AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA

.158ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2017.8.4.147

© 2017, ScienceHuβ, http://www.scihub.org/ABJNA

A comparison between tree species grown in the Kingdom of Saudi Arabia on their ability and tolerance to remove some heavy metals from

the polluted soil 1-Effect on Chemical composition

Abdullah O. Al Thobaity1; Abdullah M. Al-Ansari1 and Ansary E. Moftah2

1Dept. of Botany and Microbiology; College of Science; KSU

2Dept. of Plant Production and Protection, College of Agric., Qassim Univ

a.o.u.t@hotmail.com

ABSTRACT

Pot culture experiments were conducted to investigate the remediation potentials of two native tree species namely Tamarix nilotica and Conocarpus erectus to remove Pb or Zn (0, 100, 200, 400 and 800 mg.kg

-1 soil) and Cd (10, 20, 40 and 80 mg.kg

-1 soil) from the contaminated soils in

order to find out the most effective species that can be used to clean up the environment from heavy metal pollution. Results of this study showed that the plant growth decreased significantly at high levels of Pb and Zn and at middle levels of Cd. Photosynthetic pigments were also decreased at high concentrations of the pollutants while at low concentrations chlorophyll tended to increase slightly. Results showed also that proline, Pb, Zn and Cd concentrations in plant tissues increased with increasing their levels in the soil till certain level, then decreased with further increase of the pollutant. Peroxidase and catalase activity was stimulated with pollutant increase but decreased at highest level of Pb and Cd. The results indicated that T. nilotica and C. erectus are considered good accumulator candidates of Pb, Zn and Cd metals.

Keywords: heavy metals, pollution, phytoremediatiion, proline, enzymes

INTRODUCTION

In Saudi Arabia, many sources of pollution are becoming scary because of the industrial development, oil resources, excessive use of chemical fertilizers, huge amounts of vehicles and other activities. All these sources are introducing heavy metals in the environment either in the atmosphere or in the soil and water resources (Al-Homaidan et al., 2011). Recent studies showed that potentially toxic elements of heavy metals, lead (Pb) zinc (Zn) and cadmium (Cd), are common in many sites of the region (Adham et al., 2011). These elements were found in vegetables and fruit crops produced from these sites or grown near the tailings where their concentrations were relatively high (Franco-Hernández et al., 2010). Ingestion of toxic metals contaminated food may cause severe health problems (Hezbullah et al., 2016). This has drawn interest to develop a scientifically cost-effective remedial measure to remove heavy metals from the contaminated soils. One of the established approaches was through phytoremediation ( Malik et al., 2017).

Phytoremediation is a way for cleaning the polluted sites by using trees to extract the heavy metals from the contaminated ground, and accumulate them in roots, stems and branches. Logically, repeating cycles of planting and harvesting of trees accumulated with heavy metals will eventually reduce the concentration of toxic metals in soils to an acceptable level for other uses. A green house study shown that Acacia mangium and Acaccia auriculiformis accumulated considerable amount of Pb, Cd, As and Hg from sludge treated sand tailings (Aba-Alkhil and Moftah, 2013). Moreover, phytoremediation is one of the cost-effective approaches. Using trees to remove heavy metals from the contaminated sites brings other benefits such as improving its site quality. In this regard, a tree stand was found to have ameliorated the harsh microclimate of polluted site and also have improved its soil properties (Ayangbenro and Babalola, 2017). Using timber species for phytoremediation approach has additional benefit as it also yields timber at the end of its rotation. Earlier studies showed that tree species have a great potential to remove heavy

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metals for the contaminated sites (Aba-Alkhil and Moftah, 2015). In this regard, Moftah and Al-Ghanim (2009) found that Eucalyptus rostrata seedlings were able to accumulate high amounts of Pb when grown in polluted soil of Qassim region. However, this ideal cost-effective approach has many limitations, one of them is heavy metal contaminated sites are normally adverse to establishment and growth of plants. Greening polluted sites with tree species is well undocumented, but no documentation is available on the phytoremediation potential of these tree species. Hence, this study was embarked to determine the bioaccumulation of Pb, Zn and Cd by two selected tree species namely Tamarix nilotica and Conocarpus erectus that are successfully grown on sand soil and harsh conditions of the Kingdom.

