Effect of Molybdenum, Chrome and Cadmium Ions on Metamorphosis and Erythrocytes Morphology of the Marsh Frog Pelophylax ridibundus (Amphibia: Anura)
Ilona E. Stepanyan,
Anush S. Tsarukyan
Yury P. Petrov
The effect of high concentrations of molybdenum (Mo), chrome (Cr (VI)) and cadmium (Cd) ions on the metamorphosis of the Eurasian marsh frog Pelophylax ridibundus under laboratory conditions have been studied. The effects of these metals at concentrations exceeding Maximum Permissible Concentration (MPC) on the growth, survival and erythrocytes morphology of tadpoles and young frogs have been determined. Morphometry of mature animals and tadpoles and also their erythrocytes for assessment of this effect were used. The high levels of tadpole mortality during 1-20 days of metamorphosis were detected when animals were exposed to high cadmium (2.4 mg L-1) and chrome (4.2 mg L-1) concentrations. After 20 days of metamorphosis, both the survival of tadpoles and the growth of 30 and 60 day animals, exposed to the high concentration of chrome were decreased. Cadmium treatment slightly decreased the survival of tadpoles after 20 days. The morphology of erythrocytes of Pelophylax ridibundus was also changed in experimentally polluted water. In experimental environment containing molybdenum and chrome, the area and perimeter of young frog erythrocytes slightly differed from controls. The effects of long-time exposure to cadmium on the morphology of erythrocytes (area and perimeter) were substantially different than those of chrome and molybdenum. Thus, high concentrations of cadmium and chrome in the aquatic habitats are a danger for amphibians, especially, for the early stages of metamorphosis.
to cite this article:
Ilona E. Stepanyan, Anush S. Tsarukyan and Yury P. Petrov, 2011. Effect of Molybdenum, Chrome and Cadmium Ions on Metamorphosis and Erythrocytes Morphology of the Marsh Frog Pelophylax ridibundus (Amphibia: Anura). Journal of Environmental Science and Technology, 4: 172-181.
Received: June 27, 2010;
Accepted: August 16, 2010;
Published: September 24, 2010
With the development of industry, technogenic-pollutant migration has led to the contamination of water, soil and air systems. Industrial and power plant waste, as well as transport exhaust, contain toxic substances such as heavy metals, which accumulate in soil and water biotopes.
According to participation in biochemical processes, metals could be divided
into two groups: (1) metals participating in processes of normal growth, development
and reproduction, but having a toxic effect on living organisms in high concentrations
(molybdenum, bismuth, manganese, cobalt, copper, zinc and chrome) and (2) metals
not playing an important role in biochemical processes of living organisms and
demonstrating a high toxicity with low concentrations. They can be accumulated
in an organism during a long exposition (cadmium, lead and mercury) (Elinder
and Piscator, 1978).
Cadmium and chrome are wide spread toxic contaminants. They enter water biotopes
from many sources including natural (rocks, groundwater) and industrial and
may accumulate in the organs of aquatic animals and plants. Toxic effects of
cadmium and chrome (Cr (VI)) have been poorly studied in amphibian species (Selvi
et al., 2003; Rollins-Smith et al., 2004;
Snodgrass et al., 2005; Stacy
et al., 2005; Sura et al., 2006; Mouchet
et al., 2007; Sharma and Patino, 2008).
The study of toxic effects of the cadmium, chrome and molybdenum in aquatic
organisms, particularly in amphibians, has great implications, since they are
widely spread in the polluted biotopes. Amphibians are a key component of many
ecosystems and their disappearance may complicate efforts to manage ecosystems
on a sustainable basis (Corn, 1994). Amphibians are widely
used as bioindicators to detect the presence of toxic agents in water, mainly
because their high sensitivity makes them ideal for genotoxity monitoring of
aquatic environments (Stacy et al., 2005; Sura
et al., 2006; Edwards et al., 2006;
Zhang et al., 2007; Chen
et al., 2007). The study of the effects of toxic metals on the development
of amphibians will help to identify both the degree of species survival and
the causes of the decline in their populations.
Presently, the content of some heavy metals in the soil and water biotopes
in the recreational sites on the territory of Armenia was increased up to 2
or 3 times from Maximum Permissible Concentration (MPC) (Avetisyan,
2007). There are many studies of accumulation in water and soil biotopes
of Mo, Cr (VI) and Cd and their toxic effects on plants and human development
that have been carried out in Armenia (Sagatelyan et
al., 2003, 2007; Belyaeva,
2007; Mezhunts and Navasardian, 2007; Sahakyan
and Amirkhanyan, 2007). However, there are no studies in Armenia regarding
the effects of these metals on animals, in particular lower vertebrates such
as amphibians. The Eurasian marsh frog Pelophylax ridibundus (Selvi
et al., 2003). Rana ridibunda is a widespread species, which
inhabits all humid and freshwater habitats in the territory of Armenia. This
species, due to wide distribution, high sensitivity and short period of development
is an ideal model to study the toxic effect of heavy metals.
