1. Introduction
Recent studies report that exposure to manganese (Mn) results in neurotoxicity and/or Parkinson’s disease (PD) in welders [
1] and nurses [
2]. This phenomenon has aroused widespread concern on a global scope [
3,
4,
5]. Manganese-induced clinical neurotoxicity is associated with a motor dysfunction syndrome commonly referred to as manganism [
6], which is related with significantly higher reactive oxygen species (ROS) generation [
7]. It is therefore great concern about the environmental risk posed by manganese waste mines because most of the tailings have been left without any management and have become the main source of heavy metal contamination of agricultural soils and crops in the mining areas [
8]. Soil heavy metal pollution poses high carcinogenic and non-carcinogenic risks to the public, especially to children and those living in the vicinity of heavily polluted mining areas [
9,
10]. Multiple pathways of health risk due to heavy metal exposure in China were reviewed by Zhuang
et al. [
11], with risks coming from the intake of home-grown rice and vegetables. The use of polluted groundwater and pond water also poses potential risk to human health [
12], for instance, the high manganese concentration (1.21 ppm; U.S. Environmental Protection Agency reference, <0.05 ppm) in water used by local residents had caused a markedly below-average performance in tests of memory [
13] due to human nervous system damage cuased by excessive intake of manganese [
14]. Chai
et al. [
15] found that arsenic and manganese were the largest contributors to human health risks for the local people drinking groundwater in the **angjiang watershed. The neurotoxicologic effects of water manganese in children [
16] due to their significantly higher Mn concentrations in blood (9.5 μg/L) and hair (12.6 μg/L) was observed [
17]. Furthermore, water Mn concentrations of 0.66 μg/L [
18] (
i.e., higher than the WHO guideline of <0.4 μg/L) may lead to higher infant mortality [
19].
In China, mining activities alone have resulted in the creation of about 3.2 million ha of wastelands; and this figure keeps on increasing at a rate of 46,700 ha per year [
20], due to the rapid expansion of the mining industry in order to meet the demands of rapid economic growth. Unlike organic compounds heavy metals cannot be degraded, so the clean-up of heavy metals usually requires removal or immobilization [
21]. Recently, a number of instances of food contamination and of health problems amongst local residents caused by heavy metals in the environment have been reported in China [
22]. Although much effort and attention has been paid to the problem by governments at every level, only a low rate of restoration of metalliferous mine wastelands has so far been achieved, making it crucial to develop efficient techniques for the restoration of wastelands associated with heavy metals [
20].
Compared to treatments that involve engineering or physicochemical techniques, phytoremediation is a low-cost and environmentally benign “green” technology that is also easily translated to large-scale applications [
23]. Plants not only have the capacity to remove pollutants from mine wastelands or to render them harmless [
24], but they also maintain the biological activity and physical structure of soils [
25]. The two main categories of processes for the phytoremediation of soils contaminated with heavy metals are phytoextraction and phytostabilization [
26]. Phytoextraction usually uses plants to take up heavy metals from the soil and to translocate them from the roots to the above-ground parts [
27]. Obviously, in order to achieve effective soil decontamination, the basic requirement for phytoextraction is that plants should exhibit high productivity, and that they should be able to accumulate and tolerate high concentrations of heavy metals [
28]. For this reason, the selection of hyperaccumulators that are effective for specific heavy metals has become a priority [
29]; however, most of those that have been selected are herbs that have low biomass and shallow root systems, so that their ability to achieve substantial heavy metal accumulation and to deliver remediation effects at the deeper levels within soils are quite limited. By contrast, phytostabilization uses plants to retain heavy metals within the soil or within roots, or to reduce heavy-metal mobility and bioavailability [
26]. Planting trees on mine wastes is promoted as a sustainable and ecologically sound solution for the phytostabilization of heavy-metal-contaminated soils [
26,
30]; and because natural restoration processes are rather slow [
31], it is also used to accelerate the recovery of vegetation [
32]. The benefits and advantages that result from tree planting include: soil stabilization and improvement [
30], the accumulation or partial removal of heavy metals [
26], carbon sequestration [
33], renewable bioenergy production without detriment to the food chain or to human health [
21], the provision of wildlife habitats and the conservation of biodiversity [
34] and, perhaps most conspicuously, landscape amelioration and beautification.
