Environmental Pollution 145 (2007) 516e528

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Transfer and accumulation of metals in a soiledietewood mouse

food chain along a metal pollution gradient
Damien Rogival a,*, Jan Scheirs b, Ronny Blust a
a
Department of Biology, Ecophysiology, Biochemistry and Toxicology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
b
Department of Biology, Evolutionary Biology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
Received 5 December 2005; received in revised form 18 April 2006; accepted 19 April 2006

Non-essential metal transfer through a soildietwood mouse food chain is more important than essential metal transfer.

Abstract

We studied the accumulation and transfer of As, Cd, Cu, Pb and Zn in the compartments of a soiledietewood mouse (Apodemus sylvaticus)
food chain at five sites located along a metal pollution gradient. We observed a clear gradient in metal exposure at increasing distance from the
smelter in all compartments of the food chain for the non-essential metals. The gradient was less clear or absent for the essential metals in acorn
and mice target tissues. Regression analysis showed overall strong relationships within the soilediet and dietewood mouse compartments for
the non-essential metals, while relationships for the essential metals were weak or absent. Total metal in soil appeared as a better predictor for
the diet metal content than the available metal fraction. Our results suggest a more important transfer of non-essential elements through the food
chain than essential elements, which is probably a consequence of homeostatic control of the latter group.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Heavy metals; Transfer; Food chain; Cadmium; Apodemus sylvaticus

1. Introduction
Concentrations of metals in soils, organisms and their diet
provide information concerning their movement through the
environment, accumulation and potential toxicological effects
(Torres and Johnson, 2001). Many factors can influence the
transfer of metals to various compartments within the food
chain. First, the type of metal is an important variable in all
transfer routes. The transfer of non-essential metals, such as
Cd and Pb can differ greatly from essential metals, such as
Zn and Cu, due to the efficient homeostatic regulation of es-
sential metals in organisms (Sheffield et al., 2001). Second,
several physico-chemical (e.g. total metal concentration and
speciation) and edaphic factors (e.g. pH and total organic con-
tent) are known to influence the transfer of metals from soil to
* Corresponding author. Tel.: þ32 3 2653779; fax: þ32 3 2653497.
E-mail address: damien.rogival@ua.ac.be (D. Rogival).
plant (Salomons and Förstner, 1984). The total soil concentra-
tion of a metal does therefore not necessarily indicate its avail-
ability to plants and other soil organisms living in it (Sharma
and Shupe, 1977). These influences may therefore complicate
the identification of patterns of uptake and accumulation. In
order to cope with this problem, extraction procedures that
give an indication of metal bioavailability have been devel-
oped. Several authors have urged for more attention to metal
availability when analysing metal transfer within a food chain
(e.g. Crommentuijn et al., 2000). Third, transfer of metals to
terrestrial mammals depends on factors such as the species
and its diet, season and age (Hunter et al., 1987). Fourth, the
route of exposure is also important. Although absorption of
some metals by inhalation is more efficient than from the gas-
trointestinal tract, oral exposure by ingestion of contaminated
food is the dominant source of metals for wild mammals
(Hunter et al., 1987; Ma, 1989). Oral exposure can also occur
by direct ingestion of contaminated soil, as a result of burrow-
ing activity and grooming (Beyer et al., 1994).