The aim of this study is to detect the toxic effects of high concentration (exceeding Maximum Permissible Concentration, MPC) of cadmium, chrome and molybdenum ions on the development during metamorphosis and morphology of erythrocytes of Pelophylax ridibundus.
MATERIALS AND METHODS
Eggs of Pelophylax ridibundus (Selvi et al.,
2003) were collected from a clean pond located near Vohchaberd village (Kotaik
region) in April 2008 and incubated in glass containers with water from the
natural habitat, up to a hatching of tadpoles. After hatching, the one-day tadpoles
were placed into 40 L glass containers each with 30 animals. The tadpoles were
kept in the laboratory under natural conditions of light-dark period (16:8 h),
water temperature (25°C) and constant concentration of studied ions. Control
group of tadpoles was incubated in dechlorinated tap water. One group of tadpoles
was incubated in dechlorinated tap water+cadmium; second group of tadpoles was
incubated in dechlorinated tap water+molybdenum and third group of tadpoles
was incubated in dechlorinated tap water+chromium (VI). Experiments were done
in two stages: exposing of animals in toxic solutions from one-day larvae to
10-days tadpoles and from 20-days tadpoles to young frogs (conditionally 60-days).
Numbers of the survived animals were calculated up every 10 days at all stages
of experiments. For modeling conditions with high content of heavy metals, toxicity
tests were done using the following concentrations of heavy metals: Cd (2.4
mg L-1), Mo (78.9 mg L-1) and Cr (VI) (4.2 mg L-1),
chosen as exceeding Maximum Permissible Concentrations (MPC) for water fishery
objects (State norms 184.108.40.2060-98). Solutions of Cd, Mo and Cr (VI) were prepared
from CdCl2; Na2MoO3 and K2Cr2O7
Measurements of the basic linear characteristics of the body (length of a body - L.b and length of a tail - L.cd) for 30 and 60-day animals were performed.
A blood films were made from a drop of blood according to the method of blood
cells smear (Carmena, 1971). The smears were fixed with
96% methanol and stained with 4% Giemsa stain. Two smears on each individual
have been prepared. Erythrocytes were estimated using to the geometric morphometrics.
The blood smears were examined under a light microscope with digital chamber
(Nikon Digital Camera COOLPIX 4500, USA) with the maximum optical zoom and objective
x20. All erythrocytes images were 1024x768 pixels. The area and perimeter of
erythrocytes were measured using Image J v.1.41 (Wayne Rasband, National Institute
of Health, Maryland, USA, http//rsb.info.nih.govij).
A polarization ratio was measured as Rp/Ra ratio according to Kuzminykh
and Petrov for a spreading of cultivated cells (Kuzminykh
and Petrov, 2004; Petrov et al., 2007). At
least 700 cells were analyzed per each specimen. A software package Origin 6.1
(OriginLab Corporation, USA) was used for obtain of curves of regression.
Metamorphosis of the marsh frog Pelophylax ridibundus under laboratory conditions proceeds on average for 60 days (control group). The dynamics of survival of tadpoles during the period of metamorphosis in tap water (control), as well as in molybdenum, cadmium and chrome solutions are shown in Fig. 1. All tadpoles incubated at studied concentrations of cadmium and chrome has died by tenth day of development. In contrast, molybdenum increased the survival of tadpoles.
For fitting of experimental data on survival rate the Fit Sigmoidal option
from Origin 6.1 was used. The S-curves of control and molybdenum data are shown
in Fig. 2. The initial parts of fitting curves coincide with
||Dynamics of the survival of Pelophylax ridibundus.
Control experiments, Mo treatment, Cd treatment, Cr (VI) treatment
||Fitting of the experimental value by S-curve. (1) Control
and (2) Mo treatment. Arrows indicate the experimental values with marked
deviation from the theoretical ones
||Fitting of the experimental values of the survival of Pelophylax
ridibundus by two S-curves (I and II). 1-control curve, 2-Mo-curve
Further, despite obvious correlation between control and experimental groups,
the experimental values (arrows) considerably differ from theoretical ones.
This suggests that the processes described by S-curves have changed approximately
after 30 days. The most successful fitting of experimental values in this part
(later 30 day) can be with the use of different S-curve (Fig.
