Accumulation of heavy metals in plants grown on mineralised soils

of the Austrian Alps

W.W. Wenzel*, F. Jockwer
Institute of Soil Science, Universitat fur Bodenkultur, Gregor-Mendel-Strae 33, A-1180 Vienna, Austria
Received 15 September 1997; accepted 22 June 1998
Abstract
A eld survey of higher terrestrial plants growing on 18 metalliferous sites of the Austrian Alps was conducted to identify species
accumulating exceptional large concentrations of Cd, Cu, Ni, Pb and Zn in shoots. Minuartia verna (Caryophyllaceae) was con-
rmed and Biscutella laevigata (Brassicaceae) newly identied as hyperaccumulators of Pb (>1000 mg kg
1
Pb in shoots). Metal
concentrations in shoots exceeded those in roots. Cadmium concentrations detected in shoots of Thlaspi rotundifolium ssp. cepaei-
folium (up to 108 mg kg
1
), Cardaminopsis halleri (up to 80 mg kg
1
), Biscutella laevigata (up to 78 mg kg
1
) and Minuartia verna
(up to 59 mg kg
1
) may represent hyperaccumulation. In addition, populations of Thlaspi goesingense (up to 12400 mg kg
1
Ni)
and Thlaspi rotundifolium ssp. cepaeifoliium (up to 1934 mg kg
1
Pb) were conrmed to be hyperaccumulators. The strategy of
neighboring Thlaspi rotundifolium ssp. cepaeifolium populations to tolerate high Pb and Zn was found to switch from hyper-
accumulation at moderate and large, to exclusion at very large metal concentrations in soil. Metal partitioning in soils determined
by sequential extraction gives evidence for large proportions of potentially mobile fractions. Metals stored in shoots typically do
not exceed the soil metal pool in fractions 1 (exchangeable) and 2 (readily mobile). There is evidence that hyperaccumulation may
be associated with depressed translocation of K from roots to shoots. Under natural conditions metals accumulated in shoots are
annually recycled to the soil, hyperaccumulators do not necessarily rely on metal phases less soluble than fractions 1 and 2.
These should be considered in future development of phytoremediators, because after extraction of these phases by harvesting
shoots, further metal removal may be limited by dissolution kinetics of oxides and silicates. #1999 Elsevier Science Ltd. All rights
reserved.
1. Introduction
Transfer of potentially toxic heavy metals from soils
into shoots of higher terrestrial plants is typically low
compared to those of macronutrients (Adriano, 1986).
Therefore, those species that accumulate exceptionally
large concentrations of heavy metals in their tissues
have attracted scientists to study evolutionary, ecologi-
cal and physiological aspects involved in the process.
Baker and Brooks (1989) dened plants accumulating
>1000 mg kg
1
of Cu, Co, Cr, Ni or Pb, or >10.000
mg kg
1
of Mn or Zn as hyperaccumulator species.
Hyperaccumulation of metals has been found in tempe-
rate as well as in tropical regions throughout the plant
kingdom, but is generally restricted to endemic species
growing on mineralised soil and related rock types
(Baker and Brooks, 1989). While hyperaccumulators
have been used to identify mineralised rocks and ores,
there is more recently interest in their potential use for
decontamination of heavy metal polluted soils. In order
to develop such technologies of phytoremediation, a new
eld of research is quickly evolving (Wenzel et al., 1998).
Baker and Brooks (1989) reviewed hyperaccumu-
lators of Co (26 species), Cu (24), Mn (8), Ni (145), Pb
(4), and Zn (14). There is a relatively large number of
species hyperaccumulating Ni available for potential use
in phytoremediation, in both tropical and temperate
climatic zones. Hyperaccumulators of Cu and Co are
restricted to tropical areas, and there are only few spe-
cies with the ability to bioconcentrate Cr, Mn, Pb, Zn
or other metals (Ernst, 1974; Baker and Brooks, 1989).
Sites with anomalous high concentrations of heavy
metals in soils due to parent material, or mining and
metal processing, some dating back to prehistoric times,
are abundant in the Austrian Alps. This is evident from
locally increased levels of Pb, Zn, Cd, Ni, As, and Cu in
0269-7491/99/$see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0269-7491(98)00139-0
ENVIRONMENTAL
POLLUTION
Environmental Pollution 104 (1999) 145155
* Corresponding author. E-mail: wazi@edv1.boku.ac.at.
soils (Wenzel and Alge, 1993) and stream sediments
(Thalmann et al., 1989). Therefore, this general area has
the potential to support hyperaccumulator species for a
range of metals. Due to the abundance of mining sites,
neo-endemic processes, as proposed by Reeves and
Brooks (1983) for Thlaspi rotundifolium ssp. cepaeifo-
lium and Alyssum wulfenianum growing on mine tailings
and contaminated river gravels derived from PbZn
mines in the Cave de Predile (Raibl) area, Northern
Italy, are likely to occur. Indeed, a hyperaccumulator of
Ni, Thlaspi goesingense has been identied by Reeves
and Baker (1984). This species is endemically restricted
to few serpentinitic sites in East Austria. Sieghardt
(1987) reported on Pb and Zn concentrations in roots
and shoots of ve species grown on Pb/Zn contaminated
mining deposits in Bleiberg, South Austria, but found no
evidence for hyperaccumulation as dened by Baker and
Brooks (1989). Other information on metal accumula-
tion in plants grown on metalliferous sites in Austria is
given by Ernst (1974) who reported, Zn, Pb, Cu and Ni
concentrations in leaves of Silene cucubalus and Armeria
maritima var. serpentinii from a site in Kraubath, Styria,
did not exceed 160 mg kg
1
in dry matter.
A signicant part of the published work on metal
hyperaccumulators is based on the analysis of herbar-
ium specimens (e.g. Reeves and Baker, 1984), which
implies that soilplant relations under natural condi-
tions have received less critical scrutiny. The results
reported below pay special attention to soilplant rela-
tionships of metal hyperaccumulator species from sev-
eral metal enriched soils.
This study was specically conducted to address the
following points:
1. to conrm and identify metal accumulator species
and populations in the Austrian Alps;
2. to collect information on soil conditions, e.g. on
pH and nutrient supply, under which these hyper-
accumulator species naturally occur;
3. to investigate heavy metal speciation in these soils
and the relationship this may have to heavy metal
accumulation in the plants;
4. to evaluate the results with special reference to
their potential use in developing technologies for
phytoremediation of metal-contaminated soil in
temperate climates.
