Applied Soil Ecology 77 (2014) 917

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Applied Soil Ecology

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Soil properties and metal accumulation by earthworms in the Siena

urban area (Italy)
Francesco Nannoni, Sara Rossi, Giuseppe Protano
Department of Environment, Earth and Physical Sciences, University of Siena, Via del Laterino 8, I-53100, Siena, Italy

a r t i c l e i n f o a b s t r a c t

Article history: This paper reports the results of a study focused on the metal (Cd, Co, Cr, Cu, Ni, Pb, Sb, U and Zn) distri-
Received 31 October 2013 bution in soils and uptake and accumulation by earthworms Nicodrilus caliginosus (Savigny) from urban,
Received in revised form 10 January 2014 peri-urban, green-urban and non-urban zones of Siena municipality (central Italy). The main goal was
Accepted 12 January 2014
to dene the inuence of soil properties and metal soil contents on the uptake of these contaminants
by earthworms. Data indicated that Cd, Cu, Pb, Sb and Zn soil contents increased in the following order:
Keywords:
non-urban < green-urban < peri-urban < urban soils, suggesting that vehicular trafc affects the distribu-
Metals
tion of these metals. Pb and Sb were the main soil contaminants and their highest enrichments were
Soil properties
Earthworms
found in urban sites where stop-and-go trafc occurs. Concentrations of these trafc-related metals in
Urban soils earthworms showed a distribution pattern similar to that in soil, suggesting that soil contamination inu-
Italy enced the uptake of Cd, Cu, Pb, Sb and Zn by N. caliginosus. There were signicant positive correlations
between Cd, Pb and Sb earthworm concentrations and their soil contents. The lack of correlation for Cu
and Zn could be due to the physiological regulation of these elements by earthworms. Statistical analysis
pointed out that the uptake and accumulation of Cd, Cu, Pb, Sb and Zn by earthworms were affected by
some soil physicochemical properties such as the organic carbon and carbonate contents that are able to
rule the bioavailability of metals in soils.
2014 Elsevier B.V. All rights reserved.

1. Introduction
In recent years, the interest for urban areas has increased due to
environmental and health risks associated with the contamination
by toxic substances such as metals (i.e., Cd, Pb and Sb). In the urban
settings several human activities, such as industrial plants, vehic-
ular trafc, home heating and waste incineration, spread metals
in the atmosphere, seriously affecting air quality. Atmospheric dry
and wet depositions are the major pathway of these contaminants
to the surface soils, and contribute to the change of soil geochem-
istry as a result of fractionation and accumulation of metals through
sorption and precipitation/coprecipitation reactions. The extent
and features of these reactions depend on the chemical forms of the
elements and the physicochemical properties of the soil (Essington,
2004; Sparks, 2003).
The mobile and available fraction of metals in soil can be partly
transferred to the hydrosphere (surface waters and groundwater)
and biosphere (plants and animals). Therefore, soil acts as both a
sink and source of metals in urban areas, thus affecting their bio-
geochemical cycle in ecosystems.
Corresponding author. Tel.: +39 0577 233950; fax: +39 0577 233945.
E-mail address: giuseppe.protano@unisi.it (G. Protano).
Many studies dealt with the metal distribution in soils of urban
areas, highlighting that the highest concentrations of these inor-
ganic contaminants are generally located close to the main roads,
as well as in residential areas and commercial districts (Cicchella
et al., 2008; Imperato et al., 2003; Yang et al., 2011). Several
researches also used some plants as bioaccumulators of metals and
bioindicators in order to monitor levels of contamination of urban
environments (Dong-Sheng and Peart, 2006; Malizia et al., 2012;
Rucandio et al., 2011). Furthermore, few researchers studied metal
uptake and accumulation in soil organisms, such as earthworms in
urban areas (Kennette et al., 2002; Pizl and Josens, 1995).
Earthworms are soil-dwelling organisms commonly used in
environmental and ecotoxicological studies. Several authors report
that earthworms are able to accumulate metals such as Cd, Pb and
Zn, and there is a fairly consistent correlation between the concen-
trations of these contaminants in earthworm tissue and those in soil
(e.g., Dai et al., 2004; Hobbelen et al., 2006). Earthworms are one of
the few soil organisms available as biological indicators of metals
contamination in urban environments, therefore these organisms
are considered by several authors (e.g., Lanno et al., 2004; Suthar
et al., 2008) as bioindicators of soil quality providing important
information for the assessment of environmental risks.
Moreover, earthworms are a source of food for terrestrial orga-
nisms, especially birds, reptiles and small carnivorous mammals
(Spurgeon and Hopkin, 1996). The consumption of earthworms by