3). Thus, the experimental data can adequately be described by a combination
of two S-curves-S-curve I (up to 30 days) and S-curve II (after 30 days).
The tadpoles, exposed in water environment with Cr (VI) and Cd during of ten days of metamorphosis have shown 90% mortality. Twenty-day tadpoles were exposed in water environment with Cr (VI), Cd and Mo and their number was taken as 100%. The results are shown in Fig. 4. The fitting curves are presented as S-curves. The Mo-and Cd-curves coincide and vary synchronously with the control. In the case of chrome treatment, the sharp decline of values and absence of synchronism with other variants (Mo, Cd) is observed.
||Effect of Mo, Cr (VI ) and Cd to the survival of the Pelophylax
ridibundus after 20 days of the metamorphosis. The number of twenty
days old tadpoles is 100% (1). Fitting was made with use of S-curves
||Effect of Mo, Cr (VI) and Cd on the Pelophylax ridibundus
body and tail growth. (a) Thirty day and (b) conditionally 60-day old
animals. Control values are 100%. Values: Mean±Error
During metamorphosis, the size of the body and tail of tadpoles changes and
legs develop. From 30-day, the size of body and tail of experimental tadpoles
are changed. The body and tail of 30-day (tadpoles) and 60-day frogs (young)
were measured to compare these important stages of metamorphosis. The results
are presented in Fig. 5, where absolute values of control
are shown as 100%. According to body and tail measurements, Cr (VI) causes a
significant decrease in tadpoles body growth (30 day) and frog body growth (60
day), but does not affect the development of frog tail. Cadmium delayed the
growth of 30 day tadpoles and young frogs (60 day). Molybdenum has not affected
body and tail sizes in 30 day tadpoles as well as in 60 days frogs.
The morphological analysis of erythrocytes of P. ridibundus at the final
stage of metamorphosis (60-day) was used to estimate the effect of Cd, Mo and
Cr (VI) at the cell level. The effects of Mo, Cd and Cr (VI) on morphological
cell parameters of erythrocytes (area and perimeter) are presented in Fig.
||Effect of Mo, Cr (VI) and Cd on erythrocytes morphology of
conditionally 60-days old Pelophylax ridibundus. A-Area, B-Perimeter,
C-Polarization coefficient (Rp/Ra). Values: Mean±Error
There is a small decrease in the area and perimeter of erythrocytes of frogs
incubated in water containing of Cr (VI) and Mo in contrast to control. However,
no differences of Rp/Ra ratio between the erythrocytes of P. ridibundus incubated
in tap water (control) and in mediums containing chrome and molybdenum were
found. The significant differences in morphology of erythrocytes are observed
after incubation of tadpoles in cadmium-containing medium. The area of these
erythrocytes is increased approximately 1.5 times, while the perimeter is 1.2
times that of the control ones. Erythrocytes of frogs incubated with cadmium
are polarized more strongly than in other cases.
Molybdenum and chromium are necessary microelements in small concentrations
and are toxic in high doses. The metabolism of these metals in aquatic vertebrates,
particularly in amphibians, is poorly studied under environmental pollution.
The chemical analysis of soils and freshwater bodies from industrial and recreational
territories of Armenia indicated the presence of compounds of Cd, Cr (VI) and
Mo at concentrations 2 or 3 times higher than the maximum permissible concentrations
(Avetisyan, 2007; Sagatelyan et
al., 2007; Mezhunts and Navasardian, 2007; Sahakyan
and Amirkhanyan, 2007). In present study we have demonstrated that each
of the mentioned metals has a specific effect on the survival and metamorphosis
of the marsh frog Pelophylax ridibundus. At the studied concentrations
exceeding MPC, molybdenum promotes the survival of tadpoles and young frogs.
Ions of chromium and cadmium showed a strong toxic effect, increasing the animals
mortality from hatching and up to 10 days of development. However, tadpoles
after 20 day of development became more resistant to toxic effect of cadmium
and chromium (Fig. 1). After chromium treatment the viability
of both tadpoles and young frogs (60 day) was decreased (Fig.
1-4). The analysis of the theoretical curves of survival
of frogs from hatching up to the young frog stages indicates a two-stage process.
Both stages are described by the S-curve.
The study of the morphometric parameters of tadpoles and young individuals
of P. ridibundus shows two polar processes during the metamorphosis.
These processes are body size increase and synchronous tail reduction. This
means that process of cells proliferation is accompanied by a local process
of the programmed cell death. It is possible to assume that both processes should
be relatively autonomous and synchronous. This assumption is in close accord
with data received on Eurasian green toad Bufo viridis (Sahin
and Balcan, 2006). Authors have shown programmed cell death of skeletal
muscle tissues of the regressing tail of tadpole.