2. Materials and methods
2.1. Plant and soil sampling
Based on existing information on previous mining
activities or anomalous high levels of heavy metals in
plants or soils, we selected 18 locations (Fig. 1) having
high soil concentrations of Ni and Cr (group 1), Pb, Zn,
and Cd (group 2) and Cu (group 3). A control group
(group 4) was sampled to collect information on plant
soil relations for the same or similar plant species under
otherwise comparable ecological conditions (Table 1).
In total, 160 plant samples of 58 species, including
roots, shoots, leaves, stems, seeds, and blooms were
collected in spring and summer 1993 (Table 1). A com-
posite soil sample from the main rooting zone (typically
010/20 cm) was taken from each location.
2.2. Plant and soil analysis
Soil samples were air-dried and passed through a 2-
mm mesh screen. The screened fraction was analysed
using standard procedures (Table 2). For estimation of
total metal contents, soils were digested by aqua regia. A
Fig. 1. Map of Austria, showing locations of study sites. Full details of numbered sites are given in Table 1.
146 W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155
heavy metal fraction possibly related to phytoavailability
was extracted by 1 MNH
4
NO
3
(Table 2). Soils containing
<50 g kg
1
CaCO
3
were extracted sequentially for seven
metal fractions (Table 3) by a modied (Karczewska et
al., 1994) method of Zeien and Bru mmer (1991).
The plants were dried and ground in a metal-free mill.
The pulverised plant material was conducted to an open
vessel acid digestion using a mixture of HNO
3
, H
2
SO
4
and HClO
4
in the ratio 10:1:0.5 (Edelbauer, 1978).
Metal determinations were made using DCP-OES and
graphite furnace atomic absorption (GFAA) spectro-
photometry.
3. Results and discussion
3.1. Soils
Characteristics of the soils used in this study are
shown in Table 4. Due to serpentinitic parent material,
Table 1
Location of study sites and plant species collected
Site No. Location Species collected
High Ni/Cr sites
001 Kirchkogel near Kirchdorf a.d.Mur (Stmk); ditch 19, 49, 54
002 Kirchkogel near Kirchdorf a.d.Mur (Stmk); crest 3, 4, 15, 18, 54
003 Kraubath (Stmk); serpentine quarrel 13, 22, 49
014 Redlschlag (B); serpentine quarrel 1, 11, 24, 26, 49, 54
022 Bernstein (B); serpentine quarrel 38, 49, 54
High Zn/Pb/Cd sites
004 Bleiberg-Kreuth (K) 22, 43
005 Arnoldstein (K); Gailitz river banks 3, 9, 37, 39, 49
006 Arnoldstein (K); Gailitz river banksBBU 9, 37, 49, 55
007 Arnoldstein (K); Gailitz river banksrailway bridge 33
011 Arnoldstein (K); Bo schungswald 14
High Cu sites
019 Hochko nig (S); mining deposits 2, 12, 16, 17, 34, 35, 49, 51, 58
020 Hu ttschlag (S); Schwarzwand 29, 31, 40, 44, 45, 57
021 Mu hlbach (S); mining deposits 5, 8, 21, 25, 47, 48, 49, 50, 56
Low metal sites
008 Weibriach (K); Go sseringgraben 6, 9, 30, 41
013 Hochobir (K); Bo schung 53
016 Hochobir (K) 9, 23, 25, 27, 46, 49, 50
017 Hochkail (S); top of mountain 28, 36, 42, 49, 52, 57
018 Hochkail (S); river banks 7, 10, 20, 32
1. Achillea nobilis, 2. Adenostyles alliariae, 3. Alyssum montanum, 4. Alyssum transilvaticum, 5. Arabis cf. hirsuta, 6. Arabis pumila, 7. Athyrium lix-
femina, 8. Betula pendula, 9. Biscutella laevigata, 10. Blechnum spicant, 11. Calamagrostis spec., 12. Campanula barbata, 13. Cardaminopsis arenosa,
14. Cardaminopsis halleri, 15. Cerastium arvense, 16. Dactylis glomerata, 17. Deschampsia cespitosa, 18. Dianthus carthusianorum, 19. Silene dioica,
20. Dryopteris dilatata, 21. Equisetum arvense, 22. Erysimum crepidifolium, 23. Erysimum spec., 24. Euphorbia cyparissias, 25. Galium spec.,
26. Genista pilosa, 27. Gymnocarpium robertianum, 28. Hieracium glaucum, 29. Juncus tridus, 30. Kernera saxatilis, 31. Larix decidua, 32. Lastrea
limbosperma, 33. Lepidium campestre, 34. Leucanthemum vulgare, 35. Lychnis us-cuculi, 36. Melampyrum pratense, 37. Minuartia verna, 38. Pinus
sylvestris, 39. Plantago lanceolata, 40. Poa spec., 41. Polygala amarella, 42. Potentilla erecta, 43. Reseda lutea, 44. Rhododendron ferrugineum,
45. Saxifraga stellaris, 46. Scrophularia canina, 47. Sedum spurium, 48. Sedum telephium, 49. Silene cucubalus, 50. Silene nutans, 51. Silene rupestris,
52. Stellaria palustris, 53. Thlaspi arvense, 54. Thlaspi goesingense, 55. Thlaspi rotundifolium ssp. cepaeifolium, 56. Thymus praecox, 57. Vaccinium
myrtillus, 58. Vaccinium vitis-idea.
Table 2
Methods of soil solid phase analysis (SSR=solution:soil ratio, cm
3
g
1
; ET=extraction time, h)
Property Method description Analytical device Reference
pH 0.01 M CaCl
2
, SSR 2.5:1, ET 2 h ORION Combined Electrode Blum et al. (1989)
CaCO
3
dissolution by 10% HCl Scheibler Apparatus Blum et al. (1989)
Total C and N dry combustion CNS Total Analyzer Blum et al. (1989)
OC total Ccarbonate C calculated Blum et al. (1989)
Exchangeable cations 0.1 M BaCl
2
at soil pH, SSR 20:1, ET 2 h DCP-OES Blum et al. (1989)
CEC at soil pH sum of exchangeable Ca, Mg, K, Na calculated Blum et al. (1989)
Total metals and P aqua regia DCP-OES, GF-AAS Blum et al. (1989)
Extractable metals 1 M NH
4
NO
3
, SSR 2.5:1, ET 2 h GF-AAS Pru e (1994)
W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155 147
the rst group of soils is exceptionally high in total (T)
Ni and Cr. High levels in the parent material and long-
term deposition of Zn, Cd and Pb from smelter activ-
ities lead to anomalous large concentrations of these
metals in soil group 2. The soils of group 3 contain large
amounts of Cu due to historical mining and smelter
activities. The soils of control group 4 display no
exceptional high metal concentrations (Table 5).