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http://dx.doi.org/10.1016/j.apsoil.2014.01.004
10 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917
predators may determine a transfer and accumulation of metals in
the food chain (Edwards and Bohlen, 1996), and provides a route
through which these contaminants may be transferred to higher
trophic levels.
Uptake and accumulation of metals by earthworms depend on
both total concentrations and chemical form of these contaminants
in soil and the soil properties (e.g., Peijnenburg et al., 1999). In fact,
mobility and bioavailability of metals in soil and their uptake by soil
organisms are affected by many factors, including physicochemical
properties such as pH, cation exchange capacity, organic carbon
and carbonate contents (Lanno, 2003; Morgan and Morgan, 1988;
Peijnenburg, 2002).
The present study investigated the relationship between soil
contamination by metals and their accumulation in the earthworm
Nicodrilus [=Allolobophora (Eisen) = Aporrectodea (Oerley)] caligi-
nosus (Savigny) (Minelli et al., 1995). The research was carried out in
the Siena municipality (central Italy), where vehicular trafc is the
main potential source of contamination, since other human sources
(e.g., industrial and waste emissions) are rather limited or absent.
Aims of the study were: (i) to assess the inuence of vehicu-
lar inputs on metal distribution in soil; (ii) to determine which
soil properties among pH, organic carbon and carbonate contents,
cation exchange capacity, iron and manganese oxides and grain
size, mostly affect the uptake of metals by earthworms; (iii) to
dene the relationships between earthworm uptake and accumu-
lation and metal concentrations in soil. This study was conducted
in urban and peri-urban environments and considered main soil
properties as possible factor of inuence of metal levels in earth-
worm tissue. Moreover, the research addressed the presence of
some metals such as Sb and U, which are less investigated in these
kinds of studies.
2. Materials and methods
2.1. Study area
The study area encompasses the territory of Siena municipality
and includes the urban centre of the city as well its peri-urban,
green-urban and non-urban zones.
Siena is a tourist city with a resident population of about 55,000
inhabitants. Trafc is estimated on average at about 50,000 vehicles
a day and mainly ows along a ring road in the peri-urban sector
of the city (Comune di Siena, 2005). This ring road allows the entry
and exit of vehicles in and from the urban centre, and is connected
to the important national road axes towards Florence, Rome and
Perugia. Since 1965, the historic centre of Siena is a limited trafc
zone (LTZ).
From the geological point of view, the study area extends in
the northern sector of the Siena Basin, a NWSE oriented tec-
tonic depression where the deposition of clayeysiltymarly to
sandymarly marine sediments of the NeogeneQuaternary suc-
cession occurred during Pliocene.
2.2. Sampling strategy and procedures
Taking into account the features of vehicular trafc in the study
area, 30 sampling sites were identied as follows: (i) urban sites
(n = 11) close to the most important urban roads outside the limited
trafc zone of city; (ii) peri-urban sites (n = 9) close to the ring
road and its branches; (iii) green-urban sites (n = 4) located in green
parks within or immediately outside the historic centre of Siena;
(iv) non-urban sites (n = 6) placed in uncultivated terrains in rural
zones faraway from both the urban centre and the peri-urban zone.
Soil samples (20 cm deep) were collected in the 30 selected sites.
Each soil was a composite sample consisting of three sub-samples
collected a few meters apart. In the urban and peri-urban samp-
ling sites, soil was collected near the edge of the roadway within a
distance of 2 m.
In 15 sampling sites, 150 (10 for each sites) mature (clitellated)
specimens of earthworm N. caliginosus were collected. This species,
adapted to live also in conditions modied by human activity, was
selected due to its common presence in the study area. The individ-
uals of N. caliginosus live permanently in horizontal tunnels dug in
the upper 2025 cm of soil, feed mainly on soil organic matter and
belong to the group of endogeic earthworms. Several factors, such
as body size, physiological or seasonal conditions, may inuence
the contents of chemical elements in earthworm tissues. Conse-
quently, only mature individuals were collected in this study and
the sampling period was restricted to one month. The sampling
activity was carried out in September 2007 for both soils and earth-
worms.
2.3. Laboratory treatment
In the laboratory soil samples were dried at +40 C and man-
ually sieved through a metal-free 2 mm sieve. The soil fraction
<2 mm was homogenized by quartering. Quartering procedure was
performed by separating this soil fraction into four equal portions
(quadrants), combining two of the four quadrants diagonally and
rejecting the other two. This procedure was repeated until the
amount of about 100 g was obtained. Finally, this aliquot of soil
sample was powdered in an agate mortar using a mechanical pul-
verizer. The soil samples were solubilised by acid digestion: 1 mL
HF, 2 mL HNO3 , 2 mL HCl and 1 mL HClO4 (ultrapure reagents) were
added to 200 mg of powdered soil; then the mixture was processed
in Teon bombs using a Milestone Ethos 900 microwave lab station
(EPA, 1996). The solution was ltered and diluted with ultrapure
water to a nal volume of 100 mL.
In the laboratory earthworms were identied and rinsed with
deionised water to clean them of adhering soil particles. Then earth-
worms were placed in plastic Petri dishes (two individuals in each
dish) containing Whatman No. 1 lter paper and a few drops of
deionised water to maintain moisture, and kept at 18 C for 72 h.
In order to prevent coprophagy, the lter paper was changed daily.
The individuals were subsequently depurated for an additional day
without lter paper but with a few drops of deionised water to
allow the complete egestion of gut contents. The earthworms were
killed by freezing (80 C) and freeze-dried for 48 h. The dry weight
of sampled individuals ranged from 101 to 256 mg (146 32 mg as
mean SD).
The freeze-dried earthworms were digested individually with
an acid mixture of 3 mL HNO3 70% and 1 mL H2 O2 30% (ultra-
pure reagents) in Teon bombs placed in a Milestone Ethos 900
microwave lab station. Solution was ltered and diluted to 50 mL
with ultrapure water.
2.4. Determination of soil properties
Soil particle size distribution as percent content of the sandy,
silty and clayey fractions, was determined by the hydrometer
method. Soil pH (pHH2 O ) was measured in a 1:1 (w/v) soil/water
mixture following the EPA method 9045/D. The procedure of
Hendershot and Duquette (1986) was used to determine the effec-
tive cation exchange capacity (CECe) measuring the Ca, Mg, K, N
and Al concentrations in a mixture obtained by the reaction of 2 g
of soil and 20 mL of a 1 M NH4 Cl solution. The method of Walkley
and Black (1934) was used to determine the organic carbon (Corg )
content in soil. The carbonate content as percentage content of
calcium carbonate (%CaCO3 ) was determined with De Astis cal-
cimeter.
 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917 11