0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.04.019
 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528
This paper describes the transfer of As, Cd, Cu, Pb and Zn
in a terrestrial soiledietemouse food chain at five sites along
a metal pollution gradient. The selected food chain included
the wood mouse (Apodemus sylvaticus Linnaeus) and two pri-
mary food items of the wood mouse diet, i.e. acorn (seeds of
Quercus robur) and earthworm (Dendrodrilus rubidus and
Lumbricus rubellus). The wood mouse is an abundant oppor-
tunist rodent species that occurs over most of Europe (Corbet
and Harris, 1991) and is a frequently used study organism for
monitoring metal pollution (Talmage and Walton, 1991;
Shore, 1995). The present study differs from most of the pre-
vious ecotoxicological field studies that focus on terrestrial
mammals because it reports food chain relationships for
a wider range of elements and looks at total and available
soil metal concentrations simultaneously and in conjunction
with levels in food items and in tissues of wood mice. In
this study, metal transfer to the selected compartments of the
wood chain was not measured directly, but was inferred
from a combination of different indirect measures as described
by Notten et al. (2005). First, we measured metal concentra-
tions in the different food chain compartments at the five study
sites along the pollution gradient. Therefore, concentrations of
As, Cd, Cu, Pb and Zn were determined in the soil (total and
available concentration), acorns and earthworms, and in differ-
ent tissues (liver, kidney and bone tissue) of the wood mouse.
Next, we investigated if acorns and earthworms, and subse-
quently wood mice, showed elevated levels of metals when
living on increasing levels of soil pollution. Therefore, we in-
vestigated the occurrence of positive relationships of metal
concentrations between the different food chain compart-
ments. Differences in the strength of these relationships,
measured as the coefficient of determination, were used as
a measure of the importance of the different transfer routes.
We expected positive relationships between metal concentra-
tions in the soilediet compartments. Within the soilediet com-
partments, we expected stronger relationships between the
available soil metal concentrations and the food items than
between the total soil metal concentrations and the food items,
since total soil concentration of a metal does not necessarily
indicate its availability to plants and other soil organisms. We
expected positive relationships between metal concentrations
in the dietewood mouse compartments. We expected stronger
relationships between metal concentrations in the acorn and
wood mouse, than between the earthworm and wood mouse,
since the diet of wood mouse constitutes mainly of seeds
(70%), followed by animal food (15%) and forbs (5e10%)
(Corbet and Harris, 1991). We expected a more important trans-
fer of non-essential metals through the food chain compared to
essential metals, due to the efficient homeostatic regulation of
essential metals in organisms (Sheffield et al., 2001).
2. Materials and methods
2.1. Study sites
The present study was performed along a pollution gradient originating
from a still active non-ferrous metallurgic plant in the south of Antwerp
517
(Belgium). As, Cd, Cu, Pb and Zn are the main pollutants in this area and
they form a decreasing pollution gradient away from the plant (VMM,
1999; Janssens et al., 2001; Tersago et al., 2004; Rogival et al., 2006). Metal
soil pollution in the area around the factory is mainly a consequence of histor-
ical emissions during the period 1950e1970, and to a lesser extent also of re-
cent emission. The atmospheric metal emissions from the factory have
strongly decreased during the last decades (VMM, 1999). We established
five study sites along a west-east pollution gradient: i.e. Umicore (UM),
Fort 8, Fort 7, Fort 4 and Zoersel (reference site), located at respectively
0.1, 1, 3, 8 and 27 km east of the factory (Fig. 1). All sites were deciduous
park areas where grasslands interfered with patches of deciduous forest.
2.2. Field sampling strategy
Six to ten oak trees (Quercus robur) were selected and marked within each
site: i.e. 10 at Zoersel, 9 at Fort 4, 10 at Fort 7, 9 at Fort 8, and 6 at UM. The
minimum distance between the selected trees was 100 m. This distance is
based on the fact that the home range of wood mice in deciduous park areas
varies from 250 to 5000 m2 (Corbet and Harris, 1991). We could therefore
safely assume that we had independent sample points within each site. Soil
samples, earthworms, acorn and wood mice were collected in the direct prox-
imity of every oak tree. Based on the home range and migration distances of
wood mice in deciduous park areas described in the literature (Corbet and Har-
ris, 1991), we have used the soil metal levels around each oak tree as an esti-
mate of the soil contamination within the core home range of the wood mice
captured in the direct proximity of that tree (see Torres and Johnson, 2001;
Festa et al., 2003 for a comparable approach).
Three soil samples per tree were collected according to a standardized
sampling procedure as described elsewhere (Rogival et al., 2006). Collected
soil samples were air-dried and passed through a 2-mm sieve to remove stones,
roots, and other large items. Afterwards, samples were stored in tightly closed
polyethylene jars at room temperature and protected from light until metal
analysis.
The diet of wood mice constitutes mainly of seeds (70%), followed by an-
imal food (15%) (which includes small invertebrates, such as earthworms) and
forbs (5e10%) (Corbet and Harris, 1991). These proportions can be greatly
Zoersel
Flanders
Highway
0 1 2
Sampling location
Pollution Source Kilometres
River
Scheldt Antwerp
Fort 4
UM
Non-ferrous
factory Fort 7
Fort 8
Fig. 1. Map of the study area (Belgium). The lower map is an enlarged and
detailed representation of the square in the upper map. UM, Umicore.
518 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528
affected by the abundance of different food items. Corbet and Harris (1991)
showed that seeds, including acorn, are dominant food items during most of
the year, while invertebrates, including earthworms, are mainly consumed in
late winter. Acorn is the dominant seed in our study areas during winter, which
was the period preceding the start of our capturing sessions (FebruaryeMay).
We also chose to analyze earthworms because it was the only invertebrate spe-
cies that was present in large quantities and readily available for consumption
in all of the study sites during the period before the trapping sessions. A sec-
ond reason for analyzing both the acorns and the earthworms is that metal con-
centrations may differ greatly among animal and plant food items (Hunter and
Johnson, 1982). Five earthworms and acorns per tree were collected from the
soils by digging and hand sorting. Back in the laboratory, the food items were
rinsed with ultra pure water and the earthworms were identified. Most (78%)
earthworms belonged to the species Dendrodrilus rubidus and Lumbricus ru-
bellus. Dendrodrilus rubidus was the main earthworm species collected in all
of the study sites compared to Lumbricus rubellus. Metal analysis was only
performed with these two species, since they were both found in large numbers
in all of the five sampling sites. The ingested soil present in the gut of the cap-
tured earthworms was not removed before metal analysis because that soil is
also ingested by the wood mouse when eating the earthworm. As a conse-
quence, the wood mouse is not only exposed to the metals in the tissues of
the earthworm but also to the metals of the soil in the earthworm’s gut. The
food items were stored in polyethylene jars at 20  C until metal analysis.
A minimum of three wood mice were collected in the direct proximity of
every oak tree. Wood mice were caught overnight with Sherman live traps
baited with peanut butter from mid-April till the end of May 2003. Five traps,
placed within a 20 m circle of each tree, were set in the evening (7 pm) and
checked at dawn (7 am). A total of 126 mice were collected: 21 at UM (13
males and 8 females), 29 at Fort 8 (16 males and 13 females), 29 at Fort 7
(16 males and 13 females), 23 at Fort 4 (14 males and 9 females) and 24 at
Zoersel (13 males and 11 females). The captured wood mice were transported
to the laboratory and euthanized with ether according to legal procedures
(ACUC, 1998). The mice were sexed, weighed and tissues (liver, kidney and
bones) were dissected. Liver and kidney are the main target organs for metal
accumulation in general, and bone tissue for Pb accumulation specifically
(50e70% of the total body burden) (Sheffield et al., 2001). Bone samples com-
prised the femur, tibia and fibula of both hind legs. Muscle and other tissue
attached to the bones were completely removed with the scalpel during the dis-
section and bones were cleaned afterwards with ultra pure water. The tissues
were stored at 20  C until metal analysis. Eye lenses were isolated and
stored in 10% formaldehyde for age determination as described by Vandorpe
and Verhagen (1980).
2.3. Sample preparation and metal analyses
The total metal concentrations of the soil samples were determined using
the aqua regia extraction procedure as described elsewhere (Rogival et al.,
2006).
The available metal content of the soil was determined using extraction
with 1 mol L1 ammonium nitrate solution according to the standard German
protocol DIN 19730 (DIN, 1997). Use of ammonium nitrate soil extraction as
a reliable indicator of metal availability to organisms has previously been dem-
onstrated (Wenzel et al., 2001; Pueyo et al., 2004). A 10 g dried and homoge-
nised soil sample was extracted with 25 mL of 1 mol L1 ammonium nitrate
solution for 2 h on a mechanical end-over-end type shaker rotating at
20 rpm. After settling of the undissolved residue, the supernatant solution
was filtered through a 0.45 mm cellulose acetate membrane filter. The filtrate
was acidified with 500 mL sub-boiled HNO3 and used for analysis.
Tissues (liver, kidney and bone) and food items (acorn and earthworm)
were placed in acid-cleaned polypropylene vials and dried for 24 h at
60  C. Subsequently, samples were digested with sub-boiled HNO3 (70%)
and H2O2 (30%). Using the procedure described by Blust et al. (1988), the di-
gestion was completed by heating the samples in a microwave oven. After-
wards, the digested samples were diluted with ultra pure water, and stored
at 20  C until analysed.
An internal standard yttrium solution was spiked in all of the measured
samples to correct for possible matrix effects. The total concentrations of
As, Cu, Pb and Zn in the soil samples were measured using ICP-AES (Varian
Liberty series) and of Cd using ICP-MS (Varian Ultra-Mass series). The same
metals were measured using the above mentioned ICP-MS for the extractable
metal concentrations in the soil samples, the total metal concentrations in the
food items and in the mice tissues. Only Pb was measured in the bone samples
for two reasons: (1) in the case of chronic metal exposure, as is the case in the
present study, Pb is mainly accumulated in bone tissue. Metals such as Cd, Cu
and Zn are mainly accumulated in liver and kidney, while only very small
amounts of these metals are accumulated in bone tissue (Sheffield et al.,
2001) and (2) in order to reduce the already large number of data and analyses.
All metal concentrations were expressed on a dry weight (dw) basis in mg g1
or mg g1. Analytical accuracy was determined using certified reference ma-
terial of the Community Bureau of Reference (Belgium). Standards for trace
elements in river sediment (BCR 320) were used for the analysis of the total
metal concentration in the soil samples. Olive leaves (BCR 62), mussel tissue
(CRM 278) and bovine liver (BCR 185) were used for the metal analysis of the
food items and mice tissues. An in-house reference material of The Federal
Institute for Materials Research and Testing (BAM) (Berlin, Germany); soil
sample ANEx-RV6-xxx, was used for the analysis of the extractable metal
concentrations in the soil samples. Two blank and two reference samples
were included for each batch of 20 soil, food and tissue samples. Analyses
were accepted when recoveries ranged between 90% and 110% of the certified
values. After metal analyses, the total soil concentrations were compared with
the Belgian standards for the assessment of polluted soils, including a sanita-
tion norm and a target value (VLAREBO, 2004). The sanitation norm is the
maximum allowable metal concentration, and soil remediation measures
should be considered if this concentration is exceeded. The target value is
the Belgian background metal concentration for a clean standard soil (i.e.
soil with clay content of 10% and organic matter of 2%).
2.4. Statistical analyses
A linear mixed model analyses (LMM) was performed in order to test for
differences in metal concentrations in soil and food items among sites. Mixed-
effects models provide a powerful and flexible tool for the analysis of balanced
and unbalanced group data. Second, both fixed and random effects can be an-
alysed with this technique (SAS Institute, 1999). Site was used as an indepen-
dent class variable in our LMM analysis of the soil data. Tree nested within
study area was used as a random variable. The different metals were used
as dependent variables. Differences in body mass and tissue metal content
were analysed in a comparable way, except for the fact that age, gender and
all possible interactions among the fixed independent variables were included
in the model. Age and gender were included in the model in order to account
for potential age and gender effects. The age structure of the different popula-
tions was compared using a linear mixed model analysis with tree nested
within study area as a random variable. Site, gender and the interaction be-
tween both were used as fixed effects. In order to meet the assumptions of
the latter LMM analyses, the dependent variable was always log10(x þ 1)
transformed. All previous mixed model analyses were calculated using the
PROC MIXED module in SAS (SAS Institute, 1999). The degrees of freedom
of the fixed effects F-test were adjusted for statistical dependence using
Kenward Roger formulas. Variance components were estimated by restricted
maximum likelihood (REML). Model reduction was performed by dropping
non-significant (a ¼ 0.05) fixed effects in a backward hierarchical way, start-
ing with the least significant highest order interaction term (Verbeke and
Molenberghs, 1997). We used a Tukey multiple comparisons test in order to
establish differences among sites and between genders (SAS Institute, 1999).
The relationship between total and available soil metal concentrations was
studied using Pearson’s correlations (Statsoft, 1994). Regression techniques
were used to investigate the relationships between two different food chain
compartments for each metal over all five study sites (Statsoft, 1994). All vari-
ables were log10(x þ 1) transformed in order to meet the assumptions of the
parametric test. All regression analyses were based on colocated data (e.g.
within home ranges of wood mice). Therefore, mean soil, food and tissue
metal concentrations were calculated for every selected tree and means were
used in the analyses (see Torres and Johnson, 2001; Festa et al., 2003 for
a comparable approach).
 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528 519