According to FAO (1988), the soils are Eutric and
Rendzic Leptosols, and Eutric and Calcaric Regosols
(Table 4). Soil pH ranges from slightly acidic to alka-
line. Soils of sites 4 and 7 contain CaCO
3
equivalents of
about 200 mg kg
1
, at sites 5 and 6 about 760 mg kg
1
.
Some soils (2, 15, 4, 10) are high in organic carbon
(OC), its accumulation in most other soils is low,
pointing to an early stage of development. Cation
exchange capacity is merely a function of OC; base
saturation is, consistent with pH, close to 100%.
Except for the Ni/Cr sites, Ca dominates the exchange
complex. The exceptionally high Mg saturation of group
1 soils on serpentinite is related to total Mg concentra-
tions of between 110 and 210 g kg
1
. This results in the
ratio of exchangeable Mg to Ca that ranges between
1.49 and 7.66, compared to values <0.4 in other soils.
In similar Swiss soils, Mg accounted for more than 50%
of the positive charge of soil solutions (Gasser et al.,
1994).
In that soil group within which they are present in
high concentrations, NH
4
NO
3
-extractable fractions of
Cd, Cu and Pb increase as pH
CaCl
2
decreases (Table 5).
Table 3
Sequential extraction of metals according to Zeien and Bru mmer (1991)
Binding form extracted tentatively
a
Extractant
Exchangeable 1 M NH
4
NO
3
Specically adsorbed/surface occluded 1 M NH
4
OAc, pH 6.0
Occluded in manganese oxides 1 M NH
2
OH-HCl+1 M NH
4
OAc, pH 6.0
Organically bound fraction 0.025 M NH
4
EDTA, pH 4.6
Occluded in amorphous iron oxides 0.2 M NH
4
-oxalate buer, pH 3.25
Occluded in crystalline iron oxides 0.2 M NH
4
-oxalate buer+0.1 M ascorbic acid, pH 3.25
Residual (bound in silicates) aqua regia
b
a
The fractions are operationally dened by the extractant; there is evidence that the indicated binding forms are extracted primarily, but selec-
tivity can not be assumed (Wenzel and Blum, 1997).
b
Aqua regia was used instead of a total dissolution procedure (e.g. HF/HClO
4
) as initially proposed by Zeien and Bru mmer (1991), compare
Karczewska et al. (1994).
Table 4
Characteristics of the study soils
Site No. FAO soil group pH
CaCl
2
CaCO
3
OC C:N CEC BS K
ex
Mg
ex
Mg
ex
:Ca
ex
(g kg
1
) (g kg
1
) (mmol
(+)
kg
1
) (%) (mmol
(+)
kg
1
) (mmol
(+)
kg
1
)
High Ni/Cr sites (Group 1)
001 Eutric Regosol 6.53 1 85 15 339 100 3.3 291 6.66
002 Eutric Regosol 5.72 1 117 12 482 10 3.3 286 1.49
003 Eutric Leptosol 7.76 22 13 13 148 99 0.8 104 2.51
014 Eutric Leptosol 6.55 19 13 16 208 100 4.1 180 7.66
022 Eutric Leptosol 6.53 4 19 16 196 100 2.2 168 6.82
High Zn/Pb/Cd sites (Group 2)
004 Calcaric Regosol n.d. 202 224 21 1120 100 3.7 31 0.03
005 Rendzic Leptosol 7.05 760 10 20 55 98 0.8 3 0.06
006 Rendzic Leptosol 6.80 761 9 15 40 98 0.6 3 0.09
007 Calcaric Regosol 7.36 174 12 13 184 100 3.4 13 0.08
011 Eutric Regosol 6.17 3 19 5 243 98 3.2 35 0.18
High Cu sites (Group 3)
019 Eutric Leptosol 5.53 14 13 19 46 97 1.6 7 0.20
020 Eutric Leptosol 4.83 2 8 27 22 77 0.2 3 0.24
021 Eutric Leptosol 6.84 20 44 20 146 98 2.7 21 0.17
Low metal sites (Group 4)
013 Eutric Regosol n.d. <1 44 14 156 99 18.1 34 0.33
016 Calcaric Regoso 7.27 65 57 14 398 100 2.6 17 0.05
017 Eutric Leptosol 5.24 <1 13 16 44 96 1.4 11 0.38
018 Eutric Leptosol 6.12 <1 10 14 61 86 0.9 12 0.31
n.d.=Not determined.
148 W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155
For Ni and Zn, total metal concentrations and pH
equally seem to aect NH
4
NO
3
extractability.
Pru e (1994) proposed limits of NH
4
NO
3
-extractable
metal levels in soil, above which the probability of phy-
totoxicity and adverse aects on foodchain or ground-
water is high. These limits are 0.02 Cd, 2.5 Cu, 1.0 Ni,
0.3 Pb and 5.0 Zn (mg kg
1
); above these concentra-
tions, assessment of risk and remedial needs is required.
A comparison with Table 5 shows that these limits are
exceeded in most metal contaminated soils, but not in
the control group of this study.
Fig. 2 shows the partitioning of metals between
sequentially extracted fractions (compare Table 3). In
Ni-contaminated soils (G1), more than 50% of this
metal is bonded to the residual (silicatic) fraction. Most
of the remaining Ni was extracted by oxalate (oxidic)
and Na
2
EDTA (organic), but only small relative pro-
portions were found in the most labile fractions (13).
