Russian Journal of Plant Physiology, Vol. 50, No. 3, 2003, pp. 398–406. Translated from Fiziologiya Rastenii, Vol.

50, No. 3, 2003, pp. 445–454.

Original Russian Text Copyright © 2003 by Ivanov, Bystrova, Seregin.

Comparative Impacts of Heavy Metals on Root Growth

as Related to Their Specificity and Selectivity
V. B. Ivanov, E. I. Bystrova, and I. V. Seregin
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;
fax: 7 (095) 977-8018; e-mail: ivanov@ippras.ru
Received September 20, 2001

Abstract—Two-day-old maize (Zea mays L.) seedlings were incubated on the solutions of Ag, Cd, Pb, Zn, Cu,
Tl, Co, and Hg salts (0.001 to 3 g/l). Toxicity of heavy metals was assessed as the inhibition of root growth on
the first, second, and third days, the change in the length of the lateral root zone, and the duration of lateral root
development from the first division in pericycle to emergence. For all salts under study, the ratio of the lethal
concentration to the lowest concentration slowing down root growth was about ten, and growth inhibition was
not almost enhanced in the course of three days. With concentrations calculated as g/l, metal toxicity declined
in the following order: Cu ≈ Tl > Ag > Cd > Hg > Co > Zn > Pb; for molar concentrations, the order was the
following: Tl3+ > Cu2+ > > Ag+ > Hg2+ ≈ Cd2+ > Zn2+ ≈ Pb2+ ≈ Co2+. Duration of lateral root development was
least affected by heavy metals. Metal affinity of biological compounds for SH-groups was closely correlated (r
= 0.9) with the molar concentration that inhibited primary root growth by 50%. Because of the narrow range of
effective concentrations, only slightly increasing inhibition over the exposure time, tolerant root branching, and
close relationship between the toxicity and the constant of binding to SH-groups, we conclude that the salts
under study exert nonselective inhibition and root growth is slowed down due to the general toxicity of heavy
metals rather than selective inhibition of any particular process or processes.

Key words: Zea mays - stress - mechanisms of toxicity - root - growth - biotest - heavy metals

INTRODUCTION complex metal compound often result in the complete

Because of increasing environment pollution with
heavy metals, more and more attention is paid to their
impacts on various organisms, including plants. Most
studies are focused on the effect of heavy metals on a
particular biological process. The mechanisms of the
toxic effects of heavy metals are diverse. Heavy metals
interact with several functional groups of proteins, pri-
marily SH-groups. As a result, protein conformation is
changed, and many enzymes with SH-groups in their
active centers lose their activities. In addition, cell
metabolism is also hampered by metal binding to SH-
groups of physiologically active low-molecular-weight
metabolites. Thus the heavy metal binding to SH-
groups alone can adversely affect cell metabolism in
many ways. Other mechanisms of heavy metal toxicity
include Ca displacement, binding to carboxylic groups,
etc. However, in some cases, the effects of heavy metals
are more specific, e.g., a replacement of a similar metal
ion in metalloenzymes and specific binding of metal-
containing complexes to the particular groups in mac-
romolecules, e.g., binding of cis-dichloro-diaminoplat-
inum (II) to DNA. The effects of these complexes are
apparently very specific, and trans-isomer or similar
palladium complex is not active even at a high concen-
tration. In addition, small changes in the structure of a
Abbreviations and designations: LC50—concentration inhibiting
root growth by 50% at the second day of incubation.
loss of biological activity [1]. Therefore, when analyz-
ing the impact of heavy metals, it is essential to eluci-
date how specifically they depend on the particular
metal or metal-containing compound and to discrimi-
nate the unspecific effects due to a general toxicity
characteristic of all heavy metals. Because of different
affinity for SH-groups, the concentrations producing
these unspecific effects vary for diverse metals.
The toxicity of heavy metals for crustaceans, fish,
and fungi was long ago shown to correlate with the con-
stant of metal binding to SH-groups [2]. In this case, the
toxicity seems to result primarily from unspecific metal
effects when the dissimilar effects of various metals
depend on their different affinity for SH-groups. Such
studies have not been carried out with roots, although
the toxicity of various metals for roots was compared
[3–5].
The goal of the present study was to compare
growth-inhibiting effects of various metals on seedling
roots and then to collate this evidence with the physico-
chemical properties of metal atoms and ions in order to
evaluate the selectivity and specificity of the effects of
heavy metal salts on plants. Due to high root sensitivity
to diverse factors, the relatively unsophisticated exper-
iments with seedling roots are often employed in
biotests for assessing the toxicity of heavy metals [1, 3–
7]. For many years we used the roots as a reliable test

1021-4437/03/5003-0398$25.00 © 2003 MAIK “Nauka /Interperiodica”