In order to assess the amount of FeMn oxyhydroxides in soil,
a chemical extraction procedure was employed. This procedure
based on the step B of the protocol proposed by the Standards,
Measurements and Testing Programme (formerly BCR, Bureau
Community of Reference) of the European Commission (Pueyo
et al., 2001; Rauret et al., 1999), allows determining the concen-
tration of Fe and Mn in the reducible soil fraction. The chemical
extraction was performed adding to 1 g of powdered soil 40 mL of
a 0.5 M hydroxylamine hydrochloride solution. The mixture was
shaken for 16 h at room temperature and centrifuged, and then
supernatant was ltered.
2.5. Analytical determinations
The content of Cd, Co, Cr, Cu, Ni, Pb, Sb, U and Zn in soil and
earthworm samples, as well as the concentrations of Fe and Mn in
the reducible soil fraction, were determined by inductively coupled
plasma-mass spectrometry (ICP-MS) using the PerkinElmer Sciex
Elan 6100 spectrometer.
The accuracy of metal determinations in soil was checked using
the NIST 2709 (San Joaquin soil) and 2710 (Montana soil) standard
reference materials. The metal concentrations measured in these
standards were in good agreement with the certied values with
recoveries from 94% for Sb to 100% for Pb. The precision evaluated
as repeatability of the analytical measures (n = 5) and expressed as
percent relative standard deviation (%RSD), was below 4.7% for all
elements.
The NIST 2977 (Mussel tissue) standard reference material was
used to check the accuracy of analytical determinations in earth-
worms. The recoveries were from 92% for Cd to 110% for Sb.
Precision was determined by means of ve replicate analyses of
each earthworm sample, and was below 4% (%RSD) for all the ele-
ments.
2.6. Statistical analysis
The analytical data were treated statistically using the pro-
gram Statistica 8 (Statsoft, Padova). The ShapiroWilks W test was
applied to verify the normal distribution of data. Statistical differ-
ences between datasets were determined through the Students t
test when data were normally distributed, and the MannWhitney
U test in the case of data not normally distributed, at the 5% signif-
icance level.
Pearsons correlation test was used to identify signicant corre-
lations between metal contents in soil samples (n = 30, p < 0.05).
The relationships between metal concentrations in earthworms
and soil were examined by simple linear regression analysis.
Principal component analysis (PCA) with varimax rotation
(Facchinelli et al., 2001) was used both to evaluate the relationships
between metal contents in soil and to determine the inuence of
soil properties on the metal concentrations in earthworms.
3. Results and discussion
3.1. Soil properties
Soil samples formed from clayeysiltymarly and sandymarly
marine sediments of the NeogeneQuaternary succession (Table 1).
Soils derived from the sandymarly sediments (n = 19) had high
sand contents varying from 39 to 59% (52% as median). Silt was
the dominant particle size fraction (45% as median) in soils whose
parent rock consists of clayeysiltymarly sediments (n = 11).
According to USDA (United States Department of Agriculture) clas-
sication, soil samples had sandy-loam, loam and silty-clay-loam
textures.
Soil pHH2 O varied from 7.4 to 8.6 (weakly to strongly alkaline
soils) with most of values in the narrow range 88.2 (Table 1).

Table 1
The physicochemical properties of soils collected in the non-urban, green-urban, peri-urban and urban sampling zones of Siena area.

Soil sample Sampling zone Parent rock Particle size distribution pHH2 O CECe* CaCO3 * Corg * Fe(Fe-ox) * Mn(Mn-ox) *

