concentrations of Cd,
Cu, Cr, Fe, Mn, Ni, Pb and Zn in ve mushroom species from the
island of Lesvos (Russula delica, Lactarius sanguiuus, Lactarius
semisanguiuus, Lactarius deliciosus, Suillus bellinii), all of which
are common and frequently consumed by the local population.
In addition, we examine the impact of species identity and of the
geochemical composition of the substrate on trace metal variability by comparing the metal concentrations of mushrooms
originating from serpentine and non-serpentine soils. Finally,
we undertake an estimation of the dietary metal intakes by the
consumption of the wild mushrooms concerned.

2. Materials and methods

2.1. Sampling
Mushroom samples of ve of the most common wild growing species, namely
Russula delica, Lactarius sanguiuus, Lactarius semisanguiuus, Lactarius deliciosus
and Suillus bellinii (Table 1), were collected on the island of Lesvos, NE Aegean,
Greece, in November 2009. Each species was collected from two different sites:
R. delica, L. sanguiuus and S. bellinii were collected from one site on serpentine
soils and one on volcanic, while L. semisanguiuus and L. delicious were collected
from two sites, both located on volcanic soils (Fig. 1). Sampling from serpentine
soils was based on the geological map of Lesvos (Hecht, 19721975) and
conrmed by the presence of Alyssum lesbiacum, a serpentine endemic species
(Kramer et al., 1997). Metal concentrations in the serpentine and non-serpentine
soils were obtained from the survey of Kazakou et al. (2010). Ten to fteen
individuals of each species were collected from each site, adding up to a total of
139 samples. Both types of sampling sites were in sparse pine forest and were
distant from human activities so that they could be considered as unpolluted.
Identication of each species was based on standard reference books (Phillips,
1981; Konstantinidis, 2009).
2.2. Analytical procedures
The fruiting bodies of the fungi were thoroughly cleaned with a soft tissue
from soil and substrate debris, chopped into pieces using plastic knifes and
weighed within 24 h after collection. This was followed by freeze-drying for 48 h
(to constant weight) and, nally, pulverization in an agate mortar. Freeze drying
was conducted in samples pre-frozen at  50 1C overnight, using a Labconco
FreeZone 4.5 laboratory apparatus. Operating conditions were set at  40 1C
collector temperature and at o 5 mBar vacuum level. Water content was calculated on the basis of water loss during freeze-drying. The pulverized samples were
digested with conc. HNO3 in a Mars Xpress system (CEM), according to the
US EPAs Method 3051A (2007). Metal determinations were performed in a

Table 1
Mushroom species collected with corresponding family, habitat and edibility according to the literature.
Species

Family

Habitat

Edibility

Reference

Lactarius deliciosus
Lactarius sanguiuus
Lactarius semisanguiuus
Russula delica
Suillus bellinii

Russulaceae
Russulaceae
Russulaceae
Russulaceae
Suillaceae

Associated with Pinus, mostly on neutral and calcareous soils

Associated with Pinus on calcareous soils
Associated with Pinus, mostly at grazed sites on calcareous soil
Deciduous and coniferous woods, and forests on calcareous soils
Coastal pine forests of Mediterranean Europe

Good
Excellent
Excellent
Good
Moderate

Basso (1999)
Basso (1999)
Basso (1999)
Galli (2003)
Galli (2000)

Fig. 1. Mushroom sampling sites on the island of Lesvos (Ru de R. delica; La de L. deliciosus; La sa L. sanguiuus; La se L. semisanguiuus; Su be S. bellinii).

186

M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

Perkin-Elmer 5100ZL atomic absorption spectrometer with Zeeman background

correction. The graphite furnace technique was used for the determination of Cd,
Cr, Ni and Pb, while ame AAS was used for the determination of Cu, Fe and Zn.
All chemicals used in the determinations were of Suprapur grade supplied by
Merck, and the water used was from a Milli-Q water purication system
(18.2 MO cm). Sample handling in the laboratory was carried out in a Class 100
NUAIRE (NU 154-524E) laminar ow hood to avoid contamination.

means are signicantly different from each other. Gabriel and Games-Howell tests
were used in the cases of equal or unequal variances, respectively.
A cluster analysis was performed to investigate the relationships among the
species with regard to metal concentrations. Between groups linkage was used as
the clustering method and squared Euclidean distance as the measure of distance.
Before the analysis the data were standardized to their z scores in order to
eliminate scale effects.