 COMPARATIVE IMPACTS OF HEAVY METALS
for screening numerous and diverse compounds for
their biological activities (see review [1]). In these stud-
ies, straightforward methods were worked out for
assessing the specificity and selectivity of growth
response to any chemical by the evaluation of time-
dependent deceleration of root growth and by compar-
ing the toxicity of the particular compound with its
physicochemical properties, composition, and struc-
ture.
The impact of all chemicals, which specifically
inhibit cell divisions, dramatically increases with the
exposure prolongation, and finally root branching is
arrested. In contrast, effect of the compounds unable to
selectively inhibit cell divisions remains almost
unchanged with time. These compounds did not arrest
the formation of lateral roots even at the concentrations
approaching the lethal levels. In this study, we used
these simple approaches to evaluate the impact of a
large group of heavy metal salts on root growth.
MATERIALS AND METHODS
Maize (Zea mays L., cv. Diamant) seeds were steril-
ized with formaldehyde for 20 min and germinated on
filter paper moistened with tap water for two days in
darkness at 27°C. Seedlings with 20–30-mm-long pri-
mary roots were selected and transferred to petri dishes
150 mm in diameter onto filter paper moistened with
the solutions of heavy metals in distilled water at the
concentrations of 0.001 to 3 g/l (pH 6.0). The following
salts of pure-for-analysis grade were employed:
AgNO3, Cd(NO3)2 · 4H2O, Pb(NO3)2, ZnSO4 · 7H2O,
CuSO4 · 5H2O, Tl2(SO4)3, CoCl2, and HgCl2. The seed-
lings were incubated in a dark thermostat at 27°C for
further three days.
The toxic effect of heavy metal salts was assessed as
the inhibition of root growth on the first, second, and
third days following the beginning of incubation; the
change in the length of the lateral root zone; and dura-
tion of lateral root development.
To evaluate the root tolerance to heavy metals, we
calculated the tolerance index I with the formula
∆L
I ( % ) = ---------t × 100%,
∆L c
where ∆Lt and ∆Lc are the daily increments of root
length in the metal-treated and control plants, respec-
tively [6].
Duration of lateral root development (from the first
cell division in the pericycle to lateral root emergence
from the mother root) was calculated using the follow-
ing equation [7]:
[ ( L lr + 10 ) – L 0 ] × 24
T lr = 72 – ---------------------------------------------------,
∆L 24
where Llr is the length of the lateral root zone after 72 h,
L0 is the initial length of the primary root, and ∆L24 is
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50
399
the increment of the primary root length for 24 h. In
maize roots, divisions of pericycle cells, which result in
the development of lateral initials, take place soon after
elongation is over. The equation above makes it possi-
ble to calculate the duration of lateral root development
from the commencement of pericycle cell divisions to
its emergence from the primary root. During this
period, this root segment becomes distant from the root
tip due to elongation of the apical part. Therefore, the
higher root growth rate, the farther the particular lateral
root from the primary root apex. Thus, the distance
between the primary root apex and the lateral root
emerged closest to the apex depends on the rates of lat-
eral root development and primary root growth. These
are two different processes, and our goal was to eluci-
date the effect of heavy metals exactly on the rate of lat-
eral root development. It is impossible to obtain such
information, as is often attempted, measuring only the
distance between the most distal lateral root and the
apex of the primary root.
To evaluate the toxic effects of heavy metals on root
growth as related to their physicochemical characteris-
tics, we calculated the coefficients of correlation
between the most essential indices determining the
properties of metal atoms and ions and molar concen-
trations upon 50% growth inhibition (LC50 values).
Among the physicochemical properties of heavy met-
als, we chose the effective ion radius (after Shannon
and Prewitt), ion potential, electronegativity, the Mis-
ono parameter, and the affinity for SH-groups. Ion
radius determines the extent of ion hydration. These
two parameters are related reciprocally [2]. However,
when comparing the analogous compounds comprising
the chemical elements from various groups, one deals
with simultaneous changes in both ion charge and ion
radius. It is therefore more convenient to use the values
of ion potential, that is, the product of division of ion
charge by its radius (Z/r). Ion potential determines the
energy of interaction between metal ions and the neigh-
boring water molecules in solution. At Z/r < 30, a cation
may be solvated, at 95 > Z/r > 30, it would be strongly
repelled from the water molecule, and at Z/r > 95, a
strong repulsion would result in the formation of
hydroxy particles [8]. The Misono parameter is intro-
duced for covalent bonds as a quantitative estimate of
the propensity of metal cations to polarization. Upon
this parameter, metal ions are classified in strong and
mild Lewis acids (classes a and b). The former are rec-
ognized by a high charge density and low polarization
capacity (these acids form complexes by electrostatic
interactions). On the contrary, the latter manifest a low
charge density and high polarization capacity; these
acids form stable complexes by covalent interactions
[8]. Electronegativity determines the capacity of an
atom in a molecule to attach a pair of common electrons
[2]. The strength of a bond between a metal and an SH-
group is proportional to the insolubility of resulting sul-
fides and is described by a constant K1, which is a neg-
No. 3 2003
400 IVANOV et al.