The establishment of trees on mine wasteland is usually constrained by a physical soil structure that is unfavorable for water retention, aeration, and root penetration [
35], also accompanied by poor nutrient status or by heavy metal toxicity [
32]. Several studies have shown that many tree species can generally survive in metal-contaminated soils, though usually at a much reduced rate of growth [
26]. Encouragement of the proliferation of fine roots in uncontaminated zones of the soil is an important strategy to avoid heavy metal toxicity and other stresses [
36]. To improve the physicochemical and biological properties of mine wastelands and to increase tree survival and growth, applications of fertilizer and the incorporation of sand [
32,
37], along with physical manipulation of the mine wastes [
30], are effective courses of action. The use of fertilizer and sand to improve a large area of mine wasteland is expensive, however, both in terms of the costs of the materials themselves and in transportation costs. It remains unclear whether, as an alternative or complementary approach, the manipulation of local mine wastes with contrasting physical and chemical properties could be used to create heterogeneity in wastelands and thereby facilitate tree survival and growth.
The **angtan manganese (Mn) mine is one of the most important metal mines in Hunan Province. It has been in operation for more than 80 years and left large areas of abandoned mine wastelands. These wastelands still exist and are grouped into mine tailings and mine sludge. Mine tailings are characterized by low nutrient status and a silt-like texture [
29], while mine sludge represents a nutrient-rich substrate that is favorable for revegetation, which major disadvantages in terms of plant establishment are its degree of compaction and its anoxic nature [
38].
When grown on mine tailings, the herb species
Phytolacca acinosa has been found to be a Mn hyperaccumulator [
29]. In addition, on account of its fast growth rate and high adaptive capacity, the tree species
Koelreuteria paniculata has demonstrated a potential for phytoremediation [
39] and has been successfully established on mine tailings, with a
ca. 96% survival rate. Nevertheless, the mechanism of the phytoremediation of mine tailings brought about by
K.
paniculata trees is uncertain; and furthermore, it is unclear whether local waste manipulations, for example, through mixing mine tailings with mine sludge, might facilitate the survival and growth of
K.
paniculata in mine sludge. The objectives of the study reported here were therefore: (1) to investigate the limitations of mine sludge, compared to mine tailings, in the growth of
K.
paniculata seedlings; (2) to evaluate whether local waste manipulations of mine tailings and mine sludge could improve the survival rate and growth of
K.
paniculata seedlings; and (3) to determine the heavy-metal phytoremediation mechanism of
K.
paniculata in Mn mine wastelands.
2. Materials and Methods
2.1. Experimental Materials
Samples of mine tailings and mine sludge for pot-grown plants were collected from the **angtan Mn mine (27°53′–28°03′ N, 112°45′–112°55′ E), located in the northern part of the city of **angtan, Hunan Province, China. This region has a subtropical monsoon climate, with mean annual rainfall of 1431 mm and mean annual air temperature of 17.4 °C. Mine tailings are the abandoned wastes produced as a result of mineral processing and their area amounts to 134 ha. Mine sludge is the final mixture discharged into the tailing pond following the preliminary treatment of the manganese ore powder and its area is about 100 ha. Samples of about 50 kg each of mine tailings and mine sludge were randomly collected and transported to the laboratory for chemical analysis and for use in the experimental growth media.
Soils for use in a control experiment were sampled from a Cinnamomum camphora plantation in the campus of the Central South University of Forestry and Technology (CSUFT) (28°08′ N, 113°00′ E), Changsha City, Hunan Province, China. This site is hilly and also experiences a subtropical monsoon climate. The soil is red in color and it is derived from a plinthitic horizon soil parent material of the Quaternary period, and classified within Alliti-Udic ferrosols, which corresponds to acrisol in the World Reference Base for Soil Resources.
K. paniculata seedlings were sourced from the Zhuzhou nursery garden of CSUFT (27°54′ N, 113°09′ E). All seedlings were 1-year-old and of similar size: ca. 75 cm high, with a basal diameter of ca. 0.5 cm. The seedlings were selected immediately prior to being transplanted into experimental pots.