2.3. Analytical quality control

3. Results and discussion

The performance of the analytical method for the determination of metals was
assessed according to the guidelines of EURACHEM/CITAC (2000). Blank samples
were included in every digestion batch. In all cases, blank values were below the
limits of detection of the corresponding analytical techniques. The trueness of
metal determinations was assessed by the analysis of the BCR 278, Mussel Tissue
Reference Material, certied by the Community Bureau of Reference, which has
been used regularly for the laboratorys internal quality control of the method. For
all metals the measured values were within the acceptance range of the certied
value (certied value 72 sd). Since the certied reference material used did not
closely match the samples matrix, the trueness was additionally checked by
means of recovery tests in spiked samples, which resulted in recovery rates of 92
103% in all cases. The precision of the analytical procedure was estimated with 14
duplicate tests (10% of the total sample size). The overall precisions, expressed as
reproducibility standard deviation, were 0.09, 0.19, 0.23, 0.08, 0.12, 0.18, 0.15 and
0.08 for Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn, respectively. Finally, the limits of
detection, calculated as three standard deviations of nine independent replicates
of a dilute sample and expressed in dry mushroom weight, were 0.02, 0.01, 0.13,
1.41, 0.22, 0.02, 0.06 and 0.13 mg kg  1 for Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn,
respectively.

The descriptive statistics for water and heavy metal content of

the wild-growing edible mushrooms from Lesvos are shown in
Table 2.
3.1. Water content
The water content of the studied mushrooms was on average
90.6%, ranging between 80.0% and 96.8% (Table 2). These values
were similar to the 8595% water content range mentioned in the
recent reports on the composition and nutritional value of wild
mushrooms from Europe (Kalac, 2009), Northern Greece (Ouzouni
and Riganakos, 2007; Ouzouni et al., 2009) and Turkey (C
- ayr
et al., 2010). They also conrmed the mean water content of 90%,
which is often used for freshdry weight conversions (Kalac,
2009).
3.2. Metal concentrations and bioconcentration factors

2.4. Statistical analysis

All statistical tests were performed with SPSS 16.0. Concentrations below the
limit of detection were recorded only for Pb and they were replaced by the half of
this value (i.e. the limit of detection).
A two-way ANOVA was used to test the signicance of the species and of the
type of substrate as well as their interaction on metal content and on BCFs for the
species collected from sites with different geochemical background. The signicant interactions were further examined for simple main effects, that is the effect
of each factor separately for each level of the other factor. All other differences
were tested by one-way ANOVA. The Levenes test was used to examine the
homogeneity of variance of groups. Should a signicant difference be assessed by
the ANOVA, post hoc multiple comparisons tests were run to further detect which

All metal concentrations are expressed on a dry weight basis,

unless otherwise specied. The overall concentrations in the
mushroom samples are shown in Table 2. The ranges of concentrations spread over one to two orders of magnitude, not only
among the various species but even within the same species, as
for Fe and Pb in S. bellinii, with both the overall minimum and
maximum concentrations occurring in this species. This nding is
in accordance with the literature, since a two-fold variability of
trace metals levels in mushrooms is often encountered (Michelot
et al., 1998; Gadd, 2007; Novackova et al., 2007). Furthermore,

Table 2
Water content (%) and metal concentrations (mg kg  1 dw) in wild growing edible mushrooms from Lesvos.
Species