80 effects of various heavy metals on the morphology of

(‡)
the seedling root system.
The range of concentrations of heavy metal salts
60 inhibiting root growth is rather narrow. For maize seed-
Increment, mm

ling roots, the lethal concentrations exceeded only 10–

20-fold the lowest concentrations that visibly slowed
40 down root growth (Fig. 1). When seedlings were kept
on the solutions of heavy metal salts at lethal concen-
trations, roots stopped growing within the initial 24 h.
20 No root hairs were visible. After 72 h, roots lost turgor
and often became brownish, apparently because of cell
death. Few lateral roots developed almost did not elon-
0~
gate.
~
2 3 4 5 6 At the concentrations inhibiting root growth by
–log[C]Me, M 50%, root apices became thinner and longer. Root hairs
80 developed closer to the apex, as compared to the control
(b) roots; this evidence indicated a shortening of the grow-
ing region.
60 As a whole, the toxic effect of heavy metals on root
Increment, mm

growth did not considerably change with time; how-

40
20
0~
~ seedlings and in the seedlings incubated in the presence
0 1 2 3
–log[C]Me, g/l
Fig. 1. Toxic effects of heavy metals on the daily increment
(from 24 to 48 h) of the primary root length as related to the
negative logarithm of metal concentration in (a) mol/l and
(b) g/l.
䊉—Cu, 䊊—Tl, 䉲—Ag, 䉮—Cd, 䊏—Hg, 䊐—Co, 䉬—Pb,
䉫—Zn.
ative logarithm of solubility [2]. The corresponding
values were obtained from [2, 8].
In most publication, the concentrations of heavy
metal salts are usually given as g/l. To compare activi-
ties of various metals, one must compare molar concen-
trations as well. Therefore in Fig. 1 we give molar and
weight per volume concentrations of both salts and
metals themselves.
The experiments were run in several replications
(see the tables) and produced comparable data. The
data are presented as means and their standard errors.
RESULTS
Effect of Heavy Metals on the Morphology
and Growth of the Primary Root
By analyzing the data collected in the tables, we dis-
cern several most general features characterizing the
ever, an insignificant enhancement of root growth inhi-
bition was observed during prolonged exposure to
some metals (Tables 1, 2). To illustrate, sublethal con-
centrations of Cu and Ag reduced the 24-h root growth
increments to 75 and 65% of the control, respectively;
whereas during 48–72 h, the corresponding values were
22 and 34%. The growth increment increased within
the second and third days of incubation in the control
4 of 0.01 g/l ZnSO4 (Table 1).
To compare the toxic effects of various heavy met-
als, we related the root growth increment to the metal
concentration in solutions (g/l). Cu and Tl were most
toxic, and Pb was the least toxic (Fig. 1b). As a whole,
the toxicity assessed for the concentration of 0.01 g/l
diminished in the following order: Cu ≈ Tl > Ag > Cd >
Hg > Co > Zn > Pb. This order of toxicity is not invari-
ant and is somewhat affected by the metal concentra-
tion in solution.
We obtained the similar order of toxicity when using
50% inhibition as a criterion for arranging heavy metal
effects, with an exception of Ag, which manifested the
toxicity close to those of Cu and Tl (Fig. 1b).
However, from the standpoint of a plant physiolo-
gist, it seems essential to compare the effects of molar
concentrations as well (Fig. 1a). At the similar ion con-
centration in solution, e.g., 10–6 M, metal toxicity
diminished in the following order: Tl3+ > Cu2+ >
Ag+ >Hg2+ ≈ Cd2+ > Zn2+ ≈ Pb2+ ≈ Co2+; this range was
similar to that observed for 50% inhibition. In general,
the toxicity series for metals and ions are in a good
agreement.
The curves in Fig. 1 were used for calculating LC50
values for cadmium and lead nitrates inhibiting root
growth for 48 h. The values of salt concentrations of
0.03 and 0.33 g/l, respectively, corresponded to the data
from direct experiments (Table 2). These concentra-

