Ernst, 1974. A restricted number of plant species which have evolved metal resistance mechanisms
are able to colonize these polymetallic soils Ernst et al., 1992. In the analysis of the impact of metal
mixtures on plant performance, metal-resistant ecotypes can at least partly help to overcome the
disadvantage of sensitive plants on metal-contam- inated soils. In addition to many short-term ex-
periments with one metal Sanita´ di Toppi and Gabbrielli, 1999, only a few experiments on soils
artificially contaminated with one metal have been extended to a full life-cycle of plants Sheppard et
al., 1993. Metal-resistant plants offer an excellent opportunity to analyse the impact of complex
metal mixtures on the performance of plants dur- ing a full life-cycle. In addition, such plants can be
exposed to metal mixtures in a realistic environ- mental soil setting, i.e. with differences in pH,
organic matter, Ca content and other soil parame- ters because these plants are also adapted to low
levels of major nutrients in orogenic soils Ernst, 1974. The few studies on combination toxicology
of heavy metals analysed plants which were artifi- cially exposed to enhanced concentrations of these
elements in hydroponics for a short period Wal- lace and Abou-Zam Zam, 1989; Wallace and
Berry, 1989; Sharma et al., 1999. For our study we have selected the perennial herb Silene 6ulgaris
Caryophyllaceae being characteristic for many metal-enriched soils in Europe Ernst, 1974. In
contrast to many other metal-resistant plants, S.
6 ulgaris has nearly no symbiosis with arbuscular
mycorrhizal fungi Ernst et al., 1990; Pawlowska et al., 1996; Hildebrandt et al., 1999. Therefore
the roots are directly exposed to the metal concen- tration of the soil and changes of metal speciation
and benefits for the host by mycorrhizal fungi can be excluded Dueck et al., 1986; Hildebrandt et
al., 1999.
With regard to morphological and ecological parameters, we have shown that a Zn- and Cd-re-
sistant ecotype of S. 6ulgaris reacted quite differ- ent in the various phases of its life-cycle Ernst
and Nelissen, 1999. Here, we report on the phys- iological responses of this ecotype to an exposure
to combinations of the heavy metals Cd, Cu, Fe, Mn, Pb and Zn throughout a full life-cycle of the
Zn- and Cd-resistant plants Ernst and Nelissen, 1999. We tested the following hypotheses:
1. As soon as the metal concentration of the soil solution exceeds the Zn- and Cd-resistance of
the metal-resistant ecotype, disturbance of the nutrient uptake will occur, partly being visible
by discoloration.
2. The metal concentration in young seedlings is an indicator for the survival chance up to seed
maturity with a negative relationship between metal concentration and survival.
3. Due to low Cu concentration which diminishes the performance of this ecotype for Cu by
50, i.e. EC
50
Schat and Ten Bookum, 1992, Cu-enriched soils will strongly impair the
metabolism which will be expressed by an enhanced synthesis of phytochelatins PCs.
2. Materials and methods
2
.
1
. Soils and growth conditions Seeds of a Zn- and Cd-resistant ecotype of S.
6 ulgaris Moench Garcke were collected from a
population of the mine tailings at Plombie`resBel- gium with high EC
50
-values for Zn and Cd, and low values for Cu Verkleij and Prast 1989; Schat
and Ten Bookum 1992. Pots of 750 ml volume were filled with 1.3 kg air-dried metal-enriched
soils collected on the following sites Fig. 1: Zn – Pb-mine tailings at WildemannD 1, 2, at
BlankenrodeD 3, 4 and Plombie`resB 16, the
Fig. 1. Sampling sites of soils numbers 1 – 16 and seeds of the Cd-and Zn-resistant ecotype of Silene 6ulgaris Plombie`res, 16
for this study in Central Europe.