3. Results to the factory, were 9, 95, 4, 3 and 11 times higher than the re-
spective concentrations at the reference site, Zoersel. Accord-
3.1. Metal concentrations in food chain compartments ing to the Belgian environmental standards, the soils at UM
and Fort 8, the sites nearest to the metallurgic factory, highly
3.1.1. Total and available metal concentrations in the soil exceeded the target value for all metals, and exceeded the san-
All total soil metal concentrations differed significantly itation norm for As, Cd and Pb (Fig. 2) (VLAREBO, 2004). At
among sites (Fig. 2). Total soil concentrations of As, Cd, Cu, Fort 7, the total soil Cd concentration exceeded the target value
Pb, and Zn at UM and Fort 8, the sites nearest to the factory, and still slightly exceeded the sanitation standard. The total soil
were significantly higher than those at the other sites. Total concentrations of Cu and Pb at this site still exceeded the target
soil concentrations of these metals at UM, the site adjacent value, but were similar to the target value for As and Zn. A

120 0.16 28 6
c b d

Available [Cd] in soil (µg g-1 dw)

Available [As] in soil (µg g-1 dw)
0.14 c

Total [Cd] in soil (µg g-1 dw)

Total [As] in soil (µg g-1 dw)

100 24 5
d
c b
0.12
20
80 4
0.10 c
16
60 0.08 3
12
0.06
40 a 2
a 8
b a 0.04
b
20 c b 1
a 0.02 4 b ab
a a
0 0.00 0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

240 1.0 350 18

c
Available [Cu] in soil (µg g-1 dw)

Available [Pb] in soil (µg g-1 dw)

b
c 16
Total [Cu] in soil (µg g-1 dw)

Total [Pb] in soil (µg g-1 dw)

b 300 c
200 0.8 14
250 c
c 12
a
c 0.6
200 ac 10
60
a
b 150 8
a 0.4
40 a a 6
a 100
b
0.2 b
4
20 a a
50
2

0 0.0 0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

630 18
b
600
Available [Zn] in soil (µg g-1 dw)

16
Total [Zn] in soil (µg g-1 dw)

14
c
240
c ab 12
200 ab
ab 10
160 8
120 b
b 6
a
80 4

40 a 2

0 0
UM Fort 8 Fort 7 Fort 4 Zoersel

Fig. 2. Mean total (white bars) and available (black bars) metal concentrations (mg g1 dw  SE) in the soils of the five study sites along a metal pollution gradient
(UM (Umicore), n ¼ 21; Fort 8 and Fort 7, n ¼ 29; Fort 4, n ¼ 23; Zoersel, n ¼ 24). Total metal concentration: As: F4,41 ¼ 60.43, p < 0.001; Cd: F4,40 ¼ 54.44,
p < 0.001; Cu: F4,40 ¼ 19.65, p < 0.001; Pb: F4,40 ¼ 17.46, p < 0.001; Zn: F4,40 ¼ 43.84, p < 0.001. Available metal concentration: As: F4,40 ¼ 14.10, p < 0.001;
Cd: F4,40 ¼ 24.48, p < 0.001; Cu: F4,40 ¼ 5.04, p ¼ 0.002; Pb: F4,36 ¼ 3.88, p ¼ 0.01; Zn: F4,40 ¼ 2.95, p ¼ 0.03. Bars with the same letter do not differ signif-
icantly from each other (Tukey multiple comparisons test, a ¼ 0.05). F (ndf, ddf ), F distribution with numerator degrees of freedom (ndf ) and denominator degrees
of freedom (ddf ). Dotted line (bottom), target value (see text); dashed line (top), sanitation norm (see text). Both values are according to the Belgian standards for
the assessment of polluted soils.
520 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528

comparison of the soil data with several international target Zoersel and Fort 4, but did not differ significantly from Fort 7.
values revealed the same results as describes here above (e.g. Available Zn concentration in the soil at UM was 4 times higher
Andersen et al., 1994). All soil concentrations at Fort 4 and compared to Zoersel but did not differ significantly from the
Zoersel, the sites furthest away from the non-ferrous plant, rest of the sites.
were around or below the target value, except Pb at Zoersel. Total and available metal concentrations in the soil showed
Available soil concentrations differed significantly among positive relationships for all measured metals (As: n ¼ 134,
sites for all metals (Fig. 2). The soils at UM and Fort 8, the sites r ¼ 0.46, p < 0.001; Cd: n ¼ 128, r ¼ 0.85, p < 0.001; Cu:
nearest to the factory, had significantly higher available As, Cd n ¼ 134, r ¼ 0.51, p < 0.001; Pb: n ¼ 134, r ¼ 0.37,
and Cu concentrations than those at the other sites. Available p ¼ 0.01; Zn: n ¼ 134, r ¼ 0.51, p < 0.001).
concentrations of these metals at UM, the site adjacent to the
factory, were 3, 63 and 5 times higher than the respective con- 3.1.2. Metal concentrations in selected food items
centrations at the reference site, Zoersel. Available Pb concen- All measured metal concentrations in the acorns, except for
trations at UM and Fort 8 were significantly higher compared to Zn, differed significantly among sites (Fig. 3). Concentrations