MATERIALS AND METHODS

The present study was carried out at Qassim University, KSA during 2016/2017 to determine the ability of two tree species grow in the Kingdom of Saudi Arabia to extract Pb, Zn and Cd from polluted soil. Six month old seedlings of Tamarix nilotica and Conocarpus erectus were transplanted in plastic pots (30 cm×50 cm, diameter and depth) filled with sand and peat moss (2:1 proportion), pH 7.2; one plant in each pot. Before planting, soils were mixed with fine metal powders of PbCl2, ZnCl2 or CdCl2 to bring Pb or Zn levels of 00, 100, 200, 400 and 800 mg.kg

-1 soil,

and Cd to 10, 20, 40 and 80 mg.kg-1

soil).

To evaluate the efficiency of phytoremediation, it was necessary to homogenize the soil because the concentration of distributed contaminants in the soil plays a fundamental role in such studies. A completely randomized design with 10 replicates for each treatment was used in this experiment. Seedlings were left to grow in a greenhouse under natural light and temperature conditions. Plants of uniform height within species were selected and located in lines with a spacing of 2 m between lines and 1 m between pots to avoid mutual shading.

Three weeks after transplanting, all seedlings were fertilized 3 times, at 30 day intervals, with the complete water-soluble fertilizer “Sangral” compound fertilizer (20N-20P-20K, plus micronutrients) at the rate of 2g/kg soil. All pots were irrigated every 3 days to field capacity. Pots were weighed and supplied with amounts of water equal to evapo-transpiration losses. Plants were harvested 6 months after transplanting. The following parameters were determined:

Biomass determination: Six months after treatments, plants were harvested and shoots and roots were carefully taken separately, washed with tap water and twice with deionized water, then the fresh weight and dry weights (after oven drying for 72 h at 70°C) were determined. All dry samples were milled, air dried, and stored until metal content and other determinations were performed.

Chlorophyll content: Chlorophyll content was determined at the end of the experiment following the method of Metzner et al. (1965). Briefly, fresh leaves (0.4 g) were selected randomly from the middle part of each plant, washed with deionized water, and homogenized in a porcelain mortar with 80% acetone solution. The homogenate was centrifuged at 16,000×g for 1 min twice and the measurement of chlorophyll content was done by direct determination of the absorbance at wavelengths 663 and 646 nm at the clear supernatant fraction using spectrophotometer (Shimadzu 1240 UV–Vis).

Free proline: Concentrations of free proline were determined according to Bates et al. (1973). Leaf samples (approximately 0.2 g) are homogenized in 10 mL of 3% (v/v) aqueous sulfosalicylic acid. The homogenate was filtered through Whatman 41 filter paper. The filtrate, was acidified with glacial acetic acid and ninhydrin (1 mL each) and was heated in water bath at 100 °C for 1 h. The mixture was extracted with 5 mL toluene and the upper (toluene) phase decanted into a glass cuvette and the absorbance was measured at 520 nm. Proline concentrations were calculated using proline standards (0–50 μg/ml) in identical manner (Misra and Saxena, 2009).

Contaminant analysis: At the end of the experiment, Lead, zinc and cadmium and concentrations in plant samples were measured as follows:

Plants were harvested by removing from the soil. The plants were washed with deionised water to remove the soil particles. To determine the amount of Pb, Zn and Cd in plant tissues, dry samples of roots and shoots were dissolved in 20 mL of 1 M HCl and diluted to 50 mL with deionised water. The concentrations of the metals in the extracts were determined by atomic absorption spectrometry.

Enzyme analysis

Peroxidase activity: peroxidase activity was determined as described by Urbanek et al (1991) in a reaction mixture (0.2 mL) containing 100 mM

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phosphate buffer (pH 7.0), 0.1 μM EDTA, 5.0 mM guaiacol, 15 mM H

2O

2 and 50 μL enzyme extract.

The addition of enzyme extract started the reaction and the increase in absorbance was recorded at 470 nm for 1 min. Enzyme activity was quantified by the amount of tetra-guaiacol formed using its molar

extinction coefficient (26.6 mM-1

cm-1

).