Lithology %Sand %Silt %Clay cmol(+) kg1 % % mg kg1 mg kg1

SI-0 a Non-urban Clayeysiltymarly sed. 45 37 18 7.5 21.5 16.7 0.39 1658 210
SI-0 b Non-urban Clayeysiltymarly sed. 46 36 18 8 24.3 17.6 0.61 1775 152
SI-1 Urban Sandymarly sed. 59 29 12 7.6 23.5 19.7 0.75 1291 101
SI-2 Peri-urban Sandymarly sed. 39 33 28 7.7 24.2 13.8 0.22 2506 231
SI-3 Peri-urban Clayeysiltymarly sed. 36 45 19 7.7 24 19.7 0.26 2053 241
SI-4 Urban Sandymarly sed. 47 32 21 7.8 28.3 22.7 1.41 2825 350
SI-5 Urban Sandymarly sed. 46 41 13 8.6 24.5 32.5 2.04 634 286
SI-6 Green-urban Sandymarly sed. 58 29 13 8.4 18 28.6 0.52 1344 173
SI-7 Urban Sandymarly sed. 53 33 15 7.4 28.4 12.8 4.07 1023 361
SI-8 Peri-urban Clayeysiltymarly sed. 25 45 30 8.5 28.8 22.5 2.85 1371 176
SI-9 Peri-urban Clayeysiltymarly sed. 27 43 30 8.1 30.3 17.6 2.98 1975 329
SI-10 Urban Sandymarly sed. 47 28 24 8.1 28.2 17.6 1.63 1603 211
SI-11 Urban Sandymarly sed. 54 30 16 8.1 28.4 18.6 1.71 1313 267
SI-12 Urban Sandymarly sed. 50 34 16 8.2 23.3 20.6 0.57 1198 143
SI-13 Urban Sandymarly sed. 58 32 11 8.6 30.1 31.4 1.97 1424 165
SI-14 Peri-urban Clayeysiltymarly sed. 18 49 32 8.2 28.1 27.4 2.87 932 269
SI-15 Peri-urban Sandymarly sed. 59 28 13 7.8 31.2 21.6 1.51 1056 370
SI-16 Green-urban Sandymarly sed. 46 31 23 8.2 27.1 17.6 1.96 997 234
SI-17 Urban Sandymarly sed. 52 34 14 8 27.2 15.7 2.01 901 120
SI-18 Green-urban Sandymarly sed. 49 32 19 8.1 29.5 21.6 1.68 1165 222
SI-19 Non-urban Clayeysiltymarly sed. 24 46 30 8.1 29 21.6 1.72 2004 312
SI-20 Peri-urban Clayeysiltymarly sed. 19 52 29 8 23.6 22.9 0.6 1582 162
SI-21 Non-urban Sandymarly sed. 54 29 17 7.5 26.5 21.5 2 1549 160
SI-22 Non-urban Clayeysiltymarly sed. 10 51 39 8 21.4 25.5 0.59 1655 154
SI-23 Non-urban Clayeysiltymarly sed. 11 50 40 8 22.4 22.5 0.69 2422 140
SI-24 Peri-urban Clayeysiltymarly sed. 18 45 37 8.1 21.9 24 0.74 2065 291
SI-25 Peri-urban Sandymarly sed. 59 30 11 8.1 26.1 18.2 1.7 1154 248
SI-26 Urban Sandymarly sed. 48 33 19 8.2 28.6 18.7 1.74 1343 201
SI-27 Urban Sandymarly sed. 51 32 17 8.1 30.9 17.9 1.91 1212 170
SI-28 Green-urban Sandymarly sed. 55 30 16 8 24.7 16.1 1.5 1276 240
*
CECe: cation exchange capacity effective; CaCO3 : carbonate content; Corg : organic carbon content; Fe(Fe-ox) and Mn(Mn-ox) : Fe and Mn contents in the reducible soil fraction,
furnish an evaluation of the FeMn oxyhydroxides amount in soil.
12 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917

Table 2
Metal contents in soils of Siena area grouped by sampling zone (n = number of soil samples).

Soil sampling zone Cd Co Cr Cu Ni Pb Sb U Zn

mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1

Non-urban (n = 6)
Minimum 0.1 10.1 59.2 21.9 36.6 16.3 0.78 1.4 68.7
Maximum 0.17 15.4 81.6 24.7 60.5 37.5 1.3 2.5 90.1
Mean 0.15 12.5 71.7 23.2 43.6 23 0.99 1.8 77.6
Median 0.15 12.4 71.9 23.4 41 21.9 0.89 1.6 76.2
Standard deviation 0.03 2.3 7.6 1.1 8.7 7.8 0.25 0.49 9
Green-urban (n = 4)
Minimum 0.05 6.8 47.6 32.2 24.8 35.5 1.2 1.2 57.2
Maximum 0.23 10.2 58.9 55.8 30.9 77.6 3.5 1.4 120
Mean 0.16 8.2 54.4 39 28.4 54.2 2.4 1.3 90.7
Median 0.18 7.9 55.5 34a 28.9 51.8a 2.5a 1.3 92.7
Standard deviation 0.08 1.4 5.3 11.3 2.7 17.5 0.91 0.12 31.6
Peri-urban (n = 9)
Minimum 0.06 8.2 55.8 26.2 32.9 28 1.1 1.1 67.3
Maximum 0.35 15.4 92 82.1 55.6 259 8.9 2.6 121
Mean 0.22 11.7 76.3 45.4 45.3 81.9 3.1 1.6 96.8
Median 0.23a 10.9 79.8 42.3a 44.7 46a 2.6a 1.5 95.3a
Standard deviation 0.08 2.2 12.9 15.7 7 72.4 2.4 0.46 17.7
Urban (n = 11)
Minimum 0.07 5.9 50.4 14.2 29.8 24.8 0.88 0.93 60.2
Maximum 0.48 11.7 75.3 101 48.7 178 14.8 2.7 207
Mean 0.27 8.4 64.7 43.7 41 105 5.1 1.5 122
Median 0.25a 8.1 66.4 39.1a 40.9 106a 3.8a 1.4 116a
Standard deviation 0.13 1.7 8.9 25 5.9 52.6 4.3 0.6 50.6
a
Signicant differences among the median contents of metals in green-urban, peri-urban and urban soils compared to non-urban ones (p < 0.05).