0.10 250 1.4 500

b
b c

[Cd] in earthworm (µg g-1 dw)

[As] in earthworm (µg g-1 dw)
c 1.2

[Cd] in acorn (µg g-1 dw)

[As] in acorn (µg g-1 dw)

0.08 b 200 400

b
1.0 c

0.06 150 300

0.8
c
0.6 200
0.04 100
a a
a 0.4
a b a
0.02 b a 50 100
0.2
a a a a
0.00 0 0.0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

60 100 5 1000
b
[Cu] in earthworm (µg g-1 dw)

[Pb] in earthworm (µg g-1 dw)

c a b
b
50
[Cu] in acorn (µg g-1 dw)

[Pb] in acorn (µg g-1 dw)

ab 80 4 800
c
40 ab
ab
b 60 3 b 600

30
40 2 400
20
b a
a
20 1 200
10 a
a a a
a a
0 0 0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

70 1200
a
b
[Zn] in earthworm (µg g-1 dw)

60
[Zn] in acorn (µg g-1 dw)

1000
a
50
b a b 800
a
a
40
600
30
a a
400
20

10 200

0 0
UM Fort 8 Fort 7 Fort 4 Zoersel

Fig. 3. Mean metal concentrations (mg g1 dw  SE) in acorn (white bars) and earthworm (black bars) from the five study sites along a metal pollution gradient
(UM (Umicore), n ¼ 24; Fort 8, n ¼ 30; Fort 7, n ¼ 32; Fort 4, n ¼ 29; Zoersel, n ¼ 30). Acorn: As: F4,38 ¼ 13.21, p < 0.001; Cd: F4,36 ¼ 7.84, p < 0.001; Cu:
F4,37 ¼ 3.19, p ¼ 0.02; Pb: F4,39 ¼ 25.91, p < 0.001; Zn: F4,37 ¼ 3.22, ns. Earthworm: As: F4,33 ¼ 43.79, p < 0.001; Cd: F4,33 ¼ 106.11, p < 0.001; Cu:
F4,37 ¼ 58.86, p < 0.001; Pb: F4,35 ¼ 44.14, p < 0.001; Zn: F4,34 ¼ 4.32, p ¼ 0.006. Bars with the same letter do not differ significantly from each other (Tukey
multiple comparisons test, a ¼ 0.05). F (ndf, ddf ), F distribution with numerator degrees of freedom (ndf ) and denominator degrees of freedom (ddf ). ns, non-
significant.
 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528
of As, Cd and Pb were significantly higher in the acorns from
UM and Fort 8 compared to the other sites. Concentrations of
As, Cd and Pb at the most polluted site UM were 6, 5 and 14
times higher than the respective concentrations at the refer-
ence site, Zoersel. Concentrations of Cu in the acorns from
Zoersel were higher compared to Fort 4 but did not differ sig-
nificantly from UM, Fort 8 and Fort 7.
We observed no significant differences in metal concentra-
tions between the two sampled earthworm species Dendrodri-
lus rubidus and Lumbricus rubellus (As: F1,128 ¼ 1.09,
p ¼ 0.299; Cd: F1,138 ¼ 0.03, p ¼ 0.862; Cu: F1,135 ¼ 0.09,
p ¼ 0.763; Pb: F1,136 ¼ 0.52, p ¼ 0.473; Zn: F1,133 ¼ 0.01,
p ¼ 0.959). Therefore, the two species were pooled in the
rest of the statistical analysis. All measured metal concentra-
tions in the earthworms differed significantly among sites
(Fig. 3). Concentrations of As, Cd, Cu and Pb in the earth-
worms from UM and Fort 8, the sites nearest to the metallurgic
plant, were significantly higher compared to the other sites.
Levels of Zn in the earthworms from UM were higher com-
pared to Zoersel and Fort 4, but did not differ significantly
from Fort 8 and Fort 7. Levels of As, Cd, Cu, Pb and Zn in
the earthworms from the most polluted site UM were 30, 31,
7, 22 and 3 times higher than the respective earthworm con-
centrations at the reference site, Zoersel. Comparison of the
metal concentrations in the acorns and the earthworms showed
that all metal levels were higher in the earthworms.
3.1.3. Metal concentrations in mouse tissues
Concentrations of Cd, Pb and Zn in liver and kidney dif-
fered significantly among sites, while concentrations of As
and Cu did not (Fig. 4). Hepatic and renal Cd and Pb concen-
trations at UM and Fort 8, the sites nearest to the metallurgic
plant, were significantly higher compared to the other sites.
Levels of these metals at the most polluted site UM were for
livers 45 and 19, and for kidney 18 and 12 times higher than
the respective tissue concentrations at the reference site, Zoer-
sel. Hepatic and renal Zn levels at UM, the site adjacent to the
metallurgic plant, were higher compared to Fort 4 and Zoersel,
but did not differ significantly from Fort 8 and Fort 7. Bone Pb
concentrations at UM and Fort 8 were significantly higher than
compared to the other sites (Fig. 4). Bone Pb concentration at
the most polluted site UM was 19 times higher than at the ref-
erence site, Zoersel. The kidney contained higher levels of As
and Cd than the liver, while the Cu and Zn concentrations
were comparable. The highest Pb concentrations were mea-
sured in the bone tissue, followed by the kidney and the liver.
We observed a significant increase in hepatic and renal Cd
concentrations with age (see statistics in legend Fig. 4). No
significant gender effect was observed for all the measured
metal concentrations in liver and kidney. No significant gender
and age effects were observed for the Pb concentration in the
bone tissues.
3.2. Age and body mass of wood mice
A previous publication on the same wood mice individuals
already showed no significant differences in age and body
521
mass of the wood mice among the five study sites (Rogival
et al., 2006). It is therefore unlikely that differences among
sites in tissue metal concentrations are due to site differences
in age structure or body mass. Nevertheless, these factors may
still explain some of the within site variation.
3.3. Relationships between the food chain compartments
Significant positive relationships were found between metal
concentrations in acorn and total soil concentrations for As,
Cd and Pb, and between metal concentrations in acorn and
available concentrations for Cd and Pb (Table 1). No such re-
lationships were observed for Cu and Zn. The coefficients of
determination (r2) varied between 0.32 and 0.53 (acornetotal
soil) and between 0.17 and 0.37 (acorneavailable soil). Rela-
tionships between the earthworm and total soil metal concen-
trations were significantly positive for all metals. The r2 values
varied between 0.32 and 0.86. Significant positive relation-
ships were found between the earthworm and available soil
metal concentrations for all metals, except for As. The r2
values varied between 0.12 and 0.45. Metal concentrations
in the acorn and the mouse tissues showed positive relation-
ships for Cd and Pb. The r2 values varied between 0.33 and
0.71. No such relationships were observed for As, Cu and
Zn. Significant positive relationships were found between
earthworm and mouse tissues (liver, kidney and bones) for
Cd, Pb and Zn. The r2 values varied between 0.16 and 0.71.
No such relationships were observed for As and Cu.
4. Discussion
4.1. Metal concentrations in food chain compartments
4.1.1. Total and available metal concentrations in the soil
Total soil metal concentrations at Fort 4 and Zoersel com-
pared fairly well with total concentrations of reference soils
reported in other studies, but those at Fort 7, Fort 8 and UM
were significantly higher (Andersen et al., 1994; Bak et al.,
1997; Tarvainen and Kallio, 2002). Total soil concentrations
of As, Cd, Cu, Pb, and Zn decreased with increasing distance
from the non-ferrous metallurgic factory. This corresponds
with the fact that the non-ferrous factory is one of the impor-
tant metal emitting point sources of Cd and Pb, and to a lesser
extent of As, Cu, and Zn in Flanders (VMM, 1999). Yet, metal
pollution in the area around the factory is mainly a conse-
quence of historical emissions.
While high total concentrations of As, Cd, Cu, Pb and Zn
pose potentially high exposure to local biota, conditions con-
trolling metal availability also must be considered in charac-
terizing the transfer of metals into the food web. Relatively
small fractions of the total metals in soil were extractable
with ammonium nitrate, suggesting limited availability of
metals for uptake by organisms (As: 0e0.4%, Cd: 18e38%,
Cu: 0.6e2%, Pb: 4e7%, Zn: 8e16%). Use of ammonium ni-
trate soil extraction as a reliable indicator of metal availability
to organisms has clearly been demonstrated for Cd, Cu, Pb and
Zn, while it is questionable for As (Wenzel et al., 2001; Pueyo
522 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528