The latter are thought to be related to exchangeable,
specically-bound and Mn-oxide phases (Zeien and
Bru mmer, 1991). Copper in cupriferous soils (G3) is
more evenly distributed among phases, but fractions 1,
3 and 7 contribute less than other phases. Distribution
of Cd and Zn in a low-carbonate soil of group 2 indi-
cates a high potential mobility of these metals. More
than 30% of Zn and >50% of Cd are dissolved by
extractants 1 to 3; 5 to 10% are easily exchangeable by
1 M NH
4
NO
3
. Compared to Zn, the fractionation of Cd
is characterised by a shift towards the mobile fractions.
While exchangeable Pb in the same soils is low, the sum
of fractions 13 contributes >50%. Due to the stability
of Pb-organic complexes, >30% of Pb are bound to
organic matter (fraction 4, Na
2
EDTA). The least mobile
fractions 57 account for less than 10% of total Pb.
Fig. 2. Partitioning of Ni in soils of group 1 (G1; mean of soils 1, 2, 3,
14, 22), of Cd, Pb and Zn in a soil of group 2 (G2; soil 11), and of Cu
in soils of group 3 (G3; mean of soils 19, 20, 21). Metals were extracted
sequentially according to Zeien and Bru mmer (1991).
Table 5
Total (T) by aqua regia and 1 M NH
4
NO
3
-extractable (E) heavy metals in the study soils
Site No. Cd Cu Co Cr Ni Pb Zn
T E T E T E T E T E T E T E
mg kg
1
High Ni/Cr sites
001 3.75 <0.13 15 0.06 96 <0.13 991 0.07 2380 <0.05 43 <0.25 107 0.25
002 5.80 <0.13 23 0.07 155 <0.13 1080 0.15 1900 2.55 282 <0.25 202 0.85
003 3.69 <0.13 37 0.08 114 <0.13 276 0.09 2460 0.37 7 <0.25 48 <0.15
014 3.70 <0.13 52 0.50 172 <0.13 1910 <0.05 2580 5.81 16 <0.25 61 <0.15
022 3.54 <0.13 57 0.01 196 <0.13 1570 0.06 2390 3.94 <5 <0.25 70 <0.15
High Zn/Pb/Cd sites
004 21.30 <0.13 19 0.19 9 0.34 39 0.53 21 0.26 4370 <0.25 3860 1.8
005 24.60 0.18 61 0.32 10 <0.13 25 0.09 19 0.11 2500 0.47 13 400 63.5
006 43.70 1.41 32 0.40 10 <0.13 17 0.10 16 0.19 4010 2.19 15 000 217
007 7.43 <0.13 136 0.12 52 <0.13 30 0.19 78 0.10 262 <0.25 1040 0.29
011 25.97 1.24 169 0.25 14 <0.13 25 0.18 39 0.22 2340 2.42 3190 83.2
High Cu sites
019 3.51 <0.13 8340 33.2 33 <0.13 8 <0.05 140 1.66 28 <0.25 100 0.46
020 5.57 <0.13 3260 42.6 29 0.67 160 <0.05 61 0.23 <5 <0.25 368 6.89
021 0.17 <0.13 5970 8.55 38 <0.13 28 0.10 179 0.47 67 <0.25 326 0.59
Low metal sites
008 3.29 <0.13 4 <0.05 9 <0.13 14 0.11 9 <0.05 12 <0.25 28 0.44
013 3.39 <0.13 77 0.02 43 <0.13 36 0.06 48 0.08 6 <0.25 121 <0.15
016 0.03 <0.13 15 0.07 13 <0.13 29 0.21 20 0.15 33 <0.25 63 <0.15
017 0.04 <0.13 90 <0.05 12 <0.13 20 <0.05 35 0.12 <5 <0.25 119 4.30
018 0.03 <0.13 17 <0.05 21 <0.13 19 <0.05 39 0.08 4 <0.25 15 <0.15
W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155 149
This untypically high potential mobility of Pb is due to
deposition from smelting activities in this location
(Karczewska et al., 1994).
3.2. Metal accumulation in plants
3.2.1. Nickel
Reeves and Baker (1984) identied hyperaccumula-
tion of Ni in herbarium specimens of Thlaspi goe-
singense from two serpentinitic sites in Austria. Ni
concentrations in leaves of T. goesingense ranged
between 6380 and 9010 mg kg
1
dry matter (location
Bruck an der Mur, Styria), and between 4960 and
12 400 mg kg
1
(Bernstein, Burgenland). We resampled
these sites and extended our investigations to other ser-
pentinitic soils in East Austria (Table 1). The six best-
performing specimens were collected from site 14
(Table 6), serpentine quarrel in Burgenland, the highest
concentration was 12 396 mg kg
1
in leaves, almost
equal to that previously found by Reeves and Baker
(1984). Though less pronounced, hyperaccumulation of
Ni in T. goesingense was reconrmed for site 22 (Bern-
stein), and for two of the sites in Styria near Bruck an
der Mur (Nos. 1 and 2). No other species included in
this study was found to accumulate >1000 mg kg
1
Ni
in dry matter. Other species grown on group 1 soils
displaying relatively high concentrations of Ni in shoots
were Achillea nobilis (160 mg kg
1
, site 14), Erysimum
crepidifolium (136 mg kg
1
, site 3), Euphorbia cypar-
issias (121, 131 mg kg
1
, site 14), Genista pilosa (87 mg
kg
1
, site 14), and Silene cucubalus (64 mg kg
1
, site 3).
Equisetum arvense from cupriferous soil at site 21 had
51 mg kg
1
in its shoots. Typically, Ni concentrations in
roots of these species were higher than in shoots,
whereas the hyperaccumulator Thlaspi goesingense
showed the reverse.
An elevated Ni concentration was detected in shoots of
one specimen of Thlaspi rotundifoliumsubsp. cepaeifolium
grown on site 6 (172 mg kg
1
). The soil of this Cd/
Pb/Zn site contains only 16 mg kg
1
total and 0.19 mg
kg
1
NH
4
NO
3
extractable Ni. This may be evidence for
a non-specic metal tolerance mechanism in this species.
3.2.2. Cadmium, lead and zinc
Brown et al. (1994) reported Cd concentrations in
Thlaspi caerulescens shoots of up to 1800 mg kg
1
dry
matter when grown for 5 weeks on a soil containing
1020 mg kg
1
Cd. Baker et al. (1994) found mean Cd
concentrations of 164 mg kg
1
in T. caerulescens popu-
lations that occurred naturally on mine wastes at sites in
the Southern Pennines, UK. Other data on plants accu-
mulating Cd under natural conditions are virtually not
available. As a result, Cd was not included in the review
of metal hyperaccumulators by Baker and Brooks
(1989), and a lower limit for hyperaccumuation yet has
not yet been dened.