A positive correlation was found between pHH2O and carbonate urban soils compared to non-urban ones (p < 0.05; Table 2). For
content (r = 0.61, p < 0.001). Soil samples had a moderately high Cd and Zn the differences were signicant only for peri-urban and
to high CECe (1831.2 cmol(+) kg1 ) which increased with increas- urban soils (p < 0.05).
ing of organic carbon content (r = 0.7, p < 0.001). The organic carbon Co, Cr, Ni and U soil contents were rather homogeneous in
content varied from 0.2 to 4.1%, and the carbonate content was in all the sampling zones. As consequence, no signicant differ-
the range 12.832.5% as CaCO3 (highly calcareous soils). ence occurred between the median contents of these metals in
the non-urban, green-urban, peri-urban and urban soils (p < 0.05;
Table 2).
3.2. Metals in soils
The highest accumulation levels were found for Sb and Pb in
urban soils as highlighted also by EF median values of 2.9 and
Table 2 reports the main statistical indices (minimum, maxi-
2.8, respectively (Table 3). These relevant enrichments are due
mum, mean, median and standard deviation) of Cd, Co, Cr, Cu, Ni,
to concentrations usually in the range 215 mg kg1 for Sb and
Pb, Sb, U and Zn contents in soils (as mg kg1 dry weight) grouped
70180 mg kg1 for Pb. Also Cd, Cu and Zn were accumulated in
by sampling zone (non-urban, green-urban, peri-urban and urban).
urban soils as their contents (0.070.48 mg kg1 , 14.2101 mg kg1 ,
In order to evaluate the anthropogenic input of metals in soils,
60.2206.8 mg kg1 , respectively) were usually higher than the
the enrichment factor (EF) was calculated as the ratio between the
respective local geochemical background. However, the enrich-
element content in soil sample and the maximum value of its local
ment factors of these metals generally were between 1.1 and 2.8
geochemical background. The local geochemical background corre-
(EF = 1.4 for Cd, 1.6 for Cu, 1.3 for Zn, as median value).
sponds to the natural variability of the element in soils of the study
Analytical data revealed a marked Sb and Cu accumulation in
area. The local geochemical background of Cd, Co, Cr, Cu, Ni, Pb, Sb,
peri-urban soils (EF = 1.9 and 1.7, respectively) given by contents
U and Zn was evaluated using the range of the element contents in
in the range 1.18.9 mg kg1 for Sb and 26.282.1 mg kg1 for Cu.
the non-urban soils (Table 3).
Similar abundances and enrichment levels of Sb and Cu were also
As shown in Tables 2 and 3, analyses of Cd, Cu, Pb, Sb and Zn
found in soils collected in the green-urban sites. In these latter soils,
contents in soils revealed a distribution pattern characterized by
a Pb accumulation (EF = 1.4) was documented due to concentrations
the following features: (i) contents in the green-urban, peri-urban
ranging between 35.5 and 77.6 mg kg1 .
and urban soils were usually higher than those in the non-urban
soils; (ii) concentrations and EF median values in soil varied in
relation to the sampling zone increasing as the following order: 3.3. Metals in earthworms
green-urban < peri-urban < urban soils. Moreover, the statistical
analysis indicated that there were signicant differences among Table 4 reports the main statistical indices (minimum,
the Pb, Sb and Cu median contents in green-urban, peri-urban and maximum, mean, median and standard deviation) of metal

Table 3
Local geochemical background of metals in soil (minimummaximum in mg kg1 ) and median value of their enrichment factor (EF) in green-urban, peri-urban and urban
soils of Siena area.

Element Cd Co Cr Cu Ni Pb Sb U Zn

Background (mg kg1 ) 0.10.17 10.115.4 59.281.6 21.924.7 36.660.5 16.337.5 0.781.3 1.42.5 68.790.1
EF green-urban soils 1.1 0.5 0.7 1.4 0.5 1.4 1.9 0.5 1
EF peri-urban soils 1.3 0.7 1 1.7 0.7 1.2 1.9 0.6 1.1
EF urban soils 1.4 0.5 0.8 1.6 0.7 2.8 2.9 0.5 1.3
 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917 13

Table 4
Metal concentrations in specimens of Nicodrilus caliginosus of Siena area grouped by sampling zone (n = number of earthworms).

Earthworm sampling zone Cd Co Cr Cu Ni Pb Sb U Zn

mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1 mg kg1

Non-urban (n = 50)
Minimum 0.65 3.4 0.71 8.1 2.2 0.32 0.01 0.04 214
Maximum 4.8 9.9 2.4 21.2 11 1.8 0.29 0.22 912
Mean 1.9 5.2 1.7 13.8 6 1.2 0.13 0.1 479
Median 1.9 4.8 1.7 13 5.3 1.3 0.12 0.08 456
Standard deviation 0.71 1.2 0.37 3.9 2.2 0.43 0.06 0.05 170
Green-urban (n = 20)
Minimum 0.77 1 1.2 11.7 1.2 0.78 0.04 0.03 184
Maximum 4.1 5.9 2.7 34.9 19.4 2.1 0.38 0.13 1040
Mean 2 2.7 1.8 22.8 6.6 1.4 0.17 0.07 594
Median 1.6 2.2 1.7 20.2a 6 1.3 0.16a 0.07 565a
Standard deviation 1 1.4 0.45 6.7 4.9 0.38 0.09 0.03 188
Peri-urban (n = 30)
Minimum 1.2 2.5 1.1 11.4 0.29 0.52 0.12 0.03 198
Maximum 4.2 12.1 4.1 29.1 20.9 3.7 0.87 0.7 1548
Mean 2.6 5.6 2.2 18.9 5.4 1.9 0.24 0.1 648
Median 2.6a 4.5 2.1 18.8a 4.1 2.1a 0.22a 0.08 578a
Standard deviation 0.71 3 0.72 4.9 4.6 0.84 0.14 0.12 311
Urban (n = 50)
Minimum 0.75 1.4 1.3 9 1.4 0.86 0.07 0.03 407
Maximum 5.6 9.3 3.4 31.7 13.6 11.4 3 0.13 1586
Mean 3 4.1 1.9 19.9 5.9 3.1 0.52 0.07 869
Median 2.7a 3.8 1.8 19.8a 5.7 2.5a 0.32a 0.07 808a
Standard deviation 1.2 1.9 0.47 6.7 3.4 2.2 0.61 0.02 275
a
Signicant differences among the median contents of metals in green-urban, peri-urban and urban earthworms compared to non-urban ones (p < 0.05).