0.35 0.35 80 80
70 d d 70
a
0.30 0.30 60 60

[Cd] in kidney (µg g-1 dw)

[As] in kidney (µg g-1 dw)
[As] in liver (µg g-1 dw)

[Cd] in liver (µg g-1 dw)

50 50
a a
0.25 0.25
c
a 30 c 30
0.20 0.20
a 25 c 25
0.15 a 0.15 20 20
a
a a b
0.10 a 0.10 15 15
10 10
0.05 0.05 b a
5 a 5
a
0.00 0.00 0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

30 30 1.8 14
b
a 1.6 c 12
25

[Cu] in kidney (µg g-1 dw)

[Pb] in kidney (µg g-1 dw)

25 a a a a
[Cu] in liver (µg g-1 dw)

[Pb] in liver (µg g-1 dw)

1.4
a a a 10
20 a 20 1.2 b
a c
1.0 8
15 15
0.8 6
10 10 0.6
a 4
b a b
0.4
5 5 2
0.2 a a

0 0 0.0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

140 140 35

120 b b b b b b 120 30 c
[Zn] in kidney (µg g-1 dw)

[Pb] in bone (µg g-1 dw)

[Zn] in liver (µg g-1 dw)

100 100 25
a a a a c

80 80 20

60 60 15

40 40 10
b

20 20 5 a a

0 0 0
UM Fort 8 Fort 7 Fort 4 Zoersel UM Fort 8 Fort 7 Fort 4 Zoersel

1
Fig. 4. Mean metal concentrations (mg g dw  SE) in liver (black bars), kidney (white bars) and bone tissue of wood mice from the five study sites along a metal
pollution gradient (UM (Umicore), n ¼ 21; Fort 8 and Fort 7, n ¼ 29; Fort 4, n ¼ 23; Zoersel, n ¼ 24). The following results relate to site effects. Liver: As:
F4,38 ¼ 2.65, ns; Cd: F4,39 ¼ 60.20, p < 0.001; Cu: F4,36 ¼ 0.69, ns; Pb: F4,36 ¼ 12.16, p < 0.001; Zn: F4,37 ¼ 3.17, p ¼ 0.02. Kidney: As: F4,33 ¼ 1.94, ns;
Cd: F4,37 ¼ 49.08, p < 0.001; Cu: F4,33 ¼ 0.48, ns; Pb: F4,38 ¼ 21.54, p < 0.001; Zn: F4,36 ¼ 5.07, p ¼ 0.002. Bone: Pb: F4,37 ¼ 69.98, p < 0.001. The following
results relate to age effects. Liver: Cd: F1,101 ¼ 7.09, p ¼ 0.009. Kidney: Cd: F1,101 ¼ 39.28, p < 0.001. Bars with the same letter do not differ significantly from
each other (Tukey multiple comparisons test, a ¼ 0.05). F (ndf, ddf ), F distribution with numerator degrees of freedom (ndf ) and denominator degrees of freedom
(ddf ). ns, non-significant.

et al., 2004). Wenzel et al. (2001) reported that ammonium ni- and non-contaminated sites (Table 2). Analogous to other
trate soil extraction has rather low extraction efficiency for As, studies (Stefanov et al., 1995; Angelova et al., 2004), we ob-
which may indicate that this extraction is rather unsuitable to served higher concentrations of As, Cd and Pb in the acorns
determine available As concentrations in soil samples. The at the most polluted sites Fort 8 and UM compared to our ref-
low extraction efficiency for As is supported by the very low erence site, Zoersel. No differences in acorn metal levels were
available As concentrations measured in the present study. observed for Zn and only small differences for Cu despite the
fact that soil concentrations varied considerably among sites
4.1.2. Metal concentrations in selected food items for these two metals. In contrast to these results, Stefanov
All metal concentrations in the acorns from the five sam- et al. (1995) and Angelova et al. (2004) observed higher Zn
pling sites were within the range reported in the literature and Cu concentrations in seeds from polluted sites compared
for seeds and nuts of different plant species from contaminated to seeds from reference sites.
 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528 523