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50 No. 3 2003

 COMPARATIVE IMPACTS OF HEAVY METALS 401

Table 1. Effect of various salts of heavy metals on the daily increment in the primary root length, mm
Exposure period, h
Salt 0–24 24–48 48–72
water 0.01 g/l 0.1 g/l water 0.01 g/l 0.1 g/l water 0.01 g/l 0.1 g/l
CuSO4 · 5H2O 55 ± 3 (10) 41 ± 2 (7) 6 ± 2 (4) 66 ± 4 (10) 28 ± 4 (7) 2 ± 1 (4) 59 ± 5 (10) 13 ± 4 (6) 2 ± 2 (4)
Tl2(SO4)3 57 ± 4 (4) 29 ± 4 (4) 16 ± 1 (3) 64 ± 1 (4) 13 ± 4 (4) 2 ± 1 (3) 62 ± 5 (4) 13 ± 6 (4) 1 ± 0 (3)
AgNO3 51 ± 4 (4) 33 ± 5 (3) 7 ± 1 (4) 58 ± 2 (4) 23 ± 3 (3) 0 (4) 56 ± 6 (4) 19 ± 1 (3) 0 (4)
Cd(NO3)2 · 4H2O 54 ± 2 (8) 42 ± 5 (4) 11 ± 2 (4) 63 ± 3 (8) 44 ± 4 (4) 1 ± 0 (4) 58 ± 3 (8) 51 ± 5 (4) 1 ± 0 (4)
HgCl2 52 ± 3 (5) 37 ± 2 (4) 12 ± 3 (4) 61 ± 4 (5) 40 ± 1 (4) 3 ± 2 (4) 61 ± 5 (5) 39 ± 4 (4) 7 ± 3 (4)
CoCl2 · 6H2O 55 ± 2 (6) 58 ± 3 (4) 42 ± 2 (5) 61 ± 3 (6) 64 ± 6 (4) 30 ± 3 (5) 62 ± 5 (6) 48 ± 4 (4) 21 ± 3 (5)
ZnSO4 · 7H2O 53 ± 3 (8) 47 ± 1 (4) 37 ± 5 (3) 66 ± 4 (8) 52 ± 4 (4) 52 ± 6 (4) 61 ± 3 (8) 74 ± 1 (4) 48 ± 6 (4)
Pb(NO3)2 57 ± 3 (6) 54 ± 6 (6) 47 ± 3 (4) 59 ± 3 (6) 60 ± 7 (6) 48 ± 1 (6) 64 ± 4 (4) 65 ± 3 (4) 43 ± 7 (4)
Note: Means and their standard errors are given; the number of independent experiments is given in parentheses.

Table 2. Effect of cadmium and lead nitrates on the daily increment in the primary root length, mm
Concentration, g/l Exposure period, h
Treatment
salt metal 0–24 24–48 48–72
Water 49 ± 2 62 ± 3 58 ± 2
Cd(NO3)2 · 4H2O 0.03 0.01 39 ± 2 30 ± 1 27 ± 3
Pb(NO3)2 0.33 0.2 38 ± 3 32 ± 1 32 ± 3
Note: Means from five independent experiments and their standard errors are given.

Table 3. Effect of heavy metal salts at various concentrations on the length of the lateral root zone and the duration of lateral
root development
Length of the lateral root zone, mm Duration of lateral root development, h
Salt
water 0.01 g/l 0.1 g/l water 0.01 g/l 0.1 g/l
CuSO4 · 5H2O 77 ± 8 (10) 62 ± 8 (6) 33 ± 9 (4) 51 ± 3 (10) 50 ± 1 (6) 54 ± 5 (4)
AgNO3 65 ± 5 (4) 42 ± 1 (3) n.d. 55 ± 3 (4) 55 ± 3 (3) n.d.
Cd(NO3)2 · 4H2O 71 ± 8 (8) 56 ± 9 (4) 24 ± 3 (4) 50 ± 2 (8) 52 ± 4 (4) 53 ± 3 (4)
HgCl2 70 ± 7 (5) 58 ± 2 (4) 34 ± 3 (4) 48 ± 2 (5) 48 ± 5 (4) 50 ± 2 (4)
CoCl2 · 6H2O 74 ± 5 (6) 70 ± 9 (4) 35 ± 4 (5) 49 ± 2 (6) 53 ± 2 (4) 66 ± 2 (5)
ZnSO4 · 7H2O 75 ± 7 (8) 71 ± 5 (4) 66 ± 5 (4) 48 ± 2 (8) 47 ± 1 (4) 43 ± 1 (4)
Pb(NO3)2 67 ± 4 (4) 66 ± 8 (4) 60 ± 4 (4) 53 ± 1 (4) 53 ± 1 (4) 53 ± 1 (4)
Note: Means and their standard errors are given; the number of independent experiments is given in parentheses. The length of the lateral
root zone was measured on the third day of incubation; n.d.—no data were obtained.

tions were further used to study heavy metal distribu-
tion in various root regions [9].
Effect of Heavy Metals on Lateral Root Development
In the seedlings exposed to heavy metal salts at con-
centrations below lethal ones, lateral roots appeared at
the same time as in the control roots, that is, at about
50 h since the beginning of incubation (Table 3). The
duration of lateral root development was affected by
heavy metals to considerably lesser extent than the pri-
mary root growth. To illustrate, at 0.1 g/l concentration
of Cu, Cd, and Hg salts, a growth increment of the pri-
mary root through the second day was only 2–5% of the
control treatment while the time of lateral root develop-
ment remained almost unchanged (Fig. 2). Laterals
were often clustered, mostly at the convex side of the
root. The cause of this phenomenon has not been eluci-
dated as yet.