concentrations in specimens of N. caliginosus grouped by sampling 3.4. Soil contamination

zone.
As for soils, Cd, Cu, Pb, Sb and Zn concentrations in earthworms
of the peri-urban and urban sites were usually higher than in spec-
imens from the non-urban zone, with median values increasing as
the following order: non-urban < green-urban < peri-urban < urban
earthworms. The statistical analysis indicated that there were sig-
nicant differences among the Cu, Sb and Zn concentrations in
earthworms from green-urban, peri-urban and urban sites and
in specimens collected in non-urban sites (p < 0.05; Table 4). For
Cd and Pb signicant differences were between the contents in
earthworms from peri-urban and urban sites in comparison to non-
urban ones (p < 0.05).
In agreement with above, the highest concentrations of Pb
(2.5 mg kg1 as median value) and Sb (0.32 mg kg1 ) were mea-
sured in earthworms sampled in the urban zone. The contents
of these metals decreased in the individuals from the peri-urban
sites (2.1 mg kg1 for Pb and 0.22 mg kg1 for Sb) and the low-
est concentrations of Pb (1.3 mg kg1 ) and Sb (0.12 mg kg1 ) were
found in earthworms living in non-urban sites. A similar distri-
bution pattern in earthworms was identied also for Cd and Zn
concentrations. The Cu contents were comparable for individuals
collected in green-urban, peri-urban and urban soils (usually from
10 to 30 mg kg1 ), and higher than those in specimens living in the
non-urban zone.
In contrast, there were no signicant differences among the Co,
Cr, Ni and U concentrations in earthworms collected in the different
sampling zones (p < 0.05; Table 4).
The bioaccumulation factor (BAF) was used to estimate the
bioavailability of metals for earthworms. It was calculated as the
ratio of the element concentration in earthworm (in mg kg1 dry
weight) to the element content in soil (in mg kg1 dry weight).
BAF median values dene the following rank for the analyzed
metals: Cd (11.2) Zn (5.9) Co (0.52) Cu (0.49) > Ni (0.12) Sb
(0.09) > U (0.05) Pb (0.03) Cr (0.03). BAF values were constantly
above 1 for Cd (8.839.6) and Zn (3.510.7); the range 0.20.8 was
found for Cu and Co, whereas the lowest BAF values (<0.1) distin-
guished metals such as Cr, Pb, Sb and U.
Analytical data revealed that Sb and Pb were the main soil
contaminants in the study area. These metals were signi-
cantly enriched in urban and peri-urban soils compared to the
background, suggesting that their soil contents are presumably
inuenced by vehicular trafc. The highest Sb and Pb contents
were measured in urban soils near trafc lights, crossings and
roundabouts, places strongly characterized by stop-and-go trafc.
In these conditions the input of the trafc-related metals increases
as the fuel combustion and the wear of some vehicle components
(i.e., brakes, clutches and tires) increase.
It is noteworthy that Pb concentrations were lower in peri-urban
soils than in urban ones. This nding could be attributed to the fol-
lowing reasons: (i) most of the peri-urban roads were built and/or
improved recently (from the late 1980s to the present) and conse-
quently the peri-urban zones of Siena city were less affected by
the release of Pb from vehicles fuelled with leaded gasoline (in
Italy the abrogation of leaded gasoline occurred in 2001); (ii) traf-
c in peri-urban zones usually does not involve frequent stops and
departures; (iii) in the peri-urban zones there are no effective barri-
ers to dispersion of contaminants, differently from the urban zones
where roads are often bordered by buildings.
Furthermore, Sb and Pb were accumulated in green-urban soils.
This result could be attributable to the wide atmospheric diffu-
sion of these metals released by vehicular trafc and home heating
during the cold period (Petaloti et al., 2006; Wang, 2009). On the
other hand, the United States Environmental Protection Agency
has identied Pb and Sb as pollutants that may be released during
combustion processes (US Public Law, 1990).
The Cd, Cu and Zn soil concentrations appeared less inuenced
by vehicular trafc as these metals were slightly enriched in urban
and peri-urban soils. On the other hand, the vehicular origin of Cd,
Cu and Zn is well known (e.g., Adachi and Tainosho, 2004; Huang
et al., 1994). These metals are associated to the unburned fraction of
fuels as well as present in tires and other vehicle components. The
Cu accumulation in green-urban soils could be related to the use of
some Cu-bearing products in agriculture and gardening, together
14 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917
Fig. 1. Principal component analysis loading plot for the metal soil contents.
with inputs from other human sources, such as vehicular trafc and
home heating.
In the study area, Co, Cr, Ni and U contents were rather uniform
in green-urban, peri-urban and urban soils, and comparable with
the respective local geochemical background. This nding suggests
that the abundances of these metals in soil were mainly due to
mineralogical and geochemical features of the parent rock, as well
as the pedogenetic processes.
Based on the anthropogenic contribution to soil, Cd, Cu, Pb, Sb
and Zn can be likely referred as trafc-related metals, while Co, Cr,
Ni and U have to be considered geogenic elements. This characteri-
zation is also conrmed by statistical analysis. Pearsons correlation
analysis indicated that the Cd, Cu, Sb, Pb and Zn concentrations
in soil were correlated among them (Table 5), whereas there was