abandoned Cu-mine at MarsbergD 9, 10, the Bronze-Age smelting site at the banks of the river
Innerste near Langelsheim 11, 12, the Zn – Cu- mine tailings at KlosterrodeD 5, 6 and at
WelfesholzD 7, 8, 13, 14, 15. After chemical analysis it worked out that the sampling site of
soil 4 was a mine pit which was filled up with clay material from other origin; therefore plants grow-
ing on this soil were not incorporated in the analysis. The total and water-soluble metal con-
centration of these soils was reported by Ernst and Nelissen 1999. Due to low organic matter of
the orogenic soils, all water-soluble metals except Cu were present as free metal ions, as analysed by
a batch-column-batch procedure using the cation exchange resins Amberlite CG 120 and Chelex
100 cf. Ernst and Nelissen 1999. From water- soluble Cu 12 – 20 was bound to organic com-
plexes; only in soils 7, 9 where birch leaves were blown in from adjacent woodlands, 35 – 45 of
the water-soluble Cu was complexed.
For the analysis of seedling performance, 100 seeds were sown in each of three pots per sam-
pling site; 20 plants were harvested 14 days after emergence. In another series, 30 seeds were sown
per pot in triplicate per sampling site. Immedi- ately after emergence, they were thinned to ten
seedlings per pot and kept under the below men- tioned conditions. After 5 weeks of growth vege-
tative phase of the life-cycle, five plants per pot were harvested for growth and mineral analysis
and two plants were taken for the analysis of physiological parameters. The remaining three
plants were grown up to seed maturity which was achieved 9 months after emergence.
The plants were kept in a greenhouse at a temperature cycle of 2015°C daynight from
November to April and then at 2518°C for the rest of the life-cycle. Additional radiation was
supplied for 10 h daily from mercury iodide lamps providing a radiation flux of 235 mmol m
− 2
s
− 1
at medium plant level.
2
.
2
. Mineral elements In the seedling stage 2 weeks, 20 seedlings
from each pot were harvested, separated into root and shoot hypocotyl and two cotyledons. The
shoots were washed twice for 30 s in demineral- ized water by slight brushing to remove poten-
tially adhering soil particles, dried at 60°C for 48 h and pooled to two subsamples for analysis. The
roots were discarded because it was not possible to clean them sufficiently from adhering soil parti-
cles. At harvest 5 weeks and 9 months of growth, plants from each treatment at each age
were separated into roots, stalks and leaves and at the reproductive phase additionally into calyx,
capsule and seeds. Although roots are the first target of a metal surplus, chemical analysis of
roots grown in soil is impeded by a high affinity to soil particles and adsorbed metals Ernst,
1995. Therefore it was necessary to clean the roots first mechanically by a brush, with the risk
of loosing fine roots which especially was the case in seedlings. As next step metals from the root
surface were desorbed by a solution in 0.1 M SrCl
2
-solution for 30 min at 4°C. In the present study, SrCl
2
was used because the more effective PbNO
3 2
Harrison et al., 1979 would not allow Pb analysis of the roots.
The various plant parts and roots were dried at 60°C for 48 h. Plant material 50 – 100 mg per
sample, if present was mineralised in Teflon bombs at 140°C with aqua regia HNO
3
HCl, 3:1, vv over night and the diluted solution analysed
by atomic absorption spectrometry Perkin Elmer AAS 1100 or at low concentration of Cd, Cu and
Pb by graphite furnace AAS Perkin Elmer AAS 2100. LaNO
3 2
was added to enhance atomiza- tion of Ca and Mg. Phosphorus was determined
by a spectrophotometric method after formation of a blue ascorbic acid-phosphorus complex
Chen et al., 1956. Carbon and N were analysed by column chromatography after burning the
sample in pure oxygen Kirsten, 1979 in a Perkin Elmer CHN analyser. Mature leaves of Populus
nigra L. were used as internal laboratory reference material. Chemical analysis of soils are described
by Ernst and Nelissen 1999.
2
.