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50 No. 3 2003

402 IVANOV et al.

(‡) 100 (‡)

120

100 80

80
60
60
40
40

20 20
I, % of control treatment

0 0

I, % of control treatment
Cu Cd Hg Co Zn Pb Cu Cd Hg Co Zn Pb

(b) (b)
120 120

100 100

80 80

60 60

40 40

20 20

0 0
Cu Ag Cd Hg Co Zn Pb Cu Ag Cd Hg Co Zn Pb
Fig. 2. Comparative toxic effects of heavy metal salts on Fig. 3. Comparative toxic effects of heavy metal salts on
( ) the daily increment (from 24 to 48 h) of the primary ( ) the daily increment (from 48 to 72 h) of the primary
root length and (䊏) duration of lateral root development. root length and (䊏) the length of the lateral root zone.
Salt concentration was (a) 0.1 g/l and (b) 0.01 g/l. Histo- Salt concentration was (a) 0.1 g/l and (b) 0.01 g/l. Histo-
grams were built using the data from Tables 1 and 3. grams were built using the data from Tables 1 and 3.

The length of the lateral root zone diminished in the
presence of heavy metals to 34–95% of the control
value depending on salt concentration (Table 3). Heavy
metals reduced this zone length to a lesser extent than
the root growth increment (Fig. 3). This indirect evi-
dence presumes that heavy metals primarily affect the
growing root tip.
DISCUSSION
Comparing Heavy Metals by Their Toxicity
Our experiments demonstrated that heavy metals
dramatically differed in their toxicity (Table 1, Fig. 1).
In addition, the toxicity range depended on the way of
calculating concentrations. This fact may provide an
explanation to disagreement in the previously pub-
lished toxicity ranges for barley [10], ryegrass [11], and
wheat [12]. Plant species may also differ in the effi-
ciency of detoxification.
Our evidence on the effects of toxic metals on maize
root growth presumes that the metals under study can be
divided into three toxicity classes: high (Cu, Tl, and Ag),
moderate (Cd and Hg), and low (Pb, Co, Zn) toxicity.
Mechanism of the General Toxic Effect of Heavy Metals
The mechanisms of heavy metal toxicity are very
diverse [13–15]. Metals interact with various functional
groups of proteins, nucleic acids, and polysaccharides
and with numerous low-molecular-weight compounds.
Metals differ in their reaction capacities. Long ago an
attempt was made to relate the toxicity of particular
metals to their physicochemical properties. Thus, a
well-defined relation was established between the tox-
icity of heavy metals and the strength of their binding
to SH-groups, when the effects of heavy metals on
enzymes in vitro were studied in various species of
aquatic invertebrates and fish [2]. We are not aware of
similar evidence for higher plants. Our data demon-
strated that the affinity of metals for SH-groups was

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50 No. 3 2003

 COMPARATIVE IMPACTS OF HEAVY METALS 403

(a) r = 0.13 100 (b) r = 0.61

0.12 80

Ion potential, 1/nm

Ion radius, nm
60
0.10
40

0.08 20

0
2.1 (d) r = 0.66

Electronegativity
(c) r = 0.33
0.40
Misono parameter, nm

2.0
1.9
1.8
0.32
1.7
1.6
1.5
0.24
1.4
3 4 5
70 –log[C]Me, M
(e) r = 0.82
60

50
K1

40

30

20
3 4 5
–log[C]Me, M

Fig. 4. Correlation between the molar concentrations of heavy metals inhibiting root growth by 50% and the main physicochemical
characteristics of atoms and ions of heavy metals: (a) ion radius; (b) ion potential; (c) Misono parameter; (d) electronegativity; and
(e) metal affinity for SH-group expressed as the constant of stability.
䊉—Cu, —Ag, —Cd, —Hg, 䉱—Co, —Pb, —Zn.

significantly positively correlated with molar concen-
trations exerting 50% growth inhibition (r = 0.82).
However, no correlation was observed with ion radius,
ion potential, the Misono parameter, and electronega-
tivity of metals (Fig. 4).
Some researchers compared the impacts of several
heavy metals on the growth of roots of one and the same
plant species [10–12]; however, they did not relate tox-
icity to the physico chemical properties of these metals.
When we processed the data from [10–12] in the same
way as shown above, the stability of the complexes
between metals and SH-groups was closely related to
the extent of root growth inhibition, in spite of the fact
that different plant species and conditions of root incu-
bation were used (Fig. 5). In addition, when we ana-
lyzed the data for Lolium perenne [11] and Triticum
aestivum [12], we found a significant correlation (r =
0.9) between the concentrations producing 50% growth
inhibition and electronegativity (Fig. 5). The latter
index determines the easiness, with which electrons
transfer when forming covalent and ionic bonds, and
therefore the correlation observed is in line with the
suggestion that a toxicity of heavy metals depends on
their capacity to form strong covalent bonds. Similar
evidence was previously obtained with fungi and ani-
mal tissues [2].
Apart from interactions with SH-groups, the toxic-
ity of heavy metals also depends on their reactions with
other functional groups and on the mobility and other
ion properties. Hence the relative toxicity of particular