no correlation among the trafc-related metals and the geogenic
ones. Likewise, the principal component analysis showed that the
rst factor grouped the trafc-related metals, while the second fac-
tor consisted of the geogenic metals (Fig. 1). The PCA of the metal
contents provided three eigen values >1, and the three principle
components accounted for about 72% of the total variance (Fig. 1
reports the factor loading plot for the rst two factors after rota-
tion).
A contribution of vehicular trafc to the abundances of Cd, Cu,
Pb, Sb and Zn in urban soils was recognized in several studies
(Bretzel and Calderisi, 2006; Imperato et al., 2003; Madrid et al.,
2002; Manta et al., 2002). Differently from the results of the present
study, Cr and Ni are also considered as elements related to road
trafc being associated with fuel combustion emissions and the
particles derived by the deterioration of the vehicles structure and
components such as clutch and brakes (Manoli et al., 2002; Nerin
et al., 1999; Weckwerth, 2001).
3.5. Metal bioaccumulation in earthworms
In the study area the Cd, Cu, Pb, Sb and Zn concentrations in spec-
imens of N. caliginosus showed a distribution pattern similar to that
in soil, suggesting that soil contamination presumably inuenced
uptake of these metals by earthworms. This nding is consistent
with previous studies which demonstrated that the element body
burdens in earthworms increase with the increasing of soil contents
of metals (e.g., Morgan and Morgan, 1998; Neuhauser et al., 1995).
As in soil, concentrations of the geogenic metals (Co, Cr, Ni and U) in
earthworms were rather uniform regardless of the sampling zone.
The uptake, storage and excretion of metals by earthworms
seem to be controlled by ecological and physiological factors
(Edwards and Bohlen, 1996; Morgan and Morgan, 1999; Spurgeon
and Hopkin, 1996; Van Vliet et al., 2005). Moreover, the compli-
cated kinetics of metals in earthworms would also inuence the
mechanisms of accumulation in tissue.
Zn concentrations in N. caliginosus specimens tend to a fairly
constant level with increasing the element soil contents. This can
be explained from the fact that Zn is an essential element to many
metabolic processes and its internal level is physiologically reg-
ulated by earthworms. Homeostatic system operates to detoxify
excess of Zn and to release the element in order to meet the cells
physiological requirements.
The Cd bioaccumulation by earthworms could be attributed
both to inability of these soil-dwelling organisms to excrete this
non-essential element (Marino and Morgan, 1999), and its high
mobility and bioavailability in soil (Chlopecka et al., 1996; Li and
Thornton, 2001; Nannoni et al., 2011).
Due its essential role, also Cu body concentrations are physio-
logically regulated by earthworms (Kennette et al., 2002). However,
differently from Zn, this element showed BAF median values con-
stantly <1 reecting that Cu levels in earthworms are relatively
lower compared to the soil contents. In agreement with the results
of other studies, this evidence could be probably explained by the
fact that Cu is already toxic at low concentrations (Neuhauser et al.,
1984; Spurgeon et al., 1994).
The low Pb and Sb concentrations and BAFs in earthworms of
the study area probably reect the low mobility of these metals
in soil, where they are mainly bound to inorganic components
such as silicates and Fe-oxyhydroxides as well as organic matter.
The low BAF values indicated that earthworms do not accumulate
U. This nding agreed with the few data about the U concentra-
tions and uptake by earthworms (Di Lella et al., 2005; Oliver et al.,
2008). An accumulation of uranium was found only in earthworms
exposed to U-treated soils with concentrations of the element up
to 600 mg kg1 (Giovanetti et al., 2010).
In accordance with other Authors (Maleri et al., 2008; Sample
et al., 1999) there was no bioaccumulation of Co, Cr and Ni by earth-
worms sampled in the study area. However, data about the uptake
of these elements by earthworms are lacking and only few studies
dealt with their toxicity to these invertebrates (Lock and Janssen,
2002; Maleri et al., 2008; Manerikar et al., 2008).
3.6. Relationship between metals in earthworms and soil
properties
Principal component analysis was used to determine the inu-
ence of soil physicochemical properties on metal concentrations in
earthworms. The results of PCA (Fig. 2) indicated that the uptake
and accumulation of Cd, Cu, Pb, Sb and Zn by specimens of N. caligi-
nosus was affected by the content of organic carbon in ingested soil.
This nding supports the hypothesis that the digestion of the soil
organic matter by the enzymes of the gastrointestinal tract of earth-
worms, can release the chemical elements adsorbed by the organic
compounds. In fact, earthworms are able to extract both exchange-
able and non-exchangeable fractions sequestered by organic
compounds. On the other hand, literature reports that organic
matter is an important factor controlling the behavior of several
metals in soil (Adriano, 2001; Kabata-Pendias, 2010). Organic com-
pounds such as humic substances, may act as absorbent for metals
through sorption reactions such as ionic exchange and organic
complexation. In agreement, many studies indicated that Cu, Pb,
Sb and Zn are associated with the organic fraction in soil, forming
 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917 15