Water content

Cd

Cr

Cu

Fe

Mn

Ni

Pb

Zn

Russula delica

Mean
Sd
Median
Range
N

89.7
2.7
90.4
80.893.5
29

0.64
0.58
0.51
0.072.12
29

0.57
0.81
0.077
0.022.84
29

25.0
4.7
24.7
19.134.4
29

24.6
10.6
20.7
14.962.7
29

5.4
2.5
4.8
2.414.7
29

2.43
2.85
0.23
0.0510.21
29

0.078
0.049
0.073
0.0290.209
29

39.9
9.0
39.6
23.660.8
29

Suillus bellinii

Mean
Sd
Median
Range
N

94.9
0.9
94.9
93.296.8
28

0.06
0.04
0.05
0.020.18
28

0.32
0.56
0.15
0.042.74
28

9.7
5.2
9.2
3.524.1
28

37.7
28.1
32.8
11.7135.8
28

10.5
6.0
10.2
2.825.3
28

2.87
3.62
0.49
0.0611.12
28

0.191
0.130
0.190
0.0270.519
27

52.5
15.4
57.8
26.378.8
28

Lactarius semisanguiuus

Mean
Sd
Median
Range
N

89.6
2.9
90.6
80.093.2
30

0.16
0.10
0.15
0.060.61
30

2.03
1.21
2.11
0.034.23
30

9.3
3.7
8.2
4.624.0
30

47.9
20.8
42.9
16.999.3
30

5.1
1.5
4.9
1.97.8
30

2.88
1.79
2.73
0.076.67
30

0.097
0.052
0.107
0.0310.187
30

70.4
22.0
64.0
39.0136.4
30

Lactarius deliciosus

Mean
Sd
Median
Range
N

88.9
1.8
89.1
85.391.8
24

0.15
0.06
0.14
0.060.25
23

0.04
0.01
0.04
0.010.07
23

6.9
1.4
7.0
3.910.6
23

29.8
7.6
27.8
20.352.9
24

5.7
1.3
5.7
3.37.9
24

0.24
0.05
0.22
0.110.34
23

0.126
0.043
0.129
0.0300.182
23

81.1
15.4
78.5
62.8120.8
24

Lactarius sanguiuus

Mean
Sd
Median
Range
N

89.9
2.6
90.5
84.594.4
28

0.21
0.12
0.16
0.080.59
25

0.28
0.40
0.07
0.021.81
28

7.0
2.5
6.2
4.015.4
28

34.6
9.3
32.4
21.651.1
28

4.7
1.2
4.3
3.07.8
28

0.59
0.53
0.33
0.162.46
28

0.076
0.034
0.077
0.0310.162
28

103.2
23.4
105.0
57.4179.0
28

M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

the variability in metal concentrations has been found consistently higher in mushrooms than in plants (Kalac, 2010).
In comparison to mushrooms collected from unpolluted sites
in other European countries and Asia Minor, the mushrooms from
Lesvos contain relatively low levels for all the metals studied.
The only likely exception to this is the Cr content in L. semisanguiuus, which is consistently higher in mushrooms from both
sampling sites (n 30 individuals) than in other Lactarius species
in the present or in previous surveys. Fig. 2 depicts the concentrations found in the current survey along with values reported in
the literature for the same species and/or other species of the
same genus (Michelot et al., 1998; Sesli and Tuzen, 1999, 2006;
Demirbas-, 2001; Is- iloglu et al., 2001; Carvalho et al., 2005; Cocchi
et al., 2006; Sesli, 2006; Sesli and Dalman, 2006; Sesli and Tuzen,
2006; Konuk et al., 2007; Ouzouni and Riganakos, 2007; Ouzouni
et al., 2007, 2009; Tuzen et al., 2007; Colak et al., 2009; Ouzouni
et al., 2009; Campos and Tejera, 2011; C
- ayr et al., 2010).
The usual metal contents of European mushrooms, as reviewed
by Kalac (2010), are also shown.

187

Furthermore, the Cd and Pb contents in the mushrooms from

Lesvos did not exceed the statutory limits of 0.2 and 0.3 mg kg  1
fresh weight, respectively, for edible mushrooms (corresponding
to 2.0 and 3.0 mg kg  1 dry weight, assuming a mean water
content of 90%), established by the EU (Commission Regulation
No 1881/2006, 2006). The only marginal exceptions were two
samples of R. delica, which had Cd concentrations just above the
limit ( 2.1 mg kg  1).
According to the literature, a number of factors affect metal
content in the fruiting bodies of mushrooms, thus accounting for
the observed scatter of concentrations. These factors include
species-dependence, substrate composition, the age of the mycelium and the interval between fructication events, metal pollution at the sampling site and biochemical and chemical
parameters of the substrate, such as pH or carrier molecules
(Gast et al., 1988; Lepsova and Mejstrk, 1988; Gadd, 1993;
Michelot et al., 1998; Kalac and Svoboda, 2000; Nikkarinen and
Mertanen, 2004; Kalac, 2010). On the contrary, the age and size
of the fruiting body, and atmospheric deposition are considered

10000.000
Cd

Cr

Cu

Fe

1000.000

C (mg/Kg DW)

100.000
10.000
1.000
0.100
0.010

Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.
Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.
Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.
Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.

0.001

10000.000
Mn

Ni

Pb

Zn

1000.000

C (mg/Kg DW)

100.000
10.000
1.000
0.100
0.010

Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.
Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.
Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.
Ru de
Ru spp.
La de
La sa
La se
La spp.
Su be
Su spp.
Various spp.

0.001

Fig. 2. Comparison of metal levels in mushrooms from the island of Lesvos () with mushrooms from Europe and Asia Minor (). White bars represent usual metal
concentrations in various European species from unpolluted areas reported by Kalac (2010). Wherever data were reported in fresh weight, a mean water content of 90%
was considered to convert the values to dry weight. Abbreviations are as follows: Ru de R. delica; Ru spp. other Russula. species; La de L. deliciosus; La sa L. sanguiuus;
La se L. semisanguiuus; La spp other Lactarius species; Su be S. bellinii; Su spp. other Suillus species; various spp. various species.

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M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

of lesser importance (Kalac and Svoboda, 2000; Kalac, 2010).

