geochemistry and genotoxicity of the heavy metals in mine-abandoned areas and wasteland in the hetai...

ORIGINAL ARTICLE

Geochemistry and genotoxicity of the heavy metalsin mine-abandoned areas and wasteland in the Hetaigoldfields, Guangdong Province, China

Li Miao • Yueliang Ma • Ruisong Xu •

Wen Yan

Received: 30 May 2010 / Accepted: 27 June 2011 / Published online: 30 July 2011

� Springer-Verlag 2011

Abstract The article presents the geochemical and

biogeochemical characteristics of heavy metal elements

within the soil–plant system in the Hetai mine-abandoned

areas and wasteland, China. The cytogenetic toxic effects

of gold mine wastelands environment on the chromosome

and micronucleus were analyzed by genetic methods. The

results showed that abundances of Au and Au-associated

heavy metal elements such as Cu, As, Cd, Pb and Hg in

soils, plants and the pigments in those abandoned mine

areas were much higher than those in the background

region. The cell structures of the plants in the abandoned

mine areas were anomalous and aberrant, and there were

many nano-metal particles diffused in the cells. Moreover,

the heavy metal elements in those abandoned mine areas

had entered the ultrastructure and caused toxicities to the

organism.

Keywords Heavy metal elements � Environmental

biogeochemistry � Genotoxicity � Hetai

Introduction

Resources and environment are the essential conditions

needed for human beings to survive, breed and develop.

Now, people pay more and more attention to these prob-

lems such as lack of resources, environmental pollution and

ecological deterioration. The problem of the environment

and ecosystem caused by mining has become an important

field for environmental science research. Mining and pro-

cessing metal ore can be a significant source of heavy metal

contamination to the environment and have negative

impacts on the environment and ecosystem (Dudka and

Adriano 1997; Navarro et al. 2008; Zhuang et al. 2009).

Heavy metals are known to be extremely toxic at trace

concentrations and, unlike organic pollutants, they are not

biodegradable. They can induce clastogenic and aneugenic

effects including mitosis and cytokinesis disturbances

(Dovgaliuk et al. 2001). Despite being a principal con-

tributor to rapid economic growth, the mining industry

produces a large amount of mine tailings and abandoned

lands as well as other environmental damages in China (Li

2006; Sheoran and Sheoran 2006). The mine tailings and

wasteland are of potential risk to human health due to the

accumulation of toxic heavy metals. In China, mining areas

with different dimensions and contamination levels were

abandoned, and only a few were submitted to recovery

programmers. Nonetheless, some of the soils and waters

near small mines are used for agriculture without any

assessment of environmental and human health risks.

Earlier studies used chemical analysis to measure and

quantify the pollutants. However, this method failed to

obtain accurate results, due to the difference in sensitivity

between various instruments and as the analysis included

those chemical compounds that are not available to the

biological system. Therefore, a biological approach is

L. Miao � W. Yan

Key Laboratory of Marginal Sea Geology,

Chinese Academy of Sciences, 510301 Guangzhou,

GD, People’s Republic of China

L. Miao � W. Yan

South China Sea Institute of Oceanology,



Y. Ma (&) � R. Xu

Guangzhou Institute of Geochemistry,



e-mail: [email protected]

123

Environ Earth Sci (2012) 65:1955–1964

DOI 10.1007/s12665-011-1176-8

useful to integrate the effects of all the bioavailable con-

taminants and their interaction (Eom et al. 2007; Ansari

and Mali 2009), and the necessity of using biological

monitors has arisen (Simkiss et al.1982; Fernandez et al.

2005). With increasing concerns as to the genotoxicity of

hazardous chemicals and pollutants in water, air and soil,

several plant system bioassays have been developed for

detecting the genotoxicity of environmental pollutants. The

tests routinely employed include the micronucleus test and

chromosomal aberration assay in root tips (Marcato-

Romain et al. 2009). Several end points can be monitored

in these fast-dividing cells, such as chromosome aberra-

tions, sister chromatid exchanges and micronuclei. Micro-

nucleus formation is the most frequently used as the most

effective, and the simplest indicator of DNA damage. The

micronucleus test is sensitive to both mitoclastic and

clastogenic agents that can reflect different forms of envi-

ronmental stress (Burgeot et al. 1995). The assay has been

successfully used with reliable results for genotoxicity tests

of environmental pollutants and different chemicals

(Marco et al. 1990; Kirsch-Volders et al. 2003).

The micronucleus test on Vicia faba root tips exposed to

pollutants has been widely adopted for the determination of

the genotoxicity of contaminated soil and water (Chen et al.