Table 5
Correlation analysis of metal contents in soils of Siena area.

Cd Co Cr Cu Ni Pb Sb U Zn

Cd 1
Co 0.139 NS
1
Cr 0.158 NS
0.755 ***
1
Cu 0.246 NS
0.179 NS
0.256 NS
1
Ni 0.003 NS
0.532 **
0.711 ***
0.236 NS
1
Pb 0.698 ***
0.377 *
0.327 NS
0.310 NS
0.199 NS
1
Sb 0.426 *
0.266 NS
0.294 NS
0.459 *
0.134 NS
0.538 **
1
U 0.054 NS
0.648 ***
0.459 *
0.071 NS
0.406 *
0.155 NS
0.043 NS
1
Zn 0.542 **
0.092 NS
0.085 NS
0.327 NS
0.065 NS
0.567 **
0.696 ***
0.276 NS
1
*
p < 0.05; ** p < 0.01; *** p < 0.001; NS = Not signicant.

by earthworms. This nding agrees with the strong capacity of car-

Fig. 2. Principal component analysis loading plot for the metal earthworm con-
centrations and soil physicochemical properties (CECe = cation exchange capacity
effective; CaCO3 = carbonate content; Corg = organic carbon content; Fe-ox, Mn-
ox = Fe and Mn contents in the reducible soil fraction).
stable complexes (Adriano, 2001; Burt et al., 2003). As a result, in
the soils of the study area the organic matter could play a relevant
role in the accumulation of contaminants. Moreover, the digestion
of organic compounds in the intestinal tract of earthworms could
be an important mechanism for increasing the bioavailable fraction
of chemical elements from ingested soil. However, further research
will be required to test this hypothesis.
PCA demonstrated that the carbonate content is another soil
property that explains the variance of Cd, Cu, Pb, Sb and Zn uptake
bonates to bind some metals. In fact, Cd, Cu, Pb and Zn may replace
Ca in the lattice of these minerals as well as form owner carbonate
phases such as cerussite (PbCO3 ), smithsonite (ZnCO3 ), malachite
[Cu2 (CO3 )(OH)2 ] and otavite (CdCO3 ; Kabata-Pendias, 2010). It fol-
lows that in soils contaminated by metals, a substantial portion
of Cd, Cu, Pb and Zn can be contained in carbonate minerals such
as calcite. The bioavailability of the carbonate-bounded metals is
due to the acid solubilization of these mineralogical phases in soil.
This weathering process mainly occurs in the rizosphere where
plant roots produce and exude organic acids reducing soil pH and
creating the conditions for carbonate destabilization. The metals
released into the soil solution through this mechanism can be
absorbed by earthworms via dermal and intestinal routes.
The effect of the cation exchange capacity on the metal concen-
trations in earthworm tissue was negligible. A similar result was
found in few studies examining the role of CEC on the metal uptake
by these soil organisms (Bradham et al., 2006).
As shown in Fig. 2, there was no correlation between metal con-
centrations in earthworms and soil pH likely due to the narrow
variability of pH values usually from 8 to 8.2. Therefore, this result
did not allow establishing the inuence of pH on metal uptake by
earthworms. On this matter Janssen et al. (1997) found a signi-
cant effect of pH on Cd, Pb and Zn contents in earthworms living
in contaminated soils. Also Peijnenburg et al. (1999) indicates that
soil pH is an important soil property affecting the metal concentra-
tions in Eisenia andrei, as well as Morgan and Morgan (1999, 1988)
highlight a dependency of Pb concentrations in earthworms from
pH, but not for Cd and Zn.
Principal component analysis showed that there were no corre-
lations of metal concentrations in earthworms with the sand, silt
and clay contents or with FeMn oxyhydroxide soil contents. This
nding suggested that these soil properties seemed unable to affect
the metal accumulation in N. caliginosus.
The linear regression analysis demonstrated the existence of
statistically signicant positive relationships between the Cd,
Pb and Sb concentrations in earthworm and their soil contents

Fig. 3. Scatter plots of Cd, Pb and Sb content in soils and earthworms (mg kg1 dw) with regression analysis components.
16 F. Nannoni et al. / Applied Soil Ecology 77 (2014) 917
(p < 0.01; Fig. 3). For the geogenic elements (Co, Cr, Ni and U) there
was no signicant correlation between the two analytical datasets,
as well as for Cu and Zn although the concentrations of the latter ele-
ments increased in earthworms collected in peri-urban and urban
zones. This lack of correlation could be due to the physiological reg-
ulation of Zn and Cu by N. caliginosus. The absence of correlation
between Co, Cr and Ni concentrations in earthworms and in soils is
in agreement with the results of similar studies (Maleri et al., 2008;
Sample et al., 1999).
4. Conclusions
This study demonstrated that vehicular trafc presumably inu-
enced the levels of Cd, Cu, Pb, Sb and Zn in soils of Siena peri-urban
and urban zones, as a result of the higher trafc densities in these
areas. Pb and Sb were the main soil contaminants and their highest
enrichments were found in urban sites where stop-and-go trafc
occurs.
The concentrations of Cd, Cu, Pb, Sb and Zn in specimens of
N. caliginosus showed a distribution pattern similar to that in soil,
suggesting that soil contamination inuenced the uptake of these
trafc-related metals by earthworms. There were signicant posi-
tive correlations between Cd, Pb and Sb earthworm concentrations
and their soil contents. The lack of correlation for Cu and Zn could
be due to their physiological regulation by these soil-dwelling
invertebrates.
The results of the present study showed that some soil proper-
ties such as organic carbon and carbonate contents are important
factors affecting the accumulation of Cd, Cu, Pb, Sb and Zn by
earthworms. This nding suggests that: (i) the ingestion of organic
compounds by earthworms could be an important pathway for
the metal uptake; (ii) carbonates can release relevant amounts of
metals in soil solution by acid solubilization mainly in rizosphere.
It follows that soil properties should be considered in studies
focused on the environmental and health risks associated with
the transfer of metals to soil organisms. However, it is undeni-
ably difcult to determine the contribution of each soil property
on metal bioavailability, as soil parameters have more than one
binding mechanism for metals and often are closely intercorrelated
among them.
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