The bulk of the samples analysed in this study had
<2 mg kg
1
Cd in dry matter, a few specimens
accumulated up to 10 mg kg
1
Cd. A clearly dier-
entiated group of 15 specimens from sites 6 and 11
(group 2Cd/Zn/Pb soils) accumulated between 30 and
110 mg kg
1
Cd (Table 7). In these specimens, Cd con-
centrations were typically higher in shoots relative to
roots. The ability to bioconcentrate Cd in shoots
decreased in the order: Thlaspi rotundifolium ssp.
cepaeifolium > Cardaminopsis halleri > Biscutella lae-
vigata > Minuartia verna. The soils at these sites display
about sevenfold larger Cd pools extracted by 1 M
NH
4
NO
3
than any other soil in this study (Table 5),
indicating that large concentrations of Cd in plant
Table 7
Cd accumulation in best-performing specimens
Plant No. Species Plant part Site No. Cd (mg kg
1
)
182 Thl.rot shoots 6 108
143 Thl.rot shoots 6 101
029 Thl.rot shoots 6 101
027 Thl.rot leaves 6 89.6
183 Car.hal shoots 11 80.3
030 Bis.lae shoots 6 78.3
146 Car.hal shoots 11 76.1
162A Car.hal shoots 11 72.6
162N Car.hal shoots 11 70.3
028 Thl.rot stem 6 61.6
035 Min.ver shoots 6 59.0
026 Thl.rot roots 6 53.9
031 Bis.lae roots 6 39.9
163N Min.ver shoots 6 34.7
163A Min.ver shoots 6 32.6
Table 6
Nickel accumulation in best-performing specimens
Plant No. Species Plant part Site No. Ni (mg kg
1
)
005 Thl.goe shoots 14 12 400
012 Thl.goe shoots 14 10 500
172 Thl.goe shoots 14 9310
009 Thl.goe shoots 14 9070
008 Thl.goe shoots 14 8800
082 Thl.goe shoots 14 6860
140 Thl.goe shoots 22 5190
175 Thl.goe shoots 22 5080
062 Thl.goe shoots 1 4110
006 Thl.goe owering shoots 14 4020
174 Thl.goe shoots 2 3760
004 Thl.goe shoots 14 3720
147 Thl.goe shoots 2 3490
010 Thl.goe owering shoots 14 2730
084 Thl.goe roots 14 2690
011 Thl.goe owering shoots 14 2680
013 Thl.goe owering shoots 14 2560
060 Thl.goe shoots 2 2230
139 Thl.goe shoots 1 2160
173 Thl.goe roots 14 1870
176 Thl.goe shoots 1 1860
063 Thl.goe roots 1 1440
150 W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155
tissues may be restricted to soils that contain consider-
able amounts of easily extractable Cd. Based on the
data presented here, and on ndings of Baker et al.
(1994), it seems reasonable to classify plants having
>50 mg kg
1
Cd in shoots as Cd hyperaccumulators.
This would apply to specimens of Thlaspi rotundifolium
ssp. cepaeifolium, Cardaminopsis halleri and Biscutella
laevigata. In any case, the soilplant transfer coecients
are >1 (up to 3) and Cd accumulation in shoots exceeds
that of roots. A limit of 50 mg kg
1
for Cd is conrmed
by Cd/Zn and Cd/Pb ratios in the group 2 soils similar
to ratios between the respective limits of hyper-
accumulation, and suggested by the clear separation
from the bulk data.
Plants accumulating >10 000 mg kg
1
Zn in shoots
have been dened as hyperaccumulators (Baker and
Brooks, 1989). According to these authors, 14 hyper-
accumulator species of Zn have been identied, among
them 9 species of Thlaspi (Brassicaceae).
The best-performing specimens from this investiga-
tion are listed in Table 8. Specimens of Thlaspi rot-
undifolium ssp. cepaeifolium, Cardaminopsis halleri
(Brassicaceae) and Minuartia verna (Caryophyllaceae)
growing at sites 6 and 11 (group 2 soils) had Zn con-
centrations in shoots approaching the limit of hyper-
accumulation. Cardaminopsis halleri (L.) Hayek had
been previously identied as a Zn hyperaccumulator
before (max. conc. 13 600 mg kg
1
at a site in Ger-
many). Similarly, Thlaspi rotundifolium ssp. cepaeifolium
was found to accumulate up to 21 000 mg kg
1
Zn
(Baker and Brooks, 1989). Reeves and Brooks (1983)
found Zn levels up to 17 300 mg kg
1
in leaves of
Thlaspi rotundifolium ssp. cepaeifolium in the vicinity of
a PbZn mine in the Cave del Predile (Raibl) area,
Northern Italy. Site 6 of the present study is located
downstream of this area on the same river. Therefore,
we assume that the two populations of Thlaspi are
linked genetically due to transport along the river
Silizza (Schlitza).
Zn concentrations around 5500 mg kg
1
in shoots of
Minuartia verna at site 6 (Table 8) are comparable to
those reported by Sieghardt (1987) for specimens col-
lected at site 4. For both locations, Zn concentrations in
shoots are higher than in roots, indicating that, despite
<10 000 mg kg
1
Zn in shoots, this species reacts like a
hyperaccumulator.
Reeves and Brooks (1983) reported only four hyper-
accumulator species of Pb in their review. Among them,
the highest Pb concentrations (8200 mg kg
1
) were
found in shoots of Thlaspi rotundifoliumspp. cepaeifolium
in Cave del Predile, Northern Italy (Reeves and Brooks,
1983). We found 1934 mg kg
1
Pb in shoots of the same
species grown at site 6. As mentioned, this site is
connected to Cave del Predile by the river Silitza. As
for Zn, the accumulation by the Thlaspi population
at site 6 is less pronounced than that reported for
Cave del Predile.
Table 9 lists two other hyperaccumulators of Pb that
have not been reported by Baker and Brooks (1989).