Moreover, most of the metals are distributed unevenly within a
fruiting body, with the highest contents usually found in the cap,
particularly in the hymenophore, lower in the spores and in the
rest of cap and even lower in the stipe (Garca et al., 1998; Melgar
et al., 1998; Alonso et al., 2003; Falandysz et al., 2007; Chudzyjski
and Falandysz, 2008; Kalac, 2010; Brzostowski et al., 2011a,
2011b; Falandysz et al., 2011; Gucia et al., in press; Jarzynska
et al., 2011).
The effect of two main factors, species and type of substrate,
on metal concentrations was examined in the present survey.
Since we were able to collect only three of the ve species from
the two different types of substrate (serpentine and volcanicderived soils) that dominate the geology of the western and
central part of the island of Lesvos, the data set was subjected
to two separate analyses: a two-way ANOVA to test the signicance of both species and type of substrate as well as their
interaction in R. delica, L. sanguiuus and S. bellinii and a one-way
ANOVA to test the dependence of metal concentrations on the
species, for all ve species collected in volcanic soils.
Furthermore, in order to evaluate the ability of the studied
mushrooms to accumulate or exclude metals from their substrate,
the bioconcentration factor (BCF) was calculated for each metal
(Table 3) as the ratio between the metal concentration in the
whole fruiting body and its total concentration in the underlying
soil. Metal concentrations in soils in the vicinity of the mushroom
sampling sites were obtained by Kazakou et al. (2010). BCFs are
often calculated on the basis of bioavailable or labile, that is
weak acid extractable, metal concentrations (Chudzyjski and
Falandysz, 2008; Brzostowski et al., 2011a; Gucia et al., in press;
Jarzynska et al., 2011), whereas in other cases total metal
contents in soils, like in this survey, are used (Alonso et al.,
2003; Komarek et al., 2007; Frankowska et al., 2010; Falandysz
et al., 2011). Since the bioavailable fraction of various metals in
soils is a rather small fraction of their total concentrations
(Nikkarinen and Mertanen, 2004; Chudzyjski and Falandysz,
2008) the BCFs calculated by these two methods may obviously
be quite different, as shown by Komarek et al. (2007).
If BCF values exceed unity the species is regarded as a
bioaccumulator of the metal and, vice versa, if BCFo1 the species
is characterized as a bioexclusor. In this context, the very low

BCFs found for all the analyzed metals in all the mushrooms
under consideration suggest that none of the species clearly act as
a metal bioaccumulator, with the marginal exception of the three
Lactarius species with regard to Zn and of R. delica with regard to
Cu, for which BCFs slightly above unity were found.
3.3. Effect of the type of substrate
The F test and its statistical signicance (p) for the factors
Species and Substrate and for their interaction, as calculated by
the two-way ANOVA, are shown in Table 4 and the mean
concentrations of metals for each species and type of substrate
are presented in Fig. 3.
The effect of the two factors on concentrations was not uniform
for all the metals under consideration. Signicantly higher levels of
Cd, Cr and Ni were found in the mushrooms from serpentine soils
(main effect of type of substrate signicant at po0.001) but the
accumulation of these metals varied by species, since the interaction
term was statistically signicant. Species was not a signicant factor
for Cr, in contrast to Cd and Ni, since it explained only 5.6% of the
total variance of the metal content (partial eta-squared0.056),
while the corresponding values for Cd and Ni were 53.4% and 29.9%,
respectively. This was due to the more uniform distribution of the Cr
content among the three species within each type of substrate, in
comparison with Cd and Ni. More specically, the simple main effect
analysis showed that R. delica contained more Cd and Cr when
collected from the serpentine soils, followed by L. sanguiuus, while
S. bellinii had similar Cd and Cr content in both substrates. As for Ni,
S. bellinii and R. delica had much higher contents than L. sanguiuus
in the serpentine substrate. In contrast to the previous metals, Mn
was similar or higher in mushrooms from volcanic soils, the
concentrations being higher in S. bellinii, while no difference in
relation to the type of substrate was detected in L. sanguiuus.
The type of substrate was not signicant as a main factor for Cu and
Zn but there was a strong interaction between species and substrate,
with R. delica and S. bellinii having a higher content of both metals in
the serpentine than in the volcanic sites and with the opposite trend
in L. sanguiuus. Finally, Fe and Pb concentrations were not affected
by the type of substrate whatsoever, as manifested by the lack of
signicance of both the substrate and the interaction of species  substrate terms. A strong dependence of the concentrations

Table 3
BCFs (mean values) of metals in mushrooms from serpentine and volcanic soils of Lesvos. Asterisks indicate statistically different BCFs in volcanic and serpentine
substrates at a 0.05.
Species
L. sanguiuus
R. delica
S. bellinii
L. semisanguiuus
L. deliciosus

Type of substrate
Volcanic
Serpentine
Volcanic
Serpentine
Volcanic
Serpentine
Volcanic
Volcanic

Cr

Cu

0.0006
0.0003
0.0004
0.0006
0.0013n
0.0002
0.0276
0.0004

0.23
0.25
1.07
1.01
0.44
0.36
0.37
0.27

Fe

Mn
n

0.0007
0.0005
0.0005n
0.0003
0.0008n
0.0004
0.0011
0.0007

Ni
n

0.006
0.004
0.009n
0.003
0.02n
0.004
0.007
0.008

0.004
0.001
0.002
0.003
0.002
0.003
0.038
0.003

Pb

Zn

0.003
0.002
0.003
0.004
0.009
0.006
0.003
0.005

1.63n
1.11
0.70n
0.46
0.99n
0.55
1.01
1.26

Table 4
F test (degrees of freedom in parentheses) of the two-way ANOVA and its statistical signicance (p) for species, substrate and their interaction in R. delica, L. sanguiuus and
S. bellinii from serpentine and volcanic substrates of Lesvos.