3
. Plant pigments Discoloration was analysed on two 5-week-old
plants per pot, three pots per soil. Chlorophyll concentration was determined in the upper three
Table 1 Mean plant biomass 9 1 SE and mean concentration of macronutrients in leaves of the Zn–Cd resistant ecotype Plombie`res of S.
6 ulgaris grown for 5 weeks on orogenic soils in a greenhouse
a
Biomass mgplant Site
Macronutrients mmol g
− 1
d.m. Soil number
N P
K Ca
Mg 9.0 9 5.1
a
2400
de
70
d
11 918
c
Langelsheim I 363
c
115
a
12 Langelsheim II
10.5 9 1.7
a
2530
de
62
cd
745
b
382
cd
106
a
14 21.2 9 7.5
b
Welfesholz III 2100
c
78
e
1690
fg
534
ef
385
e
24.5 9 12.1
bc
1980
c
35
b
Welfesholz IV 1750
g
15 465
ed
378
e
Blankenrode 3
25.6 9 8.2
bc
1020
a
22
a
419
a
152
a
202
cd
25.9 9 8.4
bc
3950
g
39
b
1 1900
h
Wildemann I 606
f
182
c
28.0 9 8.5
bc
1730
b
39
b
Klosterrode II 1610
f
6 443
d
529
f
Welfesholz Ib 8
30.5 9 10.5
bc
2310
cd
40
b
1450
e
380
cd
521
f
35.2 9 13.2
c
2650
e
85
f
9 1040
cd
Marsberg II 305
b
128
ab
36.4 9 12.9
c
1650
b
69
d
Marsberg I 631
b
10 352
c
137
b
Plombie`res 16
41.5 9 18.3
cd
2230
cd
57
c
1180
d
293
b
693
g
42.6 9 14.0
cd
1800
bc
32
b
5 1190
d
Klosterrode I 428
d
355
e
59.9 9 22.9
de
2850
ef
73
d
Wildemann II 2350
I
2 393
cd
173
c
Welfesholz II 13
63.3 9 33.0
def
2540
de
82
ef
2320
I
410
d
356
e
95.8 9 28.4
fg
2170
c
87
f
1790
g
471
e
215
d
7 Welfesholz Ia
a
The plants are ranked according to biomass increase. Values with different superscripts indicate significant differences at least at PB0.05. The biomass data are from Ernst and Nelissen 1999.
leaf pairs after extraction with 80 vv aqueous acetone in the presence of small amounts of
quartz sand and Na
2
CO
3
and centrifugation. The absorption of the supernatant was measured at
470, 647 and 663 nm in a Pharmacia Ultraspec III. The concentration of chlorophyll a and b and
carotinoids were calculated using the equations given by Lichtenthaler 1987.
Anthocyanins were extracted by grinding the oldest leaves of 5 week-old plants with mortar
and pestle in the presence of quartz sand. The extraction medium was methanolHCl 991, vv.
After centrifugation, absorbance spectra of the supernatant were recorded with a Pharmacia Ul-
traspec III at 528 nm Kakegawa et al., 1991, which was the wavelength with the maximum
extinction. Cyanidinchloride was taken for cali- bration of the cyanidins.
2
.
4
. Phytochelatin analysis For the analysis of phytochelatins 3 – 5 pairs of
mature leaves were excised from two 5-week-old plants per pot, three pots per soil, and immedi-
ately frozen in liquid nitrogen. After homogeniza- tion with quartz sand and centrifugation at
27 000 × g for 20 min at 4°C the supernatant was analysed by a HPLC assay using post-column
derivatization or with 20 mM monobromobimane Sneller, 1999 modified after Rijstenbil and Wijn-
holds 1996. The samples were lyophilized and stored under vacuum until detection.
2
.
5
. Statistics Correlation between the various measured
parameters were calculated and tested for signifi- cance P B 0.05 by one way ANOVA. Multiple
comparison among means based on equal samples sizes were made by application of the T-method
Sokal and Rohlf, 1995.
3. Results