2.2. Experimental Design and Growth Media
We designed six experimental treatments: (1) uncontaminated soil, as a control (C); (2) mine tailings (T); (3) mine sludge (S); (4) a 1:1 (v/v) mixture of mine tailings and mine sludge (ST); (5) a half-volume of mine tailings and a half-volume of soil (CT); and finally; (6) a half-volume of mine sludge and a half-volume of soil (CS). For treatments (5) and (6), the half-volumes of the two components were not mixed together, but were placed separately in each half of the plant pot. Therefore, for treatments (5) and (6), the CT-C and CS-C represented for the half-volume of soil of the treatments CT and CS, and the CT-T and CS-S were for the half of the pot filled with tailings and for the half filled with mine sludge, respectively. Treatments (5) and (6) were designed to test whether heterogeneity substrate could improve seedling growth and thus facilitate restoration in mine wastes. The details of the pots filled with different substrates were presented in
Figure 1.
Figure 1.
The experimental setup included six treatments, which were filled with different soil substrates in the pots. Each pot was labeled with the treatment code.
Figure 1.
The experimental setup included six treatments, which were filled with different soil substrates in the pots. Each pot was labeled with the treatment code.
For each treatment, 12 PVC pots (32 cm high, top diameter 36 cm, bottom diameter 28 cm), with holes for drainage at the bottom, were prepared, which were sufficient for three harvesting times, each with four replicates.
After measurements had been made of the basal diameter, height and fresh weight of each
K.
paniculata seedling, and of the number of roots and their length, a seedling was placed centrally in each pot and affixed using two pieces of upright cardboard so as to ensure that the roots were straightened and spread out. Each pot was filled to within 2 cm of the rim, and when filling and planting were completed, the pieces of cardboard were carefully removed. After watering, the pots were then moved into a greenhouse shaded with warp-knitted netting. The growth of the seedlings was routinely monitored. The pots were watered at 2–3 d intervals to maintain soil moisture, and before each harvest measurement. The local climate conditions around the greenhouse were similar as the subtropical moonsoon climate as described above. Average monthly rainfall and air temperature were presented in
Figure 2, based on the data recorded in microclimate observation station near the greenhouse during perdiod from 2010 to 2013.
Figure 2.
The box-plot for average monthly rainfall (a) and monthly air temperature (b).
Figure 2.
The box-plot for average monthly rainfall (a) and monthly air temperature (b).
2.3. Seedling Growth Measurements and Chemical Analysis
The pot experiments started in late March 2012. Three sets of measurements were taken, on separate groups of seedlings, in July, September and December. The measurements included determinations of survival rate, seedling height, basal diameter, fine root length and number, and shoot and root biomass. After taking measurements of height, basal diameter and leaf number, the seedlings were marked and then cut off at the base near the soil surface. The aerial parts were separated into woody shoots and leaves, respectively. The roots of the (marked) seedlings were washed carefully with tap water on 1 mm × 1 mm mesh and the root systems were collected. For each seedling, the number of roots and their length was determined. The samples of woody shoots, leaves and roots were washed thoroughly with running tap water, followed by three rinses with deionized water. They were then oven-dried at 80 °C and their dry mass was determined. After being ground into fine powder using a pestle and mortar, the samples were digested with a mixture of concentrated HNO3 and concentrated HClO4 (4:1, v/v), prior to chemical analysis.
Samples (~500 g) of soil, mine tailings, mine sludge, and the mine waste (tailings/sludge) mixture were taken before and after the experiment. pH values were measured potentiometrically with a pH meter (PXS-270, Shanghai, China), using a 1:2.5 (v/v) sample:H
2O (distilled) suspension. Total nitrogen (N) concentrations were determined by a semimicro-Kjeldahl method. Total phosphorus (P) concentrations were measured using a sodium hydroxide-molybdenum-antimony colorimetric method [
40]. Soil and mine waste samples were digested with HCl-HNO
3-HF-HClO
4. For the determination of the concentrations of metals in plant, soil and mine waste samples, the digested solutions were analyzed using a flame atomic absorption spectrophotometer (HP3510, Shanghai, China).
2.4. Data Analysis
To investigate the phytoremediation mechanism for heavy metals, the bio-concentration factor (BCF) was expressed as the ratio of the metal concentration in the above-ground part of the seedling to the total metal concentration in soil or mine wastes [
38]. The translocation factor (TF) for metals within a given seedling was calculated as the metal concentration in the shoot divided by that in the root [
26]. One-way ANOVA was used to test differences among treatments and Tukey-Kramer honest significant difference (HSD) tests were used for pair-wise comparisons of all treatments. Statistical significance was tested at the level of 0.05 using the JMP software package (SAS Institute Inc., Cary, NC, USA).