Species
Substrate
Species  substrate

F(2, 79)
p
F(1, 79)
p
F(2, 79)
p

Cd

Cr

Cu

Fe

Mn

Ni

Pb

Zn

43.54
o 0.001
36.39
o 0.001
23.30
o 0.001

2.33
0.104
35.50
o0.001
4.87
0.010

155.89
o 0.001
0.60
0.443
5.86
0.004

4.49
0.014
0.58
0.450
1.33
0.270

34.65
o0.001
44.93
o0.001
21.72
o0.001

16.89
o0.001
120.74
o0.001
18.96
o0.001

17.37
o 0.001
2.59
0.111
2.49
0.090

122.98
o 0.001
2.26
0.137
4.66
0.012

M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

189

Fig. 3. Mean metal concentrations (in mg kg  1) in L. sanguiuus (La sa), R. delica (Ru de) and S. bellinii (Su be) from serpentine (s) and volcanic (ns) soils of Lesvos (the error
bars show the 95% condence interval of the mean).

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M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

of all metals on species was revealed by the two-way ANOVA

(po0.001 for all metals). This is discussed below, along with the rest
of the species.
The substrate on which mushrooms grow seems to be the
main source of fungal trace element uptake (Lepsova and
Mejstrk, 1988; Gadd, 1993; Gadd, 2007) and the natural geology
and geochemistry of the substrate seems to have a strong impact
on trace element concentrations in mushrooms (Nikkarinen and
Mertanen, 2004). The complex geological composition of the
island of Lesvos results in signicant differences in the chemical
composition of the derived soil, hence leading to differences in
the metal content of mushrooms. According to Kazakou et al.
(2010), the serpentine soils of Lesvos contain much higher Cr
(11184863 mg kg  1) and Ni (11973326 mg kg  1) than the
non-serpentine soils (81.9 mg kg  1 and 84.4 mg kg  1, respectively). These pronounced differences are not proportionately
reected in metal concentrations in the species collected from
both substrates, as already mentioned, as well as in their BCFs.
The statistical analysis (two-way ANOVA) revealed higher BCFs
for Ni in L. sanguiuus and for Cr in S. bellinii from volcanic than
from serpentine soils, while R. delica had equal BCFs for both
metals in both types of substrate. A reverse relationship between
metal content in soils and BCFs was found in many cases in the
past and was attributed to the restriction in the efciency of
metal accumulation caused by toxic effects of metals at high
concentrations (Alonso et al., 2003 and references therein).
It appears, thus, that a proportional increase in Cr and Ni soil
levels is not a consistent trend for all mushroom species growing
on serpentine soils.
It is possible that the Cd enrichment in R. delica and
L. sanguiuus from the serpentine sites could also be attributed to
higher metal content in serpentine soils, but no sound conclusion
can be drawn since data for Cd distribution in Lesvos soils were not
provided by Kazakou et al. (2010). Nonetheless high concentrations
of Cd are a general characteristic of the chemistry of serpentine
soils (Shallari et al., 1998; Cornara et al., 2007; Oze et al., 2008 and
references therein), while concentrations of 12 orders of magnitude lower were found in soils developed over igneous rocks
(Nikkarinen and Mertanen, 2004; Doelsch et al., 2006; Sultan
et al., 2011). On the other hand, although quite low Cd content
were found in 12 species of wild edible mushrooms from granitic
soils in NW Spain, Cd levels in Agaricus macrosporus from the same
area were noticeably high (Melgar et al., 1998), underlining that
species-specic metabolic or ecological features often interfere
with the geochemical features of the substrate in determining
the metal levels in mushrooms. Moreover, this assumption is
supported by the statistical signicance of the interaction between
species and substrate found in this work.
As mentioned above, Mn content of S. bellinii and R. delica was
signicantly higher in mushrooms from volcanic soils. This difference is the opposite of the relationship in the soils themselves
(Kazakou et al., 2010) although the dissimilarity between the two
substrates was not as pronounced as for Cr and Ni. Both these
relationship can explain the higher BCFs for Mn calculated in
volcanic than in serpentine soils. Our results show that the type
of substrate did not consistently affect Cu and Zn levels in mushrooms, since no uniform trend was observed in the three species