2004). The V. faba micronucleus test was used to evaluate the

genotoxicity of olive mill wastewater generated in mills

producing olive oil in Morocco, and the data illustrated that

the micronucleus test was a very sensitive and useful method

that allowed the detection of both clastogenic and aneugenic

effects (Hajjouji et al. 2007). Manier et al. (2009) found the

micronucleus frequency scores in V. faba root cells after

direct exposure to contaminated soils were increased in a

statistically significant manner. Rizzoni et al. (1995) studied

the Tiber River by means of the V. faba micronucleus test and

the results evidenced that the Tiber River presented muta-

genic pollution. Song et al. (2006) examined the genotoxicity

effect of soil after long-term wastewater irrigation and indi-

cated that micronucleus frequencies were 2.2–48.4 times

higher compared with the control, and chemical analysis and

genotoxicity assays were valuable complements to each other

in identifying the potential ecological risks of pollutants

brought into the soil ecosystem. Recently, to evaluate the

contamination of environmental samples, it has been rec-

ommended to combine toxicity bioassays and chemical

analyses (Marinella and Damia 2003).

From a sustainable development point of view, both the

continuity of mining activities and the minimization of

possible adverse effects of these activities are required. With

this purpose, a survey was conducted in the Hetai goldfield of

Guangdong Province, China. A battery of chemical and

biological parameters was adopted to describe and assess the

integrated situations of the mine wasteland. Chemical

analysis of Au and Au-associated elements including

Ag, Cu, Pb, Zn, Hg and As was performed, and the effects on

plants growing in the mine-abandoned areas and wastelands

were also studied. Moreover, the V. faba root micronucleus

test was selected to assess the potential genotoxicity of the

mine-abandoned areas. The objectives of the present study

were to (1) investigate the accumulation and distribution of

Au and Au-associated heavy metal elements in soil–plant

systems in the Hetai gold mine-abandoned areas, (2) to

obtain more information on physiological and ecological

characteristics of indigenous plants in goldfield suffering

long-term stress of heavy metal elements and (3) to assess

the genotoxicity potential of the gold mine-abandoned areas

and wastelands. It will provide chemical values and geno-

toxicity evidences of background and anomalous data for

evaluating the quality of mine-abandoned areas and waste-

lands in the Hetai goldfield, more than 20 years after the

abandonment of the mining activity.

Materials and methods

Study area

Hetai, the largest goldfield in South China, is located in

the Yunkai metamorphic terrane, which crops out over a

broad region of western Guangdong and southeastern Guan-

gxi Provinces (23�1703000N–23�2000000N, 112�1500000E–

112�2200000E), and covers a district area of 455 km2 approx-

imately. Rocks hosting the ores were silicified and sericitized,

and the main gangue minerals in the lodes are quartz and

sericite. Sulfide and Au ores were superimposed on the len-

ticular structures, producing disseminated and veinlet ores

with mainly chalcopyrite, pyrite, pyrrhotite and native gold

assemblages (Duan et al. 1992; Zhang et al. 2001). Most soil

types in the study area were classified as latosols, which were

developed on schists and granites. Soil profiles vary greatly in

thickness throughout the region from a few centimeters to

over a meter as a result of the topography. The pH of the soils

varies from 4.4 to 5.4. These soils, presenting different stages

of weathering, contain gravel particles of schist and are rel-

atively enriched in Au and other trace elements.

The Hetai area has a subtropical monsoon type of cli-

mate, characterized by high precipitation and mild weather.

Subtropical secondary forests grow profusely with per-

centage of vegetation coverage being greater than 95%,

and the dominant vegetation species in the studied region

are P. massoniana, P. tomentosa, D. dichotoma, etc.

Sampling and chemicals analysis

During the Hetai goldfield biogeochemical survey, soils

were collected from both the gold mine-abandoned areas

and wastelands and from areas 2–15 km away to establish

1956 Environ Earth Sci (2012) 65:1955–1964

123

the background values. Soil samples were air dried, di-

saggregated and screened through a 1-mm polyethylene

sieve to remove stones, roots and other larger particles.

Representative sub-samples of material were finely ground

to pass a 200-mesh polyethylene sieve. Precautions were

taken to avoid contamination during sampling, drying,

grinding and storage. All samples were digested using

concentrated HF–HNO3 and aqua regia. Samples were then

heated to dryness in a water bath and re-dissolved in

HNO3. Heavy metal elements were analyzed by using

inductively coupled plasma mass spectrometry (ELAN

6000), and As and Hg by hydride generation spectrometry

at the Guangzhou Institute of Geochemistry, Chinese

Academy of Science. For details of the chemical treatment

and machine measurement, refer to Liu et al. (1996).

Plant samples were collected following the method

described by Brooks et al. (1995), and approximately 200 g

of the roots, stems and leaves were separately taken from

each plant where the soil samples were collected. Fresh

samples were washed using distilled water and de-ionized

water for three times, then separately dried at 60�C for

chemical analysis. The dried leaf samples were crushed

separately through an agate grinder and the crushed

material was passed through a sieve. Chemical analysis

was carried out by ICP-MS following the same analytical

procedures used in the soil analysis and results were

expressed on a dry weight basis.