Biscutella laevigata, like Thlaspi belongs to the family
Brassicaceae. Minuartia verna (Caryophyllaceae) is the
best-performing species of this study. Johnston and
Proctor (1977, cited in Barry and Clark, 1978) reported
up to 1580 mg kg
1
Pb in Minuartia shoots grown at a
metalliferous site in Trelogan, Wales. Barry and Clark
(1978) found Pb concentrations even up to 20000 mg
kg
1
in shoots of this species grown on a metal-mining
complex in the Yorkshire Pennines, England. Total Pb
concentrations in soils of Barry and Clark's study were
up to 41 250 mg kg
1
. Conversely, Sieghardt (1987)
reported that Minuartia from mining deposits of
Bleiberg-Kreuth, Austria (site 4 of this study) accumu-
lated only 814 mg kg
1
Pb in shoots, and Pb con-
centrations in roots were greater than in shoots. Despite
the proximity of sites 4 and 6 (Fig. 1) it seems that two
dierent populations have evolved. The soil of
Sieghardt's study contained between 16 700 and 24 700
mg kg
1
total Pb, and between 5885 and 10 140 mg kg
1
1 M NH
4
OAc (pH 7)-extractable Pb in the <2 mm
fraction. The soil of site 6 in this study contains only
4010 mg kg
1
total and 2.19 mg kg
1
1 M NH
4
NO
3
extractable Pb (Table 4). This points to a clear gen-
etic dierentiation between the two populations of
Minuartia; metal tolerance at site 6 is related to uptake
and transfer to shoots, while at site 4, partial metal
Table 9
Pb accumulation in best-performing specimens
Plant No. Species Plant part Site No. Pb (mg kg
1
)
035 Min.ver shoots 6 2180
143 Thl.rot shoots 6 1930
029 Thl.rot shoots 6 1660
163N Min.ver shoots 6 1430
163A Min.ver shoots 6 1290
030 Bis.lae shoots 6 1090
Table 8
Zn accumulation in best-performing specimens
Plant No. Species Plant part Site No. Zn (mg kg
1
)
029 Thl.rot shoots 6 9080
027 Thl.rot leaves 6 8860
146 Car.hal shoots 11 8570
183 Car.hal shoots 11 8220
035 Min.ver shoots 6 7700
028 Thl.rot stem 6 6440
143 Thl.rot shoots 6 5980
182 Thl.rot shoots 6 5840
162A Car.hal shoots 11 5750
163N Min.ver shoots 6 5690
163A Min.ver shoots 6 5550
162N Car.hal shoots 11 5320
030 Bis.lae shoots 6 4870
W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155 151
exclusion may be involved in tolerating the excep-
tionally high amounts of available Pb in soil. This
gives evidence that populations of the same species
may use dierent strategies to adapt to specic soil
environments.
3.2.3. Copper
We found only one report on hyperaccumulation of
Cu (>1000 mg kg
1
dry matter in shoots) in plants
grown naturally in temperate regions: Ernst (1974)
found 1074 mg kg
1
Cu in leaves of Minuartia verna at
a site in Ko nnern, Germany.
We collected a variety of species from three sites
(group 3, Table 1) in Salzburg (Fig. 1). Although the
limit of hyperaccumulation was never exceeded, we
found considerably high Cu concentrations (722 mg
kg
1
) in a specimen of Silene cucubalus (Table 10),
exceeding the concentration in its roots (654 mg kg
1
).
Ernst (1974) found up to 374 mg kg
1
Cu in leaves of
this species grown in Ko nnern, Germany. Other species
accumulating >100 mg kg
1
Cu typically had higher
Cu concentrations in roots (Table 10). Copper con-
centrations of up to 115 mg kg
1
in shoots of Equisetum
arvense clearly exceeded 1.3 mg kg
1
reported by Ernst
(1974) for a site in Clausthal, Germany.
3.3. Evidence for depressed K translocation to shoots
Accumulation and tolerance of exceptional high
levels of heavy metals in shoots is possibly related to
other physiological processes. Therefore, we explored
our data for possible relations between uptake and
translocation of heavy metals and the macronutrients
Ca, Mg, K and P. Translocation of Ca, Mg and P from
roots to shoots was apparently not aected by
accumulation of potentially toxic metals in shoots. Fig.
3 shows that K translocation from roots to shoots
seemed to be depressed in hyperaccumulators of Ni
(Thlaspi goesingense) compared to a control. Similar
results were obtained for the Pb/Zn accumulators
Thlaspi rotundifolium ssp. cepaeifolium and Biscutella
laevigata. Using raster scans across a leaf of Hausman-
niastrum robertii, Morrison et al. (1981) found reduced
K concentrations in regions containing elevated Co and
Mn. Ernst (1974) found less K in chlorotic leaves of
Agrostis tenuis grown on Zn and Pb contaminated soils
compared to a non-chlorotic control. In a pot experi-
ment, lower concentrations of K and P in Thlaspi goe-
singense shoots occured at high levels of added Zn in
soil, while Ca was not aected (Reeves and Baker, 1984).
In summary, there is evidence that K may be related to
compartmentation of toxic metals in hyperaccumulator
species, but the mechanisms are not yet characterised.
3.4. Heavy metal extraction by hyperaccumulators in
phytoremediation and in natural systems
Some authors have calculated the potential eective-
ness of hyperaccumulator species by linear extrapola-
tion of the metal removal rate from soil obtained in
short-term experiments (Baker et al., 1994; Brown et al.,
1994). This implies a constant rate of uptake over the
whole period of phytoremediation. This approach does
not account for the possibility of changes in metal par-
titioning among soil solid phase fractions, and for rela-
ted changes in solubility and availability to the plant.
Typically, uptake of metals occurs from soil solution
and particle surfcaces (Sposito, 1989), but uptake from
Table 10
Cu accumulation in best-performing specimens
Plant No. Species Plant part Site No. Cu (mg kg
1
)]
133 Equ.arv roots 21 782
170 Sil.cuc shoots 19 722
101 Sil.rup roots 19 698
166 Sil.cuc roots 19 654
108 Des.ces roots 19 632
097 Sil.cuc roots 19 595
119 Sax.ste shoots 20 300
111 Poa spec roots 20 288
168 Sil.cuc roots 21 287
109 Des.ces shoots 19 269
100 Sil.rup shoots 19 222
132 Sil.cuc roots 21 207
096 Sil.cuc shoots 19 206
131 Sil.nut roots 21 155
104 Ade.all roots 19 155
110 Sax.ste stem and owers 20 129
118 Sax.ste shoots 20 119
128 Equ.arv shoots 21 116
102 Ade.all shoots 19 105
Fig. 3. Boxplot of shoot:root ratios of K in Thlaspi goesingense
hyperaccumulating Ni (Acc., n=5) and in non-accumulating species
(Non-acc., n=22).