investigated. The same was also true for BCFs for Cu, although
higher BCFs were found for Zn in the volcanic than in serpentine
substrate, which could be attributed to the lower Zn content of the
latter type of soils (Kazakou et al., 2010). This nding is further
corroborated by the conclusion of Gast et al. (1988) that Zn and, to
a lesser degree, Cu concentrations in 21 mushroom species from
the Netherlands and Belgium were independent of soil concentrations but signicantly dependent on inter-specic differences
arising from the essential biochemical role of both metals to living
organisms. Finally, each of the three mushroom species had similar
Fe and Pb contents in both volcanic and serpentine soils and the
variance in their concentrations is explained only by speciesspecic differences, which are consistent in both types of soil,
although serpentine soils in Lesvos contain twice as much Fe and
approximately equal levels of Pb compared to the non-serpentine
ones (Kazakou et al., 2010). These relative Fe and Pb abundances in
mushrooms and soils explain the statistical evidence of higher
BCFs for Fe in volcanic than in serpentine soils and equal BCFs for
Pb in both types of soils.
From the above discussion it appears that the role of the type
of substrate in determining trace metal concentrations in mushrooms is not straightforward but its manifestation varies with the
species as well as with the metal. Furthermore, signicant
geochemical differences are more clearly reected in mushroom
content in trace metals, as in the case of Cr and Ni, while
differences of a smaller extent may be overwhelmed by a number
of other factors affecting metal uptake by mushrooms.
3.4. Effect of the species identity
Since the geochemical composition of the substrate has been
found to play a role in regulating the metal content of the
mushrooms studied here, the potential preferences of the various
species towards accumulation of specic metals was investigated
in mushrooms collected from the same type of substrate, i.e. soils
derived from volcanic rocks. The variability of metal concentrations among the species was tested by one-way ANOVA and the
results are summarized in Table 5 and shown in Fig. 4.
For all metals, signicant differences of means were found
among the species at the o0.001 level. The post hoc multiple
comparisons tests revealed some well-dened trends in the
preference of some species to accumulate or exclude metals.
R. delica had signicantly higher Cu than all the other species,
S. bellinii preferentially accumulated Mn and Pb, whereas the
highest levels of Cr, Ni and Fe were found in L. semisanguiuus, the
latter metal being equivalently abundant in S. bellinii, as well. Zn
was found in signicantly higher levels in L. sanguiuus. On the
other hand, signicantly lower concentrations of Zn were found in
R. delica, of Cd in S. bellinii and of Cu in L. sanguiuus. Apart from
these pronounced differences the distribution of the metals
among the species did not exhibit a clear pattern as subgroups
of species with non-signicantly different means often overlapped. The one-way ANOVA of the calculated BCFs (for all metals
but Cd) leads to very similar conclusions.
In accordance to our results, a preferential accumulation of Cu
but also Cd, Pb and Zn was reported in R. delica over L. deliciosus
from the C
- anakkale Province of Turkey (C
- ayr et al., 2010) but the

Table 5
F test (degrees of freedom in parentheses) of the one-way ANOVA and its statistical signicance (p) of metal concentrations in mushroom species from volcanic soils of
Lesvos.

Species

F(4, 92)
p

Cd

Cr

Cu

Fe

Mn

Ni

Pb

Zn

8.69
o 0.001

93.30
o 0.001

151.65
o 0.001

12.05
o 0.001

49.25
o0.001

61.69
o0.001

25.81
o0.001

54.36
o0.001

M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

191

Fig. 4. Mean metal concentrations (in mg kg  1) in R. delica (Ru de), L. deliciosus (La de), L. sanguiuus (La sa), L. semisanguiuus (La se) and S. bellinii (Su be) from volcanic
soils of Lesvos (the error bars show the 95% condence interval of the mean and the dot-lines the homogeneous subgroups found by the post hoc Games-Howell test).

differences between concentrations among various species were

not statistically tested. Since Cu was an order of magnitude higher
in R. delica than in L. deliciosus the deduction could be considered

as reliable, but this may not be the case for the other metals.
Furthermore, Ouzouni et al. (2009) reported high Cu but low Cd
concentrations in R. delica from West Macedonia and Epirus,

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M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

Greece. On the other hand, a minimum Cu level of 4.2 mg kg  1

was reported by Campos and Tejera (2011) for L. sanguiuus in a
survey including 15 species collected from quartzite acidic soils in
Spain, the species with the next lowest Cu content being
L. deliciosus (5.4 mg kg  1). These results were very close to ours,
as far as both the values per se and the ranking of species are
concerned. Another study on metal concentrations in 6 species of
wild edible mushrooms from a trafc-polluted area in Balkesir,
Turkey, found the lowest Cd and Cu levels in L. semisanguiuus,
and slightly higher metal content in L. sanguiuus (Is- iloglu et al.,
2001). The lowest Cu content was also found in L. deliciosus by
Carvalho et al. (2005) in a study on 6 species from Portugal and
the second lowest among 28 species by Alonso et al. (2003) in
northwest Spain.
Summarizing the above information, it appears that the three
Lactarius species under consideration do not tend to accumulate
high amounts of Cu, in contrast to R. delica, which seems to take
up Cu from its substrate more effectively. The tendency of these
species towards Cd accumulation remains ambiguous. Since the
aforementioned surveys were held in both contaminated and
non-contaminated areas, the impact of anthropogenic origin
might be a potential explanation for this discrepancy, the natural
scarcity of the metal taken into consideration.
The intra-specic variation in the selective uptake of Cr and Ni by
L. semisanguiuus was noticeable, manifesting itself as a 12 orders
of magnitude magnication of concentrations in comparison to the
remaining four species (to a much lesser extent this was also found
for Fe). The enrichment of all the three metals in L. semisanguiuus in
relation to the other Lactarius species was still valid when statistically tested in a subset of data comprising only the three Lactarius
species collected from exactly the same site, thus eliminating the
potential inuence of other environmental factors on metal levels
(F(2, 38)300.9, po0.001 for Cr; F(2, 38) 151.9, po0.001 for Ni;
F(2, 38) 31.7, po0.001 for Fe). Moreover, the levels of Cr and Ni in
L. semisanguiuus of non-serpentine provenance are twice as high
for Cr and similar for Ni compared to those in mushrooms from the
serpentine sampling sites. A high bioaccumulation capacity of
L. deliciosus for Zn was reported by Alonso et al. (2003), in a survey
comprising another 27 species of various nutritional modes from
northwest Spain. Relatively high Zn was also found for L. deliciosus in
our survey, but a consistent conclusion of the tendency of the
species regarding Zn uptake cannot be drawn since the two surveys
do not include any other species in common. Additionally, the
survey by Alonso et al. (2003) was carried out in both contaminated
and non-contaminated areas.
The highest Mn and Pb concentrations have been found in
S. bellinii. A tendency of some Suillus species to accumulate Mn
has previously been recorded (Michelot et al., 1998), but an