Ultrastructural analysis

Fresh pieces (1 9 1 mm) were cut with a razor blade from

the basal area of each leaf and fixed in 2.5% (w/v) glu-

taraldehyde in phosphate buffer (pH = 7.2). Leaf pieces

were subsequently fixed in 1% osmium tetroxide solution

in phosphate buffer (pH = 7.2), dehydrated in an ascend-

ing ethanol series and embedded in epoxy resin (Epon

812).Ultra-thin sections were cut with an ultra-microtome

and then stained with uranyl acetate and lead citrate and

studied with a transmission electron microscope (TECNAI

12) in the South China Agricultural University. The com-

positions of plant cell were measured by an energy spec-

trometer (Edax company genesis 2000, with a detection

limit of 1%).

Genotoxicity test

Fifty grams of soil (passed through a 5-mm sieve) was

extracted thoroughly with 250 ml of double distilled water at

room temperature for 24 h at 125 rpm in a mechanical

shaker. Soil aqueous extracts were prepared by 24-h

extraction with distilled (dried soil:water, 1:2) and stirred for

24 h. Then, the mixture was placed at 4�C for 24 h for

decanting. For this study, broad bean seeds of V. faba

(provided by the Huazhong Normal University) were used.

Dry seeds of V. faba were soaked for 24 h in deionized water.

Seedling coats were then removed and the seedlings were

allowed to germinate between layers of moist cotton. Once

roots had reached a length of 2–3 cm, six to eight of the

germinated seeds mentioned above with homogeneous

growth roots were exposed to soil water extract or negative

control (double distilled water and background area sam-

ples) for 5.5 h followed by a 24-h recovery period. After

exposure, root tips (meristem zones) were cut and placed

overnight in the dark in the Carnoy fixation solution con-

taining methanol and glacial acetic acid (3:1, V/V) at 4�C

and then stored in 70% ethanol. Root tips were washed with

distilled water several times and hydrolyzed with 1 M HCl at

60�C for 10 min. The root cap was removed before

squashing root tissues, and samples were stained by the

Feulgen technique; 1 mm slices of the mitotic zone from

well-stained root tips were immersed in a drop of 45% acetic

acid on a clean slide and squashed under a cover glass. The

slides were examined under a Zeiss microscope. At least

three slides were stained per replica and at least 1,000 cells

were scored from each slide. Therefore, the analysis was

conducted on an average of 5,000 cells per treatment. MCN

frequency was calculated from the number of MCN scored

divided by the total cells scored, and expressed in terms of

MCN/1000 cells. The MN% was calculated as following:

MCN ¼ Number of cells containing MCN=

Total number of cells counted� 1; 000&

Results and discussion

Ecological characteristics of plants

Plants in the Hetai goldfield along the ore vein were

characterized by leaves that were rough, lacking luster and

having a yellowish discoloration on the surface, and by

wizened and perished branches and leaves. The plants such

as P. massoniana, P. tomentosa and D. dichotoma all

presented a linear yellow and yellowish color in the cru-

shed zone of the goldfield areas, which distinguished them

from the dark glossy green plants in the background area.

The plants growing on the gold ore presented unhealthy

ecological characteristics.

Concentration of heavy metals in soils

The statistical analysis of data on heavy metal element

contents of soils collected from the Hetai gold mine-

abandoned areas and wastelands including pithead,

wasteland and soil irrigated with wastewater and the

background area are summarized in Table 1. The results

Environ Earth Sci (2012) 65:1955–1964 1957

123

showed that the abandoned goldfields had higher content of

Au compared with the background areas, whereas the

abandoned fields had higher values for some of the gold

indictor elements, notably Cu, Pb, As and Hg. The content

of Au in pithead soil, mine wasteland and soil irrigated

with wastewater sites was 114, 232 and four times higher

than that in the background area, respectively. Higher

concentrations of Co, Ni, Cu, As, Cd, Pb and Hg were

reported in the pithead site. In addition, high concentrations

of Cu, Cr, Ni and As were also found in the mine wasteland

and soil irrigated with wastewater. The soils in this area

were acidic with pH values ranging from 4.4 to 5.4, which

can be explained by the presence of high levels of pyrite

and arsenopyrite that can be easily weathered. The envi-

ronment with low pH and wet climate is in favor of the

dissolution of trace elements (Carbonell-Barrachina et al.

1999, 2004; Ciftci et al. 2005). The higher values of the

heavy metal element contents indicated that significant Au

and Au-associated heavy metal element contamination

occurred in these abandoned soils.

Heavy metals accumulated in plants

Heavy metal element compositions of the different species

of plants are summarized in Table 1. The plants in the gold

mine abandoned fields showed anomalies with high

Table 1 Heavy metal element concentrations of the soils and plants in the study area (mg/kg)