152 W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155
less soluble phases, e.g. silicates, metal oxides and car-
bonates is probably kinetically limited. Repeated crop-
ping of hyperaccumulators on metal contaminated soils
may diminish the readily mobile phases if the rate of
uptake exceeds that of metal dissolution from less
mobile compounds.
Calculated uptake rates of Pb and Zn by the best
performing individuals of Thlaspi rotundifolium ssp.
cepaeifolium (Table 7) and of Ni by Thlaspi goesingense
(Table 6) within a growing season (assumed to be 10
weeks) are in the range between 10
6
and 10
7
mol
plant
1
h
1
. Depending on crystallinity, specic surface,
pH and presence of inorganic or organic ligands, dis-
solution rates of Al and Fe hydroxides and oxides
(Sparks, 1989) calculated for the rooting zone of an
individual plant (assuming an intensively rooted soil
mass of about 5 kg plant
1
and a surface area of 2 m
2
g
1
oxide) may be orders of magnitude above or below
this plant extraction rate. Based on the molar ratios of
<0.25 (typically <0.05) between the metals of concern
(e.g. Zn, Pb, Ni) and Fe found in the oxidic fractions
(fractions 5+6) of the metalliferous soils of this study
(Fig. 2), the actual rate of release of these metals from
Fe oxides is possibly less than for Fe. This calculation
though preliminaryshows that the eectiveness of
phytoremediation may decrease with time if the metal
dissolution rate in soil drops below the potential
extraction rate by the plant. Since the extraction rates
by hyperaccumulators are sandwiched by the metal dis-
solution rates from oxides, managing soil conditions,
e.g. pH, redox potential and ligand concentrations
seems to be promising.
In contrast to the situation with phytoremediation,
metals extracted by hyperaccumulators from soil under
natural conditions are recycled. Based on the pH of the
study soils (Table 4) it seems reasonable to assume that
leaching of metals below the rooting zone is low. There-
fore, we expect only minor changes in the readily avail-
able metal pool with time. A comparison of metal
partitioning among seven sequentially extracted soil
fractions and the metals accumulated in the best per-
forming individuals of Thlaspi rotundifolium ssp. cepaei-
folium (Cd, Pb, Zn) and Thlaspi goesingense (Ni) is
shown in Fig. 4. The amount of Cd, Pb and Zn accu-
mulated in Thlaspi shoots is less or similar to the amount
of exchangeable metals in fraction 1, that of Ni is almost
equal to the sum of fractions 1 and 2 (exchangeable +
specically sorbed/surface occluded Ni). This indicates
that uptake of these metals by hyperaccumulator speci-
men of Thlaspi under natural conditions is based on
metal fractions that are considered available for non-
accumulating species as well. This underlines the con-
clusions from the previous paragraphs.
4. Conclusions
The ability of Thlaspi goesingense to hyperaccumulate
Ni up to 12 400 mg kg
1
has been conrmed. Further,
hyperaccumulation of Pb by Minuartia verna and T.
Fig. 4. Metal pools in sequentially-extracted soil fractions of the rooting zone of one plant compared to storage in shoots of Thlaspi rotundifolium
ssp. cepaeifolium (Pb, Zn, Cd) and T. goesingense (Ni). Calculation of metal pools in soil are based on a bulk density of 1300 kg m
3
, a rooting zone
extending to 20 cm depth and 187 cm (area covered by a Thlaspi of 20 g dry matter), and on Pb, Zn and Cd fractions in soil 6 (T. rotundifolium ssp.
cepaeifolium), and on Ni fractions in soil 11 (T. goesingense). The metal pool accumulated in Thlaspi shoots was calculated assuming 20 g dry matter
and concentrations in shoots of the best performing individuals of Thlaspi (Ni 12396, Zn 8855, Cd 108.2, and Pb 1934 mg kg
1
d.m., compare
Tables 6 and 7).
W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155 153
rotundifolium ssp. cepaeifolium was revealed. The rst
report of the ability of Biscutella laevigata to hyper-
accumulate lead has been made. Based on the current
data, it is proposed that Cd concentrations >50 mg
kg
1
should qualify plants as hyperaccumulator species.
Four species, Thlaspi rotundifolium ssp. cepaeifolium,
Cardaminopsis halleri, Biscutella laevigata and Minuartia
verna meet this criterion. Comparable data on Cd accu-
mulation are scarce in the literature.
The soil conditions under which hyperaccumulators
naturally grow were also studied. Soils pH ranges from
slightly acidic to alkaline, some contain considerable
amounts of carbonate. Despite such conditions favour-
ing immobilisation of metal cations, easily-extractable
(potentially mobile) metal fractions are high in these
soils. The easily extractable metal pool is large enough
to explain the amount of metals stored in shoots of
hyperaccumulators. In the potential use of such plants
in phytoremediation, one must consider the possibility
of a decreasing metal extraction rate after removal of
the readily available fractions. Preliminary calculations
indicate that the dissolution rate of less mobile metal
fractions in soil does not necessarily keep step with the
removal by plants found in natural systems.
Assuming that hyperaccumulator species under nat-
ural conditions take up metals only to an extent that
does not exceed the pool of metals in the soil which is
easily accessible for plant roots the concept of hyper-
accumulation may require considerable redenition.
This plant-available metal pool in the rhizosphere of
hyperaccumulator species is likely to be sustained by
recycling of metals via senescence of the shoot system. In
addition to genetic variation, dierential uptake of
metals by specimens of the same species may therefore
be related to the amount of readily available metals in
the soil. If this is the case, the bioconcentration of metals
in specimens that grow on soils with a small available
metal pool may not qualify them as hyperaccumulators
according to the numerical denition of Baker and
Brooks (1989). If the same specimens would be grown
on soils with a large available metal pool, metal con-
centrations in their tissues may exceed the numerical
limits of hyperaccumulation. If hyperaccumulation is
based upon the removal of the bulk of labile metal spe-
cies from a soil, followed by the recycling of this pool
via the annual process of senescence, then a number of
species may possess this ability and are not classied as
hyperaccumulators because edaphic conditions preclude
the ability of the plants to exceed the numerical limit set
for the denition of a hyperaccumulator species.