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50 No. 3 2003

404 IVANOV et al.

2.1
44
(a) r = 0.92 2.0 (b) r = 0.98
40 1.9

Electronegativity
36 1.8
K1

1.7
32
1.6
28
1.5
24 1.4
2.1
44
(c) r = 0.95 2.0 (d) r = 0.96
40 1.9

Electronegativity
36 1.8
K1

1.7
32
1.6
28
1.5
24 1.4
4 5 6 7 4 5 6 7
–log[C]Me, M –log[C]Me, M

Fig. 5. Correlation between the molar concentrations of heavy metals inhibiting root growth by 50% and the main physicochemical
characteristics of atoms and ions of heavy metals: (a, c) metal affinity for SH-groups (b, d) electronegativity.
(a, b) Data from [11]; (c, d) data from [12]. (a, b) Lolium perenne; (c, d) Triticum aestivum.
䊉—Cu, —Cd, —Pb, —Zn, 䉱—Ni.

metals is different in various species of plants and other
organisms. In addition, in some cases several metals
may selectively arrest the processes more closely
related to growth, such as ethylene synthesis inhibited
by silver [16].
The well-defined correlation between the extent of
growth inhibition and metal affinity for SH-groups,
essential for the functions of most enzymes, points out
at a mechanism of unspecific and nonselective toxicity
general for various metals, with some particular reac-
tions overlapping this mechanism. Hence we consider
heavy metals to be unspecific agents. Among the com-
mon causes of their toxicity affecting plants and ani-
mals, their interactions with SH-groups are important.
General Features of Root Growth when Affected
by Heavy Metals
The salts of heavy metals under study greatly differ
in toxicity; however, their toxic effects on root growth
manifest common features: (1) a narrow range of con-
centrations that slow down root growth (Fig. 1); (2)
insignificant changes in the inhibition extent (Table 1);
(3) root branching is not affected in spite of the strong
inhibition of the primary root growth (Fig. 2). Accord-
ing to our evidence obtained previously, these features
are characteristic of diverse chemicals, including non-
electrolytes (alcohols, phenols, and naphthols) of
unspecific action, some antibiotics with nonselective
effects on growth and cell division, various complexes
of platinum group metals that are not cytostatics, potas-
sium and sodium salts, etc. [1]. All these chemicals
slow down root growth only at the concentrations
approaching the lethal ones, through a general distur-
bance of the cell structure and metabolism. In such
cases, any specific mechanisms of growth-inhibiting
activity are unlikely. It is therefore vital to determine
how close are the concentrations under study to the
lethal ones, the point missed by most authors. The
majority of studies on the physiological effects of
heavy metals were carried out using the solutions of
heavy metals at the concentrations evidently approach-
ing the lethal ones. In such cases, it is impossible to
assess the selectivity of the toxic effect of a heavy metal
as well as any other agent, because other processes
affected by the agent may mediate the toxic effect. In
this context, especially interesting are the processes
unaffected by high concentrations of heavy metal salts.
Little temporal changes in root growth inhibition are
characteristic of all chemicals affecting cell division

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50 No. 3 2003

 COMPARATIVE IMPACTS OF HEAVY METALS 405

nonselectively [1]. On the contrary, the effect of the 140 (a) trans-[Pt(NH3)2Cl2]
specific inhibitor of cell division is dramatically
enhanced with the prolongation of exposure, even 120
though sometimes the rate of root growth is not affected 1
100 2
within the first day of incubation. We believe that the
following mechanism is at work in this case. The grow- 80
ing root tip comprises the meristem, where cells divide, 60
and the elongation zone, where cells reach their final
size due to rapid growth. At the particular moment of 40
observation, the root growth rate is determined by the
growth of elongating cells, because they grow much 20