Table 1 earthworms than the acorns. The accumulation of high tissue

Results of regression analyses for metal concentration (mg g1 dry wt) between metal concentrations by earthworms is well documented.
the dietesoil (n ¼ 37) and mouseediet (n ¼ 37) compartments
Metals taken up by earthworms either through the skin or
Variables ( yex) a b r2 from ingested soil may contribute to the high metal levels ob-
Dietesoil served (Kennette et al., 2002). The low metal concentrations
log As acornelog total As soil 1.20 0.72 0.51 *** measured in the seeds are in agreement with the tendency of
log Cd acornelog total Cd soil 0.01 0.57 0.32 ***
log Cu acornelog total Cu soil 1.81 0.13 0.02 ns
many metals to become immobilized in root and other
log Pb acornelog total Pb soil 1.80 0.72 0.53 *** below-ground storage plant tissues and undergo limited trans-
log Zn acornelog total Zn soil 2.95 0.22 0.05 ns location to aboveground structures. Therefore, seeds typically
log As acornelog avail. As soil 0.01 0.27 0.08 ns contain much lower metal concentrations than other plant
log Cd acornelog avail. Cd soil 0.04 0.61 0.37 *** parts (Torres and Johnson, 2001).
log Cu acornelog avail. Cu soil 0.06 0.05 0.003 ns
log Pb acornelog avail. Pb soil 0.79 0.41 0.17 *
log Zn acornelog avail. Zn soil 0.42 0.13 0.02 ns 4.1.3. Metal concentrations in mouse tissues
log As earthwormelog total As soil 0.82 0.87 0.75 *** The observed high tissue concentrations for Cd, Pb and Zn
log Cd earthwormelog total Cd soil 0.71 0.93 0.86 *** at the most polluted sites UM and Fort 8 are in accordance
log Cu earthwormelog total Cu soil 0.43 0.85 0.73 *** with previous studies in the same pollution gradient. Metal
log Pb earthwormelog total Pb soil 1.28 0.74 0.55 ***
log Zn earthwormelog total Zn soil 1.30 0.57 0.32 **
levels in excrements of passerine birds (Janssens et al.,
log As earthwormelog avail. As soil 0.01 0.30 0.09 ns 2001), in organs of wood mice (Tersago et al., 2004) and in
log Cd earthwormelog avail. Cd soil 0.19 0.67 0.45 *** blood of humans (Staessen et al., 2001) in the same metal gra-
log Cu earthwormelog avail. Cu soil 0.03 0.35 0.12 * dient showed the same pattern. After comparing the tissue
log Pb earthwormelog avail. Pb soil 0.47 0.49 0.24 ** metal concentrations observed in the present study with those
log Zn earthwormelog avail. Zn soil 0.60 0.39 0.15 *
reported in the literature for small rodents at contaminated and
Mouseediet non-contaminated sites (Table 3), we can conclude that only
log As liverelog As acorn 0.01 0.02 0.0002 ns
the hepatic and renal Cd levels at the most polluted sites
log Cd liverelog Cd acorn 0.08 0.61 0.37 ***
log Cu liverelog Cu acorn 2.04 0.25 0.06 ns UM and Fort 8 were comparable to hepatic and renal Cd levels
log Pb liverelog Pb acorn 0.14 0.68 0.46 *** in rodents from other heavily polluted environments. Further-
log Zn liverelog Zn acorn 1.98 0.13 0.02 ns more, the measured renal Cd concentrations in UM and Fort 8
log As kidneyelog As acorn 0.005 0.30 0.09 ns were one of the highest concentrations ever reported in kidney
log Cd kidneyelog Cd acorn 0.04 0.57 0.33 ***
of free-living wood mice (Table 3). The tissue concentrations
log Cu kidneyelog Cu acorn 1.07 0.10 0.01 ns
log Pb kidneyelog Pb acorn 0.04 0.67 0.44 *** of As, Cu, Pb and Zn at the most polluted sites UM and Fort 8
log Zn kidneyelog Zn acorn 1.63 0.002 0.00001 ns were generally much lower than the concentrations reported in
log Pb boneelog Pb acorn 0.09 0.84 0.71 *** other heavily polluted environments (Table 3). As a result we
log As liverelog As earthworm 1.30 0.07 0.01 ns hypothesize that only the high Cd tissue levels may possibly
log Cd liverelog Cd earthworm 1.00 0.85 0.73 ***
cause toxic effects in wood mice from the most polluted sites.
log Cu liverelog Cu earthworm 1.51 0.02 0.0005 ns
log Pb liverelog Pb earthworm 1.70 0.60 0.36 *** However, further research is needed to elucidate if wood mice
log Zn liverelog Zn earthworm 0.52 0.44 0.19 * from the most polluted sites encounter toxic effects of the high
log As kidneyelog As earthworm 0.04 0.003 0.08 ns Cd tissue levels observed in the present study. In contrast, it is
log Cd kidneyelog Cd earthworm 0.46 0.79 0.62 *** likely that far greater As, Cu, Pb and Zn tissue concentrations
log Cu kidneyelog Cu earthworm 0.02 0.11 0.01 ns
would have to occur in order to cause toxic effects.
log Pb kidneyelog Pb earthworm 1.47 0.59 0.35 ***
log Zn kidneyelog Zn earthworm 0.54 0.40 0.16 *
log Pb boneelog Pb earthworm 1.09 0.84 0.71 *** 4.2. Soileacorn and soileearthworm metal transfer
2 2
a, intercept; b, slope; r , coefficient of determination; avail., available. r sig-
nificant at *p < 0.05, **p < 0.01 and ***p < 0.001 level; ns, not significant. Significant positive correlations were observed between
total soil and acorn metal concentrations for As, Cd and Pb,
and between available soil and acorn metal concentrations
The metal concentrations in the earthworm species Dendro- for Cd and Pb. In contrast to our results, Torres and Johnson
drilus rubidus and Lumbricus rubellus from the five sampling (2001) did not find significant relationships for these metals
sites were within the range reported in the literature for these between the seeds of salt marsh bulrush (Scirpus robustus)
two species and other earthworm species from contaminated and the total or DTPA-extractable soil concentrations.
and non-contaminated sites (Table 2). Analogous to other Significant positive correlations were observed between
studies (Van Hook, 1974; Pizl and Josens, 1995), we observed total soil and earthworm metal concentrations for all metals.
higher concentrations of As, Cd, Cu, Pb and Zn in earthworms The relationships between available soil and earthworm metal
at the most polluted sites Fort 8 and UM compared to our ref- concentrations were significantly positive for all metals,
erence site, Zoersel. except for As. Our results support earlier reports on observed
After comparing the metal concentrations in the acorns and positive relationships between earthworms and total or avail-
earthworms at the five sampling sites, we can conclude that all able soil metal residues (Pizl and Josens, 1995; Rida and
measured metal concentrations were much higher in the Bouché, 1997; Kennette et al., 2002).
524 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528

Table 2
Mean metal concentrations (mg g1 dw) in seeds and nuts of different plant species and in earthworm species at reference (Ref) and polluted (Poll) sites reported in
the literature
Source As Cd Cu Pb Zn
Ref Poll Ref Poll Ref Poll Ref Poll Ref Poll
Seeds and nuts
Current study Quercus robur 0.02 0.08 0.22 1.03 29.3 41.8 0.28 3.79 48.6 55.3
Murillo et al., 1999 Helianthus annuus 0.01 0.06 0.08 0.24 17.8 47.2 0.27 0.42 43.6 73.6
Torres and Johnson, Scirpus cyperinus 0.03 e e e 12.7 e 0.11 e e e
2001
Bennett et al., 2000 Zizania aquatica 0.02 0.09 0.01 e 3.2 e 0.15 e 36.8 e
Folkeson et al., 1990 Fagus sylvatica e e 0.09 e 19.7 e e e 36.1 e
Angelova et al., 2003 Linum usitatissimum e e 0.52 2.30 12.0 45.2 1.50 11.3 19.8 97.4
Angelova et al., 2003 Cannabis sativa e e 0.30 1.00 5.9 8.9 1.00 7.61 17.8 73.5
Stefanov et al., 1995 Pisum sativum e e 0.45 1.13 e e 0.65 3.84 32.4 56.0
Stefanov et al., 1995 Zea mays e e 0.19 0.24 e e 0.27 5.71 22.4 26.9
Earthworms
Current study Lumbricidae sp. 6.2 189.2 12.9 395.5 10.7 74.9 34.0 757.3 310 890
Langdon et al., 2002 Lumbricidae sp. 3.2 902 e e e e e e e e
Langdon et al., 2003 Lumbricidae sp. 13 1358 e e e e e e e e
Van Hook, 1974 Lumbricidae sp. e e 3.1e9.3 e e e 4e6 e 93e502 e
Morgan and Morgan, Lumbricidae sp. e e 12.3 9e812 13 13e39 2.2 69e3667 426 466e2933
1990
Pizl and Josens, 1995 Lumbricidae sp. e e 15.6 31.2 11.1 14.9 28.9 77.5 333 1101
Morgan and Morgan, Lumbricidae sp. e e 12 42e147 14 10e16 5 71e900 330 770e1745
1999
Kennette et al., 2002 Lumbricidae sp. e e e e 9.1e12.2 21e83 0.6e12.9 24e33 168e180 207e754