Acknowledgements
This study was partly funded by the Hochschul-
jubila umsfonds der Stadt Wien, and by an award given
to the senior author (Bodenkulturpreis der Wiener
Handelskammer). We are grateful to Dipl.Ing. Herbert
Sattler for his support in plant and soil sampling, and to
Ing. Ewald Brauner for performing DCP-OES meas-
urements.
References
Adriano, D.C., 1986. Trace Elements in the Terrestrial Environment.
Springer-Verlag, New York, Berlin, Heidelberg, Tokyo.
Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which
accumulate metallic elementsa review of their distribution, ecol-
ogy and phytochemistry. Biorecovery 1, 81126.
Barry, S.A.S., Clark, S.C., 1978. Problems of interpreting the rela-
tionship between the amounts of lead and zinc in plants and soil on
metalliferous wastes. New Phytologist 81, 773783.
Baker, A.J.M., Reeves, R.D., Hajar, A.S.M., 1994. Heavy metal
accumulation and tolerance in British populations of the metallo-
phyte Thlaspi caerulescens J. & C. Presl (Brassicaceae). New Phy-
tologist 127, 6168.
Blum, W.E.H., Spiegel, H., Wenzel, W.W., 1989. Bodenzustands-
inventur. Konzeption, Durchfu hrung und Bewertung. Bundes-
ministerium fu r Land- und Forstwirtschaft, Wien.
Brown, S.L., Chaney, R.L., Angle, J.S., Baker, A.J.M., 1994. Phyto-
remediation potential of Thlaspi caerulescens and bladder campion
for zinc- and cadmium-contaminated soil. Journal of Environmental
Quality 23, 11511157.
Edelbauer, A., 1978. Traubenertrag, Mineralstogehalt von Bla ttern
und 1-ja hrigen Trieben sowie Frostanfa lligkeit der Knospen von
Vitis vinifera L. bei verschiedenen Cl

/SO
4
2
-Relationen. Z. Pan-
zenernaehr. Bodenk 141, 8394.
Ernst, W., 1974. Schwermetallvegetation der Erde. Gustav Fischer
Verlag, Stuttgart.
FAO, 1988. FAO/Unesco soil map of the world. Revised legend.
World Resources Report 60, FAO, Rome.
Gasser, U.G., Juchler, S.J., Sticher, H., 1994. Chemistry and specia-
tion of soil water from serpentinitic soils: importance of colloids in
the transport of Cr, Fe, Mg, and Zn. Soil Science 158, 314322.
Karczewska, A., Mavrodieva, R., Wenzel, W.W., 1994. The eect of
metal sources and indigenous soil pH on metal fractions in soil.
Journal of Environmental Geochemistry and Health 16, 487504.
Morrison, R.S., Brooks, R.R., Reeves, R.D., Malaisse, F., Horowitz,
P., Aronson, M., Merriam, G., 1981. The diverse chemical forms of
heavy metals in tissue extracts of some metallophytes from Shaba
Province, Zaire. Phytochemistry 20, 455458.
Pru e, A., 1994. Einstufung mobiler Spurenelemente in Bo den. In:
Rosenkranz, D., Einsele, G., Harress, M., (Eds.), Erga nzbares
Handbuch der Manahmen und Empfehlungen fu r Schutz, Pege
und Sanierung von Bo den, Landschaft und Grundwasser. 15.
Lieferung, I/94, Erich Schmidt Verlag, Berlin.
Reeves, R.D., Baker, A.J.M., 1984. Studies on metal uptake by plants
from serpentine and non-serpentine populations of Thlaspi goe-
singense Ha la csy (Cruciferae). New Phytologist 98, 191204.
Reeves, R.D., Brooks, R.R., 1983. Hyperaccumulation of lead and
zinc by two metallophytes from mining areas of Central Europe.
Environmental Pollution (Series A) 31, 277285.
Sieghardt, H., 1987. Schwermetall- und Na hrelementgehalte von
Panzen und Bodenproben schwermetallhaltiger Halden im Raum
Bleiberg in Ka rnten (O

sterreich): I. krautige Panzen. Z. Panze-

nerna hr. Bodenk 150, 129134.
Sparks, D.L., 1989. Kinetics of soil chemical processes. Academic
Press, San Diego.
Sposito, G., 1989. The Chemistry of Soils. Oxford University Press,
New York, Oxford.
154 W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155
Thalmann, F., Schermann, O., Schroll, E., Hausberger, G., 1989.
Geochemical Atlas of the Republic of Austria 1:1,000.000. Geolo-
gische Bundesanstalt, Wien.
Wenzel, W.W., Blum, W.E.H., 1997. Eect of sampling, sample pre-
paration and extraction techniques on mobile metal fractions in
soils. Advances in Environmental Science, in press. Also In:
Adriano, D.C., Chen, Z.-S., Young, S.-S., Iskolnolar, I.K. (Eds.),
Biochemistry of trace metals, advances in environmental science,
Science Reviews, Northwood, pp. 121172.
Wenzel, W.W., Salt, D., Smith, R.S., Adriano, D.C., 1998. Phyto-
remediation. A plantmicrobe based remediation system. In:
Adriano, D.C., Bollag, J.M., (Eds.), Bioremediation of Soils, SSSA
Monagraphs.
Wenzel, W.W., Alge, G., Sattler, H., 1993. Environmental soil mon-
itoring in Austria: Methodology and results, In: Varallyay, G.,
(Ed.), Proc. Int. Workshop on Harmonization of Soil Conservation
Monitoring Systems, Budapest, pp. 3548.
Zeien, H., Bru mmer, G.W., 1991. Ermittlung der Mobilita t und Bin-
dungsformen von Schwermetallen mittels sequentieller Extra-
ktionen. Mitteilg. Dtsch. Bodenkundl. Gesellsch 66, 439442.
W.W. Wenzel, F. Jockwer / Environmental Pollution 104 (1999) 145155 155