Element V Cr Co Ni Cu Zn As Ag Cd Sn Au Hg Pb

Pithead soil

Soil 96.4 68 53.8 24.8 56.2 33.1 23.8 0.9 0.3 13.7 0.142 0.3 69.6

D. dichotoma

Root 1.53 18.6 2.54 8.95 17.2 14.8 0.56 0.03 0.11 0.65 21.1 0.1 19.1

Stem 0.05 0.39 0.28 0.44 2.05 6.11 0.14 0.01 0.05 0.07 0.58 0.05 8.86

Leaf 0.14 0.65 1 1.07 3.13 24.4 0.16 0.027 0.08 0.67 0.8 0.02 45.2

P. massoniana

Root 0.3 2.45 1.03 4.03 14.9 20 0.11 0.05 0.56 0.1 27.7 0.05 11.77

Stem 0.32 0.28 1.05 2.44 21.3 16.2 0.14 0.04 0.56 0.24 2.34 0.04 6.95

Leaf 0.11 0.24 2.44 7.94 9.99 24.5 0.23 0.06 0.21 0.28 1.7 0.04 3.73

Mine wasteland

Soil 107 105.3 3.38 23.1 85.7 40.5 15.9 0.69 0.2 17.5 0.2904 0.2 38.1

D. dichotoma

Root 0.3 3.13 0.88 2.28 8.51 14.5 0.23 0.01 0.148 0.159 1.02 0.019 48.3

Stem 0.28 1.33 1.17 1.84 5.09 38.8 0.39 0.016 0.176 0.899 2.22 0.031 151

Leaf 0.44 4.25 0.99 3.27 12.5 31 0.36 0.027 0.313 0.261 0.8 0.048 2.58

P. massoniana

Root 0.16 0.22 0.45 0.61 5.59 13.8 0.12 0.019 0.302 0.189 0.9 0.046 3.32

Stem 0.11 0.34 0.91 1.45 3.38 23.9 0.21 0.014 0.159 0.453 1.12 0.055 3.66

Leaf 1.37 1.2 0.35 1.66 3.76 51.3 0.33 0.004 0.081 0.128 2.24 0.05 8.91

Soil irrigated with wastewater

Soil 73.2 39.6 3.49 17 78.4 41.9 24.2 1.05 0.2 19.2 0.005 0.28 31.7

P. massoniana

Leaf 0.42 1.45 2.6 5.23 53.5 31.8 1.32 0.089 0.225 0.362 81.6 0.011 27.6

Background area

Soil 39.55 23.7 2.15 5.9 6.25 25.05 2.75 0.65 0.15 14.45 0.001 0.1 25.9

D. dichotoma

Root 2.07 12.735 0.425 7.335 5.255 20.25 0.29 0.055 0.235 0.765 0.665 0.05 19.85

Stem 0.055 1.215 0.05 0.805 2.37 9.4 0.24 0.005 0.155 0.13 0.42 0.03 12.36

Leaf 0.16 0.995 0.125 1.145 3.47 33.55 0.175 0.015 0.24 0.535 1.47 0.015 27.25

P. massoniana

Root 0.665 18.29 0.335 8.705 3.495 22.8 0.115 0.03 0.495 0.315 0.6 0.03 2.455

Stem 0.305 1.28 0.185 1.01 3.87 27.05 0.105 0.02 0.61 0.215 1.87 0.015 4.385

Leaf 0.125 0.65 0.31 0.95 2.43 27.05 0.3 0.02 0.225 0.335 1.3 0.015 1.915


123

concentrations of heavy metal elements. Au contents in the

roots of D. dichotoma and P. massoniana collected from

the pithead (21.1 and 27.7 lg/kg, respectively) were higher

than that from the background area. Among other heavy

metal elements, Pb, Cu, Co, Ni and As were the most

absorbed and accumulated in plants tissues in the pithead

sites. In the mine wasteland, Pb had the highest concen-

trations in D. dichotoma organs, especially in the leaves

with a concentration of 151 mg/kg. Except for Au and Pb,

the main heavy metals accumulated in D. dichotoma were

Cu, Cr and As. Furthermore, less proportion of those ele-

ments was transferred to the upper parts of the plant and the

rest remained in the roots. For P. massoniana, the content

of Cu was higher than that in the background samplings.

The content of other heavy metals in P. massoniana were

not different compared to that in the background area. The

leaves of P. massoniana that grew on soil that was irrigated

with wastewater was the one with the highest Au and Cu

content (81.6 lg/kg and 53.5 mg/kg, respectively). From

the geobotanical observations of the studied region, it can

be seen that these soil and plant samples were all charac-

terized by high concentrations of Au and Au-associated

elements in the goldfield. Au and several other heavy metal

elements such as Cu, As, Pb and Hg were good pathfinders

for the ore body in this region. The significant enrichments

of Au and other heavy metal elements in plant tissues were

associated with areas of gold mine. The element concen-

trations of the leaves were mainly influenced by rocks and

soils that the plants grew on.

Pigment contents in plants

The most important photosynthetic pigment is chloroplast,

which consists of two types: chlorophyll-a and chlorophyll-

b. It has been suggested that the Chl-b/Chl-a ratio is a useful

parameter to determine the physiological conditions and

photobionts subjected to heavy metals (Chettri et al. 1998).