faster than the meristematic cells. However, the growth
by elongation proceeds only for a short period, and to
maintain root growth, uninterrupted cell transition to
elongation is necessary. Within one mitotic cycle under
usual growth conditions, the cells of the basal half of
the meristem pass to elongation, and the size of mer-
istem is maintained due to continuous cell divisions in
its apical half. Finally all cells, except the outmost api-
cal cells of the quiescent center, leave the meristem.
The life-span of cell residence in the meristem is quite
stable, and hence with cell division slowing down, the
rate of cell transit to elongation diminishes. To keep it
constant would mean to shorten the period of cell resi-
dence in the meristem. As root growth proceeds, the
rate of cell transit to elongation diminishes progres-
sively due to an exponential increase in the number of
cells in the progeny of meristematic cells that are pass-
ing to elongation at the particular time point. With the
chemicals that hamper both cell division and elonga-
tion, the inhibition of root growth will not develop with
time, because from the very beginning, growth inhibi-
tion is determined by the inhibition of elongation. In the
present study, growth inhibition was not accompanied
by dramatic time-dependent growth retardation. It fol-
lows that the chemicals under study do not manifest a
selective cytostatic effect.
Two curves in Fig. 6 illustrate root growth inhibition
by two platinum complexes: a cytostatic (cis-isomer)
and another compound (trans-isomer) affecting both
cell division and elongation. At low concentrations suf-
ficient for specific inhibition of cell divisions by the cis-
isomer, the extent of inhibition dramatically increased
with exposure duration. trans-Isomer did not produce
such an effect. Previously we used the analysis of the
curves shown in Fig. 6 in order to select highly cyto-
static compounds among platinum complexes obtained
by directed synthesis [1].
Effect of Heavy Metal Salts on Root Branching
All the salts of heavy metals under the present study
did not affect the duration of lateral root development
even when the primary root growth was drastically
inhibited (Table 3, Fig. 2). These data corroborate the
earlier evidence that demonstrated the root branching to
be exceptionally resistant to heavy metals and other
toxicants [1, 17]. With invariant duration of lateral root
development, the zone of lateral roots was shortened, as
I, % of control treatment
0
~
0 1 2 3
140 (b) cis-[Pt(NH ) Cl ]
3 2 2
120
1
100
2
80
60
40
20
0
~
0 1 2 3 4
–log[C], g/l
Fig. 6. Root growth inhibition by (a) trans- and (b) cis-com-
plexes of platinum.
The curves are derived from [5]. Exposure time of (1) 0 to
24 h and (2) 48 to 72 h.
compared to the control roots (Table 3), when the pri-
mary root growth was inhibited. These changes make
the root system more compact as a general response to
the toxic effects of heavy metals [4, 5, 18, 19].
In contrast to heavy metals, all chemicals with their
inhibitory effect dramatically enhanced during expo-
sure blocked root branching. The inhibitors with the
impact practically unaffected by the exposure duration
did not considerably change the duration of lateral root
development. The inhibition of both cell division and
elongation by heavy metal salts was also corroborated by
direct length measurements of fully-elongated cells and
by the data on the rate of cell formation [1, 17, 20, 21].
Heavy metals were reported to affect cell wall plas-
ticity, presumably because the metal ions interact with
the carboxyl groups of uronic acids, a cell wall compo-
nent [22–24]. However, we demonstrated [1] that at
concentrations approaching the lethal ones, various
chemicals, including those not reacting with uronic
acids, decreased cell length by the end of their growth.
The decrease in cell wall plasticity could result from
disordered metabolism, rather than from the direct