Ions of Cr (VI) inhibit the growth of tadpoles (Fig. 5a, b),
but do not affect decrease in tail size (Fig. 5b). This indicates
that the processes of proliferations in the bodies of animals are relatively
independent from processes in the tails. The inhibition of cell proliferation
in the body is increased during growth of tadpoles. Since, the processes of
cell proliferation up to 60th day (young frogs) were decreased only in the body
of animals, we assume that ions of chrome in the second stage do not influence
the programmed cell death in their tail.
Present data show that Cd ions do not affect cell proliferation or cells death of 30 day tadpoles. However, Cd promotes a decrease of the size of the body and of the tail of 60 day animals (young frogs). Therefore, it could be assumed that Cd, in contrast to Cr (VI), enhances the processes of apoptosis in the tail at stage II.
Similar effects of Cd and Cr (VI) are known for others species of amphibians.
The development of tadpoles of Xenopus laevis and Rana pipiens
during metamorphosis, survival of adult individuals during hibernation, as well
as oogenesis were inhibited by cadmium (Karasov et al.,
2005; Stacy et al., 2005; Gross
et al., 2007; Sharma and Patino, 2008). The
stage-dependent uptake features also related to toxic effects were reported
for high concentration of chrome during developmental stages of Brazilian Hylidae
frogs (Natale et al., 2000). The above mentioned
data indicate that cadmium and chrome exhibit high toxic effect during developmental
stages of amphibians.
In spite of the numerous data regarding the toxic effect of Cr (VI), Cd and
Mo (Natale et al., 2000, 2006;
Loumbourdis, 2005; Fenoglio et
al., 2006; Tejada-Jimenez et al., 2007;
there are insufficient data (Carmena, 1971; Vasilkov
and Gricenko, 1989) about the effects of these metals on cell parameters.
Erythrocytes are very convenient object for studying of toxicity of investigated
substances at cellular level (Witeska and Kosciuk, 2003;
Velcheva et al., 2006; Arnaudov
et al., 2008). First, they can be received easily without killing
of animals and, secondly, they are true cells. According to the note, the obtained
results with the high probability it is possible to extrapolate on any other
cells of an organism. In the some known studies (Witeska
and Kosciuk, 2003; Velcheva et al., 2006;
Arnaudov et al., 2008) various pathological changes
in the morphology of fishes and frogs erythrocytes under the influence
of increasing copper and zinc concentrations were found out.
The analysis of morphological parameters of erythrocytes of Pelophylax ridibundus has allowed us to estimate the effect of Mo, Cd and Cr (VI) at the cell level (Fig. 6). Molybdenum and chrome do not significant affect the form (Rp/Ra ratio), but they have effect on size (area and perimeter) of erythrocytes in comparison to control. This could indicate that the effects of chrome on processes of cells proliferation and apoptosis do not correlate with their direct influence on cells, if we consider the erythrocyte as a representative cell. Cr (VI) probably has a regulatory function at the level of the organism. On the contrary, the strong change of the size and the form of erythrocytes under cadmium treatment indicates that its effect on cell proliferation and apoptosis correlates with a direct influence on the structure of cells.
Cadmium is known to stimulate the deformation of cells and their damage, the
increase in numbers of apoptotic bodies (Loumbourdis, 2005).
Chrome induces the delay of growth and necrotic changes. Considering the known
toxic effect of Cd and Cr (VI) and present results, we conclude that chrome
and cadmium in high concentrations represent the greatest danger to amphibians
in the water habitats, especially at early stages of development. The effect
of some heavy metals on enzymes of human erythrocytes was shown (Al-Mustafa,
2006). However, the literature data about morphometry of erythrocytes of
frogs during their long contact with heavy metals were not found.
The physiological role of molybdenum in life activity of animals is multiform
(Tejada-Jimenez et al., 2007). Taking into account
the important role of Mo and our results, we assume that for animals in the
control experiments, the natural background of Mo was lower than physiologically
So, examination of the toxic effects of the of high concentrations of molybdenum (Mo), chrome (Cr (VI)) and cadmium (Cd) ions on the metamorphosis and erythrocytes morphology of the eurasian marsh frog Pelophylax ridibundus under laboratory conditions has shown that the chrome ions was inhibit the growth of body and the survival of tadpoles. Cadmium treatment induces the slight decrease in both the survival of tadpoles after 20 days and in their growth, as well as affects erythrocytes morphology. Molybdenum treatment increases the survival of tadpoles and changes slightly erythrocytes morphology.
The authors wish to thank Drs. M. Arakelyan, O. Glebov, D. Abramyan and N. Glebova for help at the time of preparation of the manuscript.
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