In this study, the Chl-b/Chl-a ratio was used to quantify the

sensitivity of heavy metal elements effected the chloro-

phyll-a and chlorophyll-b in the gold mine-abandoned

areas. The concentrations of pigments measured in leaves

are shown in Table 2. Chlorophyll-a, chlorophyll-b and

total chlorophyll contents in the leaves of the abandoned

goldfield were all lower than that of the background value,

especially in P. tomentosa. The correlation analysis showed

that there was a negative correlation between the element in

the soil and the pigment content in the plant (Table 3). For

D. dichotoma, there was a negative relationship between the

pigment and Cr, Cu, Au and As in the soil. For P. mas-

soniana, the pigment was negatively correlated with Cd, Ag

and Hg in the soil. Therefore, the environmental matter

contents had noticeable effects on the contents of plant

Table 2 The concentration of pigments in the plants of the study area (mg/kg)

Plant Sampling site Chlorophyll-a Chlorophyll-b Carotenoid Chlorophyll

(a ? b)

Chlorophyll-b/

chlorophyll-a

P. massoniana Pithead soil 1,070 248 291 1,310 0.23

Mine wasteland 874 354 107 1,230 0.40

Soil irrigated with wastewater 996 183 292 1,180 0.18

Background area 1,400 375 361 1,780 0.27

D. dichotom Pithead soil 803 161 213 963 0.20

Mine wasteland 745 171 302 916 0.23

Background area 972 214 300 1,190 0.22

P. tomentosa Pithead soil 896 350 148 1,250 0.39

Mine wasteland 675 192 252 868 0.28

Background area 2,230 725 396 2,950 0.33

Table 3 The correlation analysis between the element in the soil and the pigment content in the plant

Pigment D. dichotom P. massoniana

Cr Ni Cu Au As Cd Ag Hg

Chlorophyll-a -0.635 -0.478 -0.590 -0.647 -0.652 -0.700 -0.753 -0.611

Chlorophyll-a -0.683 -0.630 -0.650 -0.641 -0.798 -0.891 -0.950 -0.872

Carotenoid -0.382 -0.479 -0.359 -0.292 -0.796 -0.833 -0.912 -0.834

Chlorophyll (a ? b) -0.649 -0.512 -0.607 -0.651 -0.686 -0.779 -0.836 -0.713

Chlorophyll-b/chlorophyll-a -0.366 -0.677 -0.389 -0.182 -0.741 -0.927 -0.987 -0.984


123

pigments. It was concluded that the mineralized zone had an

impact on living organisms and the growth of plants with

high concentrations of heavy metals, such as Au, Cu, Hg

and metalloids, such as As. These elements inhibited

chlorophyll and carotenoid biosynthesis and retarded the

incorporation of these pigments into the photosystems. The

presence of heavy metal and metalloid enrichment in the

abandoned area restrained the growth of cells and resulted

in declines in pigment contents of plants. Low pH values

observed in the study region were favorable for the mobility

of the toxic elements (Au, Cu, Hg and As), which entered

the cell and changed the compositions of the pigment, and

Mg and Fe that serve as central ions of leaf pigments

gradually decreased, thus causing the unhealthy ecological

characteristics of plants in the abandoned goldfields (Sto-

bart et al. 1985; Caspi et al. 1999; Boswell et al. 2002;

Rahman et al. 2007; Cabral 2003; Carreras and Pignata

2007).

Botanic cell tissue

From the transmission electron microscope picture, it can

be seen that there are no anomalous phenomena in the

botanic cell tissue of plants vegetated in the background

area (Fig. 1a, c, e, g). Cytoarchitectures of the leaves,

which included nucleolus, mitochondrion, chloroplast,

Golgi bodies and vacuoles, were natural and visible, and

boundaries were distinct and obvious (Fig. 1a). Numerous

big starch grains and less osmiophilic droplets were found

in the stoma (Fig. 1c). The mitochondria were normal, well

distributed and very clean (Fig. 1e). There were more

chloroplasts, the grana in the chloroplast were well

developed, and the thylakoid were compactly stacked

(Fig. 1g).

The cell and chloroplast structures of the leaves in the

abandoned goldfield were deformed and some were even

broken. As Fig. 1b shows, there were less chloroplasts, and

these were distorted or even disintegrated. The cell tissues

were irregular and less compact, or less dense, and the

grana in the chloroplast were disintegrated. The starch

grains disappeared, more osmiophilic droplets appeared

and were badly distributed in the stoma; the size of the

osmiophilic droplet was increased (Fig. 1d). Serious dam-

ages in ultrastructure caused by stress were indicated by

scattered nucleoli, condensed chromatin, almost empty

nuclei with nuclear membrane disrupted and nucleoplasm

flowing into the cytoplasm, swollen and partly dissolved

cristae of mitochondria, disrupted and collapsed chloro-

plast envelopes and some dissolved thylakoids that flowed

into the cytoplasm (Fig. 1d, h).

From the observation of the cell ultrastructural changes

in the plant collected from the abandoned goldfields, it was

found that nanometal particles diffused into the cell wall,

chloroplast and mitochondria. Those particles are distinctly

different from the phenol compounds found by Pijut et al.