Fig. 5. Dendrogram of the cluster analysis on the mushroom species from volcanic
soils of Lesvos, in relation to their metal contents.

assessment of the afnity of S. bellinii for Mn or Pb could not be

made since, to our knowledge, no data of the metal content of this
species have been published yet.
The grouping of the species under consideration in relation to all
the studied metals was examined by cluster analysis (Fig. 5). The
only similarity was found between L. deliciosus and L. sanguiuus, all
other species forming single-species groups. It is worth noticing that
species not only in the same family but even of the same genus have
apparently developed diversied processes to interact with the
metal burden in their growing environment. A close relationship
between L. deliciosus and L. sanguiuus was also reported by Campos
and Tejera (2011) in a cluster analysis based upon the concentrations of Al, Zn, Cu and Rb, i.e. the metals with the higher absorption
rate from the substrate, performed on 15 species from Spain.
The authors attributed this relation to similarities regarding the
nutritional behavior of the two Lactarius species, being both ectomycorrhizae of the genus Pinus as opposed to species exerting other
nutritional strategies such as saprobes on soil organic matter.
Although in the present survey all the studied species share the
same nutritional mode (ectomycorrhizae) it appears that the interconnection between L. deliciosus and L. sanguiuus still holds. Both
species had intermediate to low levels of all the examined metals.
On the other hand the interaction of L. semisanguiuus with metals
seems to be markedly different from the other two Lactarius species
and this is probably due to its great afnity for Cr, Fe and Ni. S.
bellinii and R. delica are located between the previously discussed
groups in the dendrogram, the rst one characterized by high Mn
and Pb and low Cd content and the latter by high Cu and Cd and low
Cr, Fe, Ni, Pb and Zn concentrations.
3.5. Dietary intake
In order to calculate the potential trace element intake through
the consumption of wild edible mushrooms from Lesvos, we considered 60 kg as the weight of an average consumer, in agreement
with the EU Scientic Committee for Food Adult Weight parameter
(Ouzouni et al., 2007) and assumed a serving of mushrooms equal to
300 g of fresh weight, which contains 30 g of dry matter (Kalac and
Svoboda, 2000; Svoboda et al., 2000). The intake calculations were
made on the basis of the average metal content in mushrooms,
regardless of the type of substrate, since collection for human
consumption takes place at random, in both serpentine and volcanic
localities. The results of intake and the respective percent coverage of
recommended daily intakes (RDI) or tolerable daily intakes (TDI) are
shown in Table 6.
The great variability in metal contents of mushrooms resulted
in an accordingly great variability in metal dietary intake.
The highest intakes of the toxic metals Pb and Cd were provided
by S. bellinii and R. delica, respectively. The potential intake of Cd
through the consumption of a serving of R. delica was 32% of the
recommended by the World Health Organization value for daily
intake of the metal, reaching 53% when collected from serpentine
soils (not shown in Table 6). Nonetheless, the intake of both toxic
metals through the consumption of wild mushrooms from
Lesvos is well below their tolerable values. R. delica and
S. bellinii were the most important suppliers of Cu and Mn,
respectively. L. semisanguiuus is expected to supply the higher
amounts of Cr, reaching 30.5% of the RDI, followed by R. delica. Ni
content in one serving of R. delica, S. bellinii and L. semisanguiuus
represented 1012% of the TDI, while the Ni intake by the
consumption of R. delica and S. bellinii from the serpentine
substrate could reach 22.2% and 26.3% of the TDI, respectively
(data not shown in Table 6). Finally, the consumption of one
serving of wild mushrooms from Lesvos could cover 812% and
923% of the RDI values for Fe and Zn, contributing to the human
nutritional requirements for these essential metals.