After comparing the total soilediet and the available soile
diet relationships, comparable patterns were observed for
acorn and earthworm. First, total soilediet relationships
were significantly positive for As, while the available soile
diet relationships were not (see Table 1). The rather low ex-
traction efficiency of the ammonium nitrate soil extraction
for As, as previously reported by Wenzel et al. (2001), could
possibly explain these results. This was also supported by
the very low available As concentrations measured in the pres-
ent study. Second, the total soilediet relationships were much
stronger than the available soilediet relationships for all
metals, except for Cd in the acorn, which was comparable
(see Table 1). The total soil fraction seemed therefore a better
predictor for acorn and earthworm metal contents than the
available fraction, which was opposite to what we predicted.
We have no good explanation for this observation except for
the fact that this may question the standard use of the ammo-
nium nitrate soil extraction method for determining metal
availability to plants and soil organisms, as proposed by
DIN 19730 (1997). Previously, salt solutions such as ammo-
nium nitrate, gave no or only poor reflections of the plant-
available (e.g. Jackson and Alloway, 1991; Gupta and Aten,
1993) and earthworm-available (Bequer et al., 2005) fractions
of the soil metal content and causes for these observations
could not be explained satisfactorily.
After comparing the soileacorn relationships to the soile
earthworm relationships, higher coefficients of determination
were found for the soileearthworm relationships for all metals
(see Table 1). Furthermore, much higher metal concentrations
were measured in the earthworm than in the acorn. These
results suggest a more important transfer of metals from the
soil to the earthworm, than from the soil to the acorn. All to-
gether, the positive soileacorn and soileearthworm relation-
ships suggested metal transfer from the soil to the diet
compartment for most of the metals, which was in accordance
with what we predicted.
4.3. Dietewood mouse metal transfer
The elevated metal concentrations in the mouse tissues and
the positive relationships between the metal concentrations in
mouse diet and tissues for Cd, Pb and to the lesser extent for
Zn, indicate transfer of these metals from the diet to the mouse
compartment. No such relationships were observed for As and
Cu. Roberts and Johnson (1978) and Hunter et al. (1987) also
observed positive relationships between metal concentrations
in the estimated diets and tissue levels of wood mice, field
vole and common shrew for Cd and Pb, but not for Zn. In con-
trast to our results, Torres and Johnson (2001) found positive re-
lationships between Cu concentrations in the liver of the house
mouse (Mus musculus) and the seeds of salt marsh bulrush.
Folkeson et al. (1990) observed positive relationships between
Cu concentrations in beech nuts and liver of the yellow-necked
mouse (Apodemus flavicollis). In current study, no significant
dietemouse relationships for As and Cu were found. Based
on the increased As concentration in the diet of the wood mouse
with decreasing distance from the non-ferrous factory, we
would expect the same pattern for As levels in wood mouse tis-
sues. Selective sequestration of As in tissues other than liver and
kidney, such as hair and nails, and a relative faster rate of
Table 3
Ranges of mean tissue metal concentrations (mg g1 dw) in wood mouse and other small rodents at reference (Ref) and polluted (Poll) sites reported in the literature
Source Species As Cd Cu Pb Zn
Ref Poll Ref Poll Ref Poll Ref Poll Ref Poll
Liver
Current study A. sylvaticus 0.06 0.12 0.52 23.21 16.54 18.75 0.07 1.34 80.97 106.19
Ismail and Roberts, 1992 A. sylvaticus 1.75 5.60 e e e e e e e e
Erry et al., 1999 A. sylvaticus 0.1e0.8 1.3e5.4 e e e e e e e e
Erry et al., 2000 A. sylvaticus 0.5e0.9 0.4e8.7 e e e e e e e e

D. Rogival et al. / Environmental Pollution 145 (2007) 516e528

Hunter et al., 1987 A. sylvaticus e e 0.4 18.2 16 14e16 e e e e
Hunter and Johnson, 1982 A. sylvaticus e e 0.51 1.46 13 24 e e e e
Beardsley et al., 1978 M. agrestis e e 0.1e2 4e9 2e24 40e56 2 3 90 149
Ma, 1989 A. sylvaticus e e e e e e 0.5 e e e
Chmiel and Harrison, 1981 A. sylvaticus e e e e e e 3.5 8.5 e e
Roberts et al., 1978 A. sylvaticus e e e e e e 5.4e7.9 12e14 e e
Johnson and Roberts, 1978 A. sylvaticus e e 0.66 4.36 e e 5.37 13.0 84.8 133
Cook et al., 1990 A. sylvaticus e e e e e e e e 309 218
M. agrestis e e e e e e e e 77 80
Kidney
Current study A. sylvaticus 0.11 0.25 3.26 57.74 22.02 23.82 0.81 9.71 82.73 111.30
Ismail and Roberts, 1992 A. sylvaticus 4.90 15.4 e e e e e e e e
Erry et al., 1999 A. sylvaticus 0.2e0.6 1.6e4.9 e e e e e e e e
Erry et al., 2000 A. sylvaticus 0.4e1.0 0.5e6.7 e e e e e e e e
Hunter et al., 1987 A. sylvaticus e e 2.0 41.7 22 19e25 e e e e
Hunter and Johnson, 1982 A. sylvaticus e e 1.46 7.4 12 14 e e e e
Beardsley et al., 1978 M. agrestis e e 1e5 2e14 13e49 21e56 4 7 77 108
Ma, 1989 A. sylvaticus e e e e e e 0.8 e e e
Chmiel and Harrison, 1981 A. sylvaticus e e e e e e 0.4 9.8 e e
Roberts et al., 1978 A. sylvaticus e e e e e e 9e13 39e47 e e
Johnson et al., 1978 A. sylvaticus e e 2.19 18.0 e e 9.4 39.2 101 158
Cook et al., 1990 A. sylvaticus e e e e e e e e 338 401
M. agrestis e e e e e e e e 82 84
Bone
Current study A. sylvaticus e e e e e e 1.36 25.56 e e
Johnson et al., 1978 A. sylvaticus e e e e e e 12e34 189e672 e e
Chmiel and Harrison, 1981 A. sylvaticus e e e e e e 25 67 e e
Roberts et al., 1978 A. sylvaticus e e e e e e 12e21 189e352 e e
Ma, 1989 A. sylvaticus e e e e e e 1.9 26.3 e e
Beardsley et al., 1978 M. agrestis e e e e e e 1e5 12e17 e e