(1990). The phenol compounds in the plant cell were round

in shape with diameters of 4–6 lm. In addition to those

phenol compounds found in cells, new ones appeared in the

form of irregular shapes and nano-particles (\1 lm). The

particles were deposited in the cell walls and chloroplast

membranes and caused the roughened or broken appear-

ance of the membranes. In this study, the nanometal par-

ticles were called electron-dense deposits and their

microstructure composition was analyzed by X-ray energy

spectrometer. The concentrations of the electron-dense

deposits in the cells are summarized in Fig. 2. The results

indicated that they comprise mainly C, O, Cu, Ni, Au and

Hg, which differed from the phenol compounds. It suggests

that Au, Cu and Pb may deposit within the botanic cell

tissue in the form of a high electron-density substance.

There was a remarkable similarity or correspondence in the

anomalous elements between the geochemistry and the

botanic cell. It was illustrated that the biogeochemical

anomalies were closely related to the botanic anomalies in

the cell aspect. Concerning the toxicity of the different

metals studied in the region, the harmful elements were Au,

Ag, Cu, Hg, and As, which can change the structure of the

cell at high concentrations. Song et al. (2004) have also

studied this cell anomaly in the goldfield. The variations of

the structure of the cell as stated above were the result of

the longtime stress caused by these elements with anoma-

lous concentrations in the Hetai goldfield. The harmful

metals probably entered the cell and altered the structure of

the cell wall and plasma membranes, lowering their

selective permeability. This subsequently allowed metal

cations to enter the cytoplasm, initiating a series of

degenerative processes that probably caused severe alter-

ations to different metabolic pathways such as photosyn-

thesis or oxidative mechanisms (Cabral 2003; Carreras and

Pignata 2007). Patra et al. (2004) also found that excessive

concentrations of metals result in phytotoxicity through:

(1) changes in the permeability of the cell membrane; (2)

reactions of the sulfhydryl (–SH) groups with cations; (3)

affinity for reacting with phosphate groups and active

groups of ADP or ATP; and (4) replacement of essential

ions.

The micronucleus rates of V. faba root tip cells

All soils were subjected to the V. faba micronucleus assay

for the evaluation of genotoxicity. The results shown in

Table 4 indicate elevated micronuclei frequency in all soils

compared with control. The frequency of micronuclei was

59.6, 47.51 and 95.79% in the pithead soil, mine wasteland

and soil irrigated with wastewater, respectively, while the

average value in the control was about 11.58% MN cells.


123

The highest micronucleus frequencies were found in soil

irrigated with wastewater, which were close to the outfall

of the wastewater discharge. The micronucleus frequencies

in the gold pithead soil, mine wasteland and soil irrigated

with wastewater were five, four and eight times than that in

the background area, respectively. Moreover, it was

found that there were double and multi-micronuclei in the

gold abandoned sites. The higher the heavy metal con-

centrations, the more elevated were the micronucleus

frequencies.

Fig. 1 Transmission electron microscope (TEM) images of plant cytoarchitectures


123

Induction of chromosome aberrations

Changes were observed in the organization and morphol-

ogy of the chromosomes in the root tips exposed to soil

water extract as in Fig. 3. Figure 3a shows that there were

no anomalous phenomena in V. faba root tip cell in the

background area. The types of mitotic chromosomal

abnormalities displayed were micronuclei, budding nuclei,

chromosomal bridge, and chromosomal fragmentation

through their prophase, metaphase, anaphase and telophase

in the mitotic cycle. The screening of mitotic divisions also

revealed that micronuclei and budding nuclei were the

most dominant abnormalities in all soil water extract and

they increased as the concentration of heavy metals

increased (Fig. 3b, c). Micronuclei and fragments were the

products of damaged or abnormally acting chromosomes

during the last mitotic divisions. Chromosome fragmenta-

tion was one of the main features of abnormal cell division

and chromosomal aberration (Fig. 3d, e). Other anomalies

were formation of anaphasic bridges (Fig. 3f), accompa-

nied by chromosomal rupture, isolated chromosomes

(chromatids not migrating) and chromosomes with non-

disjunction (chromatids not separating). The chromosomal

bridges resulted from the formation of dicentric and ca-

centric chromosomal fragments from refusion. This was in

accordance with the observation that the formation of the

chromosomal bridge was accompanied by the occurrence

of chromosomal fragments. The chromosomal fragments

and chromosomal bridges observed in those experiments

indicated that the heavy metal elements such as Au, Cu,

As, Pb and Hg may affect the structure and conformation of

DNA, the main component of the chromosomes in V. faba

root tip cells.