M. Aloupi et al. / Ecotoxicology and Environmental Safety 78 (2012) 184194

193

Table 6
Estimation of trace element intake (mg) and percent coverage of recommended or tolerable upper level of metal intake (in parentheses), through consumption of one
servinga of mushrooms by a normal 60 kg consumer.
Mushroom species

Cd

Cr

Cu

Fe

Mn

Ni

Pb

Zn

Russula delica

0.0190 7 0.017
(31.8)
0.0018 7 0.001
(3.0)
0.0045 7 0.002
(7.3)
0.0063 7 0.004
(10.5)
0.0048 7 0.003
(8.1)
0.060b

0.017 70.024
(8.5)
0.0107 0.017
(5.0)
0.0012 70.00
(0.6)
0.0087 0.012
(4.0)
0.061 70.036
(30.5)
0.20

0.7507 0.140
(2.5)
0.2927 0.154
(1.0)
0.2067 0.043
(0.7)
0.2097 0.074
(0.7)
0.2787 0.111
(0.9)
30b

0.747 0.32
(5.9)
1.13 70.84
(9.0)
0.897 0.23
(7.2)
1.047 0.28
(8.3)
1.44 70.63
(11.5)
10-15

0.1627 0.074
(5.4)
0.3157 0.181
(10.5)
0.1727 0.040
(5.7)
0.1417 0.037
(4.7)
0.1537 0.044
(5.1)
3.0

0.0737 0.085
(10.1)
0.0867 0.109
(11.9)
0.0077 0.001
(1.0)
0.0187 0.016
(2.5)
0.0867 0.054
(11.9)
0.72

0.002 70.001
(1.1)
0.006 70.004
(2.7)
0.004 70.001
(1.8)
0.002 70.001
(1.1)
0.003 70.001
(1.4)
0.214b

1.207 0.27
(8.9)
1.587 0.46
(11.7)
2.437 0.46
(18.0)
3.107 0.70
(22.9)
2.117 0.66
(15.6)
12-15

TDIc

RDId

TDIe

RDIf

RDIf

TDIc

TDIc

RDIf

Suillus bellinii
Lactarius deliciosus
Lactarius sanguiuus
Lactarius semisanguiuus
Recommended or tolerable
daily intake (mg/day)
Reference
a

One serving 300 g fresh weight containing 30 g dry weight.

Calculated from the weekly or daily intake per kg body weight for a 60 kg person; TDI: tolerable daily intake; RDI: recommended daily intake.
WHO (2008).
d
WHO (1996).
e
European Commission (2003).
f
US National Research Council (1989).
b
c

4. Conclusions
This rst survey of metal contents in 5 wild edible mushrooms
from Lesvos showed low levels of the studied metals in comparison
to mushrooms from Europe or Asia Minor, probably due to the
absence of seriously polluting human activities on the island.
Furthermore, the dietary metal intake through consumption of
wild mushrooms from the island does not seem to pose any threat
to human health.
Both the substrate geochemistry and the species identity were
found to affect mushroom metal contents, although not in a
uniform way. In general, mushrooms growing on serpentine soils
had considerably higher Cr and Ni contents compared to those
from volcanic areas. However it was found that R. delica took up
both metals proportionally from the soil, S. bellinii took up only Ni,
while L. sanguiuus seemed to control metal concentrations, being
less affected by the metal levels in the soil. The elevated Cd
concentrations recorded in the mushrooms from the serpentine
sites could also be attributed to enhanced levels of the metal in
the soils, as literature data indicate, but further investigation is
needed to conrm this assumption. The calculation of the BCFs
showed that all the species under consideration acted as bioexclusors of all the metals studied, with the exception of the three
Lactarius species and of R. delica, which could marginally be
regarded as bioconcentrators of Zn and Cu, respectively.
Mushroom selectivity towards metal uptake was examined by
considering only those specimens growing on soils of volcanic
origin, so as to eliminate the effect of the substrate composition.
The statistical analysis revealed a species-specic interaction of
the studied mushrooms with the various metals, still valid in
species of the same genus. Specically, R. delica showed a stronger
afnity for Cu, S. bellinii for Mn and Pb, L. semisanguiuus for Cr, Ni
and Fe and L. sanguiuus for Zn.
Despite the large number of publications on the subject, the
complex interactions between mushroom species and metals in
the environment still remain obscure and their study is still
challenging. This is probably due to the fact that the metal
concentrations in mushrooms are inuenced by both biotic and
abiotic variables. This survey has shown that for a better understanding of the processes involved in metal uptake by mushrooms, the metal variability in the substrate should be taken into
consideration, either these metals originate from natural or from

pollution sources. In this view, the most illustrative way to

determine the metal uptake by mushrooms from their substrate
is to determine the metal concentrations in the underlying soil
and to calculate their BCFs.

Acknowledgments
This work was supported by the Research Unit of the University of the Aegean. The authors would like to thank Elisabeth
Zagle, for her participation in the analyses, Dimitris Dimou and
Dr. Elias Polemis for providing information on mushroom biodiversity on Lesvos, and Dr. Triantaphyllos Akriotis for his comments on the manuscript.

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