525
526 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528
excretion of As may account for such a lack of relationship
(Sharma and Shupe, 1977; Torres and Johnson, 2001). In anal-
ogy to our results, Ismail and Roberts (1992) found that As in
tissues of field voles, bank voles and wood mice from an As re-
finery and a reference area did not consistently reflect As levels
in soil or vegetation, indicating that these animals may not be
a reliable monitor of environmental As.
The Cu concentrations in the earthworms increased with
decreasing distance from the non-ferrous factory, while in
acorns no such gradient was observed. However, tissue Cu
concentrations in wood mice did not differ significantly among
sites. Two mutually non-exclusive possible explanations could
account for such a lack of significant differences. First, since
acorn Cu concentrations did not differ much among sites,
and because acorn is the dominant food item consumed by
the wood mouse during most of the year (Corbet and Harris,
1991), very small or no differences in Cu concentrations in
mouse tissues could be expected among sites. The clear Cu
gradient observed in earthworms was not reflected in mouse
tissues because this food item is only consumed in late winter
(Corbet and Harris, 1991). Second, it is well known that, over
a wide concentration window, the retention of Cu in mamma-
lian systems is effectively regulated by absorption-excretion to
maintain a homeostatic situation (Sheffield et al., 2001).
Therefore, the small differences in acorn Cu concentrations
among sites are most probably regulated by the homeostatic
system of the wood mouse.
We observed a positive earthwormemouse relationship for
Zn, despite the fact that Zn is under close homeostatic control
in mammals (Sheffield et al., 2001). However, the coefficients
of determination were low (liver: r2 ¼ 0.19; kidney: r2 ¼ 0.16).
Two mutually non-exclusive possible explanations could ex-
plain this observed relationship. First, total Zn concentrations
in the soils of the most contaminated sites were particularly el-
evated (approximately 10 times) compared to the reference site.
Burrowing rodents, such as the wood mouse, are mainly exposed
to environmental contaminants via the consumption of contam-
inated food, and to the lesser extent via direct ingesting of con-
taminated soil (Beyer et al., 1994). As a result, direct soil intake
together with intake via contaminated diet may result in high Zn
intake, which may possibly break down the Zn regulatory capac-
ity of the body, resulting in elevated Zn levels in tissues of wood
mouse at the most polluted sites. Second, Cd-induced changes in
Zn homeostasis may result in an increased retention of Zn in the
liver and/or kidney due to Cd accumulation and metallothionein
induction in these organs (for a review see Brzoska and Mon-
iuszko-Jakoniuk, 2001).
When comparing the acornemouse relationships to the
earthwormemouse relationships, comparable coefficients of
determination were found in the case of Pb (liver r2: 0.46 vs
0.36; kidney r2: 0.44 vs 0.35). In the case of Cd, the earthworme
mouse relationships (liver r2: 0.73; kidney r2: 0.62) were much
stronger than the acornemouse relationships (liver r2: 0.37;
kidney r2: 0.33). The earthwormemouse relationships were
significantly positive for Zn, while the acornemouse relation-
ships were not. All together, these results suggest a more im-
portant transfer of Cd from the earthworm to the mouse
compartment, than from the acorn to the mouse compartment.
Transfer of Zn from the diet to the mouse compartment only
seemed to occur from the earthworm, but not from the acorn.
Furthermore, overall transfer of metals from food to mice is
probably to a large extent determined by the small proportion
(15%) (Corbet and Harris, 1991) of animal food in the wood
mouse diet. Plant tissue compromises the largest part of the
wood mice diet (70%) (Corbet and Harris, 1991) but contains
far less metals than animal food as indicated by the metal con-
centrations of acorn and earthworm in Fig. 3. The non-essen-
tial metals were a factor 100e2500 higher in the earthworms
compared to the acorns, which is much larger than the five fold
smaller portion of animal food in the wood mice diet. The dif-
ference for the essential metals was smaller but still in favour
of the earthworms.
It should be noted that the transfer of metals to the mouse
compartment not only occurs through the oral route but also
through the respiratory route. Although absorption of some
metals from the lungs is more efficient than from the gastroin-
testinal tract, oral exposure by ingestion of contaminated food
is the dominant source of metals for wild mammals (Hunter
et al., 1987; Ma, 1989). In the present study, the metal pollu-
tion is mainly a consequence of historical emissions from the
non-ferrous factory. During the last decade, the atmospheric
metal emissions from the factory have strongly decreased
(VMM, 1999). Nevertheless, the relative contribution of expo-
sure through the oral and respiratory route for mice living in
the pollution gradient under study needs further investigation.
4.4. Overall transfer of essential and non-essential
elements
Regression analysis showed strong relationships within the
soilediet and dietewood mouse compartments for the non-
essential metals Cd, Pb, and to a lesser extent for As, while
relationships for the essential metals Cu and Zn were weak
or absent. These results were in accordance with what we pre-
dicted. Hunter and Johnson (1982) and Hunter et al. (1987) ob-
served a much greater transfer for the non-essential metal Cd
than for the essential metal Cu in a soiledieterodent food
chain. Previous studies observed stronger relationships within
a soiledieterodent food chain for the non-essential metals Cd
and to the lesser extent Pb (Sharma and Shupe, 1977; Roberts
and Johnson, 1978), while relationships for the essential metal
Zn were absent (Roberts and Johnson, 1978). In line with these
results, we observed stronger transfer of non-essential ele-
ments through the food chain than essential elements, which
was probably a consequence of the homeostatic control of
the latter group.
5. Conclusions
The present study differs from most of the previous ecotox-
icological field studies that focus on terrestrial mammals be-
cause it reports food chain relationships for a wider range of
elements and investigates total as well as available soil metal
concentrations simultaneously and in conjunction with levels
 D. Rogival et al. / Environmental Pollution 145 (2007) 516e528
in food items and in tissues of wood mice. We observed a clear
gradient in metal exposure away from the smelter in all com-
partments of the food chain for non-essential metals (As, Cd
and Pb). The gradient was less clear or absent for the essential
metals Cu and Zn in acorn and mice target tissues. The posi-
tive soileacorn and soileearthworm relationships suggested
metal transfer from the soil to the diet compartment for
most of the metals, which was in accordance with what we
predicted. Within the soilediet compartments, the total soil
fraction seemed a better predictor for acorn and earthworm
metal contents than the available fraction, which was in con-
trast to what we predicted. The positive dietewood mouse re-
lationships observed for Cd, Pb and to a lesser extent for Zn,
indicate transfer of these metals from the diet to the mouse
compartment. Within the dietewood mouse compartments,
the earthwormemouse relationships were stronger than the
acornemouse relationships, except for Pb, which were compa-
rable. This was in contrast to what we predicted. All together,
our results suggest a more important transfer of non-essential
elements through the food chain than essential elements,
which was probably a consequence of the homeostatic control
of the latter group. This was in line with our prediction.
Acknowledgements
The study was funded by the GOA project ‘Integrated wild-
life study on the effects of environmental pollution on aquatic
and terrestrial vertebrates’ (University of Antwerp). We thank
the cities of Antwerp, Mortsel and Zoersel, and AMINAL.
Damien Rogival holds an IWT-grant.
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