Conclusion

Rocks, soils and plants have obvious geochemical and

biogeochemical anomalies of Au and Au-associated heavy

metal elements in the abandoned mine areas. They were

characterized by relatively high concentrations of Cu, As,

Pb and Hg. Au and Au-associated heavy metal elements

inhibited chlorophyll and carotenoid biosynthesis and

retarded the incorporation of these pigments into photo-

systems in the Hetai the abandoned mine areas. They

induced decreased chlorophyll-a, chlorophyll-b and total

chlorophyll contents in the leaves of the goldfield, which

were all lower than that in the background values. The

correlation analysis showed that there was a negative cor-

relation between the element in the soil and the pigment

content in the plant, and that the environmental matter

contents had noticeable effects on the contents of plant

Fig. 2 Analytical results of the

electron-dense deposits in the

cell by EDX

Table 4 The micronucleus

rates of V. faba root tip cells in

the soil aqueous extracts

Micronuclei

frequency(%)

Pithead

soil

Mine

wasteland

Soil irrigated

with wastewater

Background

area

H2O

Micronuclei 59.6 ± 4.5 47.55 ± 4.2 95.79 ± 5.6 11.58 ± 1.2 6.32 ± 0.8

Double micronucleus 2.12 ± 0.5 1.06 ± 0.4 5.73 ± 0.7 0.14 ± 0.2 0

Multi-micronucleus 0.89 ± 0.3 0.13 ± 0.1 1.07 ± 0.1 0 0


123

pigments. The main damages found using transmission

electron microscope were characterized by chaos and

vacuolation of mitochondria, swollen thylakoids of chlo-

roplasts, disorder and disappearance of grana lamellae,

disintegrated chloroplasts, deforming and destroyed

nucleus, destruction of nucleoplasm, disruption of nuclear

membrane, and even the death of whole cells. Au and Au-

associated elements such as Cu and Pb were detected at

comparatively high concentrations in the cell, and they

were not phenolic substances. The micronucleus and

chromosome aberrations found indicated that the aban-

doned mine areas could induce different types of chro-

mosomal aberration and increase the micronucleus

frequency of V. faba root tip cells. It was observed that

chromosomal fragment, chromosomal bridge and chro-

mosome unwinding abnormally occurred at every stage of

mitosis. Moreover, it was found that there were double and

multi-micronuclei in the experiment. It can be concluded

that the heavy metal elements in these abandoned mine

areas have obvious teratogenic and cytogenetic toxic

effects on the organism.

Acknowledgments This work was supported in part by the Scientific

Frontier Program for Young Talents of the South China Sea Institute of

Oceanology, Chinese Academy of Sciences (Grant No. SQ200807) and

the Open Fund of Key Laboratory of Marginal Sea Geology, Chinese

Academy of Sciences (Grant No. MSG200902). The authors would like

to thank the two anonymous reviewers whose critical review and

comments greatly helped to improve the quality of the manuscript.

Fig. 3 Chromosomal aberrations induced by soil water extract in V. faba root tip cells


123

References

Ansari MI, Mali A (2009) Genotoxicity of agricultural soils in the

vicinity of industrial area. Mutat Res Genet Toxicol Environ

Mutagen 673(2):124–132

Boswell C, Sharma NC, Sahi SV (2002) Copper tolerance and

accumulation potential of Chalamydomonas reinhardtii. Bull

Environ Contam Toxicol 69:546–553

Brooks RR, Dunn CE, Hall GEM (1995) Biological system in mineral

exploration and processing. Eills Horwood Limited, Hemel

Hempstead, p 538

Burgeot T, His E, Galgani F (1995) The micronucleus assay in

Crassostrea gigas for the detection of seawater genotoxicity.

Mutat Res 342:125–140

Cabral JP (2003) Copper toxicity to five Parmelia lichens in vitro.

Environ Exp Bot 49(3):237–250

Carbonell-Barrachina AA, Porthouse JD, Mulbah CK et al (1999)

Metal solubility in phosphogypsum-amended sediment under

controlled pH and redox conditions. J Environ Qual 28:232–324

Carbonell-Barrachina AA, Rocamora A, Garcıa-Gomis C et al (2004)

Arsenic and zinc biogeochemistry in pyrite mine waste from the

Aznalcollar environmental disaster. Geoderma 122:195–203

Carreras HA, Pignata ML (2007) Effects of the heavy metals Cu2?,

Ni2?, Pb2?, and Zn2? on some physiological parameters of the

lichen Usnea amblyoclada. Ecotoxicol Environ Saf 67(1):59–66

Caspi V, Droppa M, Horvath G et al (1999) The effect of copper on

chlorophyll organization during greening of barley leaves.

Photosynth Res 62(2):165–174

Chen Y, Wang C, Wang Z et al (2004) Assessment of the

contamination and genotoxicity of soil irrigated with wastewater.

Plant Soil 261:189–196

Chettri MK, Cook CM, Vardaka E et al (1998) The effect of Cu, Zn

and Pb on the chlorophyll content of the lichens Cladoniaconvoluta and Cladonia rangiformis. Environ Exp Bot 39(1):

1–10

Ciftci E, Kolayli H, Tokel S (2005) Lead–arsenic soil geochemical

study as an exploration guide over the Killik volcanogenic

massive sulfide deposit, Northeastern Turkey. J Geochem Explor

86(1):49–59

Dovgaliuk AL, Kaliniiak TB, Blium LAB (2001) Assessment of

phyto- and cytotoxic effects of heavy metals and aluminum

compounds using onion apical meristem. Tsitol Genet 35:3–9

Duan J, He S, Zhou C et al (1992) On the structural characteristics of

Hetai gold deposit Guangdong and the gold-deposit model of

shear zone type. J Cent South Inst Mineral Metall 23:245–253

(in Chinese with English abstract)