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 50 No. 3 2003

406 IVANOV et al.
interaction of toxic chemicals, including heavy metals,
with cell wall constituents.
We conclude from the data presently obtained that
all the salts of heavy metals under study inhibit root
growth nonselectively at the concentrations approach-
ing the lethal levels. The inhibition results from numer-
ous structural and metabolic disorders produced by
heavy metal ions similar to those observed when heavy
metals affected other physiological processes. The vari-
ations in toxicity among the heavy metals under study
are primarily related to different ion affinities for SH-
groups.
ACKNOWLEDGMENTS
The authors thank Dr. N.V. Obroucheva for her crit-
ical suggestions and help in the course of this manu-
script preparation.
This work was supported by the Russian Foundation
for Basic Research, project nos. 00-04-48434a and 01-
04-06367.
REFERENCES
1. Ivanov, V.B., Root Growth Responses to Chemicals, Sov.
Sci. Rev., Ser. D, 1994, pp. 1–70.
2. Levina, E.N., Obshchaya toksikologiya metallov (Gen-
eral Toxicology of Metal), Leningrad: Meditsyna, 1972.
3. Wang, W., Root Elongation Method for Toxicity Testing
of Organic and Inorganic Pollutants, Environ. Toxicol.
Chem., 1987, vol. 6, pp. 409–414.
4. Breckle, S.-W., Growth under Stress: Heavy Metals,
Plant Roots: The Hidden Half, Waisel, Y. and Kafkafi, U.,
Eds., New York: Marcel Dekker, 1991, pp. 351–373.
5. Hagemeyer, J. and Breckle, S.-W., Growth under Trace
Element Stress, Plant Roots: The Hidden Half, Waisel, Y.
and Kafkafi, U., Eds., New York: Marcel Dekker, 1996,
pp. 415–433.
6. Willkins, D.A., The Measurement of Tolerance to
Edaphic Factors by Means of Root Growth, New Phytol.,
1978, vol. 86, pp. 623–633.
7. Ivanov, V.B., Bystrova, E.I., Dubrovsky, J.G., and
Ploshinskaya, M.E., Duration of Lateral Root Formation
in Maize Seedlings as Effected by Diverse Factors, Root
Demographics and Their Efficiencies in Sustainable
Agriculture, Grasslands and Forest Ecosystems,
Box, J.E., Ed., Dordrecht: Kluwer, 1998, pp. 777–787.
8. Metal Ions in Biological Systems, Concept on Metal Ion
Toxicity, Singel, H. and Singel, A., Eds., New York: Mar-
cel Dekker, 1986, vol. 20. Translated under the title
Nekotorye voprosy toksichnosti ionov metallov, Mos-
cow: Mir, 1993.
9. Seregin, I.V. and Ivanov, V.B., Cadmium and Lead Dis-
tribution and Their Toxic Effects on Maize Roots, Plant
under Environmental Stress. Int. Symp., Moscow, 2001,
pp. 255–256.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
10. Neiboer, E. and Richardson, D.H.S., The Replacement
of the Non-Descriptive Term “Heavy Metals” by a Bio-
logically and Chemically Significant Classification of
Metal Ions, Environ. Pollut., 1980, vol. 1, pp. 3–26.
11. Wong, M.H. and Bradshaw, A.D., A Comparison of the
Toxicity of Heavy Metals Using Root Elongation of Rye
Grass, Lolium perenne, New Phytol., 1982, vol. 91,
pp. 255–261.
12. Karataglis, S., Estimation of the Toxicity of Different
Metals Using as Criterion the Degree of Root Elongation
in Triticum aestivum Seedlings, Phyton, 1987, vol. 26,
pp. 209–217.
13. Ernst, W.H.O., Vercleij, J.A.C., and Schat, H., Metal
Tolerance in Plants, Acta Bot. Neerl., 1992, vol. 43,
pp. 229–248.
14. Ernst, W.H.O., Effects of Heavy Metals in Plants at the
Cellular and Organismic Level, Ecotoxicology, Ecologi-
cal Fundamentals, Chemical Exposure and Biological
Effects, Schuurman, G. and Markert, B., Eds., Heidel-
berg: Wiley, 1998, pp. 587–620.
15. Seregin, I.V. and Ivanov, V.B., Physiological Aspects of
Cadmium and Lead Toxic Effects on the Higher Plants,
Fiziol. Rast. (Moscow), 2001, vol. 48, pp. 606–630
(Russ. J. Plant Physiol., Engl. Transl.).
16. Nichols, R. and Frost, C.E., Post-Harvest Effects of Eth-
ylene on Ornamental Plants, Ethylene and Plant Devel-
opment, Roberts, J.A. and Turker, G.A., Eds., London:
Butterworths, 1985, pp. 343–351.
17. Ivanov, V.B., Bystrova, E.I., Obroucheva, N.V., Anti-
pova, O.V., Sobotik, M., and Bergmann, H., Growth
Response of Barley Roots as an Indicator of Lead Toxic
Effects, Angew. Bot., 1998, vol. 72, pp. 140–143.
18. Arduini, I., Godbold, D.L., and Onnis, A., Cadmium and
Copper Change Root Growth and Morphology of Pinus
pinea and Pinus pinaster Seedlings, Physiol. Plant.,
1994, vol. 92, pp. 675–680.
19. Breckle, S.-W., Growth of Tree Roots under Heavy
Metal (Pb-) Stress, Acta Phytogeogr. Suec., 1996,
vol. 81, pp. 39–43.
20. Nesterova, A.N., Effects of Lead, Cadmium, and Zinc
Ions on the Meristem Organization and Growth of Maize
Seedlings, Cand. Sci. (Biol.) Dissertation, Moscow:
Moscow State Univ., 1989.
21. Obroucheva, N.V., Bystrova, E.I., Ivanov, V.B., Anti-
pova, O.V., and Seregin, I.V., Root Growth Responses to
Lead in Young Maize Seedlings, Plant Soil, 1998,
vol. 200, pp. 55–61.
22. Lane, S.D., Martin, E.S., and Garrod, J.P., Lead Toxicity
Effect on Indole-3-Acetic Acid-Induced Cell Elonga-
tion, Planta, 1978, vol. 144, pp. 79–84.
23. Burzynski, M. and Jacob, M., Influence of Lead on
Auxin-Induced Cell Elongation, Acta Soc. Bot. Pol.,
1983, vol. 52, pp. 231–239.
24. Barcelo, J., Poschenrieder, Ch., Andren, I., and Gunse, B.,
Cadmium Induced Decrease of Water Stress Resistance
in Bush Bean Plants (Phaseolus vulgaris, cv. Con-
tender), Plant Physiol., 1986, vol. 125, pp. 17–25.
Vol. 50 No. 3 2003