Dudka S, Adriano DC (1997) Environmental impacts of metal ore

mining and processing, a review. J Environ Qual 26:590–602

Eom IC, Rast C, Veber AM et al (2007) Ecotoxicity of a polycyclic

aromatic hydrocarbon (PAH)-contaminated soil. Ecotoxicol

Environ Saf 67:190–205

Fernandez MD, Cagigal E, Vega MM et al (2005) Ecological risk

assessment of contaminated soils through direct toxicity assess-

ment. Ecotoxicol Environ Saf 62:174–184

Hajjouji HE, Pinelli E, Guiresse M et al (2007) Assessment of the

genotoxicity of olive mill waste water (OMWW) with the Viciafaba micronucleus test. Mutat Res 634:25–31

Kirsch-Volders M, Sofuni T, Aaderma M et al (2003) Report from the

vitro micronucleus assay working group. Mutat Res 540(2):

153–163

Li MS (2006) Ecological restoration of mineland with particular

reference to the metalliferous mine wasteland in China, a review

of research and practice. Sci Total Environ 357:38–53

Liu Y, Liu HC, Li XH (1996) Simultaneous and precise determination

of 40 trace elements in rock samples using ICP-MS. Geochimica

25(6):552–558 (in Chinese with English abstract)

Manier N, Deram A, Curieux FL et al (2009) Comparison between

new wild plant Trifolium repens and Vicia faba on their

sensitivity in detecting the genotoxic potential of heavy metal

solutions and heavy metal-contaminated soils. Water Air Soil

Pollut 201:342–352

Marcato-Romain CE, Guiresse M, Cecchi M et al (2009) New direct

contact approach to evaluate soil genotoxicity using the Viciafaba micronucleus test. Chemosphere 77(3):345–350

Marco AD, Boccardi P, Simone CD et al (1990) Induction of

micronuclei in Vicia faba root tips treated in different soils with

the herbicide alachlor. Mutat Res 241:1–6

Marinella F, Damia B (2003) Toxicity testing of wastewater and

sewage sludge by biosensors, bioassays and chemical analysis.

Trends Anal Chem 22:299–310

Navarro MC, Perez-Sirvent C, Martinez-Sanchez MJ et al (2008)

Abandoned mine sites as a source of contamination by heavy

metals, a case study in a semi-arid zone. J Geochem Explor

96:183–193

Patra M, Bhowmik N, Bandopadhyay B et al (2004) Comparison of

mercury, lead and arsenic with respect to genotoxic effects on

plant systems and the development of genetic tolerance. Environ

Exp Bot 52:199–223

Pijut PM, Lineberger RD, Domir SC et al (1990) Ultra structure of cells

of Ulmus americana cultured in vitro and exposed to the culture

filtrate of Ceratocystis ulmi. Phytopathology 80(8):764–767

Rahman A, Hasegawa H, Rahfuzur MM et al (2007) Effect of arsenic

on photosynthesis, growth and yield of five widely cultivated

rice (Oryza sativa L.) varieties in Bangladesh. Chemosphere

67(6):1072–1079

Rizzoni M, Gustavino B, Ferrari C et al (1995) An integrated

approach to the assessment of the environmental quality of the

Tiber River in the urban area of Rome: a mutagenesis assay

(micronucleus test) and an analysis of macrobenthic community

structure. Sci Total Environ 162:127–137

Sheoran AS, Sheoran V (2006) Heavy metal removal mechanism of

acid mine drainage in wetlands: a critical review. Miner Eng

19:105–116

Simkiss K, Taylor M, Mason AZ (1982) Metal detoxification and

bioaccumulation in molluscs. Mar Biol Lett 3:187–201

Song C, Lei L, Cheng S (2004) Gold biogeochemical anomaly and

aberrance effect of botanic cell tissue. Geochemica 33(2):

191–196 (in Chinese with English abstract)

Song Y, Wilke BM, Song XY et al (2006) Polycyclic aromatic

hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and

heavy metals (HMs) as well as their genotoxicity in soil after

long-term wastewater irrigation. Chemosphere 65:1859–1868

Stobart AK, Griffiths WT, Ameen BI (1985) The effect of Cd2? on

the biosynthesis of chlorophyll in leaves of barley. Plant Physiol

63(3):293–298

Zhang G, Boulter CliveA et al (2001) Brittle origins for disseminated

gold mineralization in mylonite: Gaocun Gold Deposit, Hetai

Goldfield, Guangdong Province, South China. Econ Geol Bull

Soc Econ Geol 96:49–59

Zhuang P, McBride MB, Xia H et al (2009) Health risk from heavy

metals via consumption of food crops in the vicinity of

Dabaoshan mine, South China. Sci Total Environ 407(5):

1551–1561


123

geochemistry and genotoxicity of the heavy metals in mine-abandoned areas and wasteland in the hetai...

Documents