Catalase activity: Catalase activity was assayed spectrophotometrically by monitoring the decrease in absorbance of H

2O

2 at 240 nm. CAT was measured

according to the method of Noctor and Foyer (1998). The enzyme was extracted in 50 mM phosphate buffer (pH=7). The assay solution contained 50 mM phosphate buffer and 10 mM H

2O

2. The reaction was

started by addition of enzyme aliquot to the reaction mixture and the change in absorbance was followed 2 min after starting the reaction. Unite activity was taken as the amount of enzyme, that decomposes 1 M of H

2O

2 in one min.

Statistic analysis

The experiment was arranged in a completely randomized design and was analyzed by analysis of variance. All data were statistically analyzed and ANOVA was tested according to Snedecor and Cochran (1980) with the aid of SPSS (1990) computer program for statistics. Differences among treatments were tested with LSD at 5% level of significance.

RESULTS AND DISCUSSION

Dry biomass: Data recorded in Figs (1 and 2) showed that heavy metal application, particularly at high concentrations, affected the fresh and dry weights of shoots and roots of both tree species. Cadmium exhibited maximum inhibitory effect on the shoot and root weights of the species, while the effect of Pb was less than that of Cd. On the other side low levels of Zn was likely to improve shoot and root biomass as compared with control plants at the first sample (60 days after treatment). In this regard 100 ppm of Zn treatment caused an increase of about 2.5% and 2.0% in fresh weights (Fig. 1) and by about 7.6% and 10.1% in dry weights (Fig. 2) of T. nilotica and C. erectus shoots, respectively, as compared

with control plants. It was clear that heavy metal (HM) treatments caused a gradual decrease in the fresh and dry weights of shoots to reach their minimum at 800 ppm of Pb or Zn and 80 ppm of Cd treatments. In this regard, 800 ppm of Pb caused a decrease in the dry weight of T. nilotica and C. erectus shoots by about 42.1% and 17.8%, respectively as compared with control. Similarly, 800 ppm of Zn caused a decreased in the dry weight by about 42.1% and 21.1%, respectively. The corresponding decrease at 80 ppm of Cd was about 44.7% and 35.7%, respectively.

Growth inhibition is a common response to HM stress and is also one of the most important agricultural indices of HM tolerance. Lead is not generally considered to be an essential element for plant growth. The effect of Pb on seedling growth seems to be different with regards to plant species, cultivars, organs and metabolic processes (Patil and Umadevi, 2014). Growth disorders causing reductions in biomass yields are commonly observed in plants subjected to high metal levels (Panich-Pat et al., 2010). In the present study, C. erectus seemed to be more tolerant to Pb, Zn and Cd than T. nilotica, thus high HM concentrations had less effect on the biomass of C. erectus than on that of T. nilotica. Inhibition of plant shoot and root growth as affected by HM treatments has been found by Ang et al. (2010). This negative effect could be attributed to the reduction in the meristem activity. The suppression of cells elongation by HM was also reported by de Souza et al. (2012).

Based on growth attributes, our results indicated that C. erectus was better adapted to contaminated soils. However, The seedling stage experiment demonstrated that the toxicity of a metal, in comparison to another may not be the same at all the stages of plant development. The toxicity of some metals may be so severe that plant growth is reduced before large quantities of the element can be translocate (Umadevi and Avudainayagam, 2013). The present study supported the findings of Malar et al. (2014) who stated that plant biomass is a good indicator for characterizing the growth performance of plants in the presence of heavy metals.

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Fig (1): Effect of Pb, Zn and Cd on the fresh weight of T. nilotica and C. erectus trees.

Fig (1): Effect of Pb, Zn and Cd on the dry weight of T. nilotica and C. erectus trees.

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Total Chlorophyll content: Data recorded in Fig (3) showed clearly that total chlorophyll contents of C. erectus was higher than that of T. nilotica. Total Chl of both tree species increased as time passed until reached its maximum after 180 days. The pigment was stimulated by low and medium HM concentrations. Only the highest concentrations of Pb and Zn resulted in a significant decrease in total Chl at any sampling time as compared with control plants, while the highest and medium concentrations of Cd caused a significant inhibition in total Chl at any sampling time.

Because changes in pigment content are related to visual symptoms of plant disorder (Zengin and Munzuroglu, 2005), Chl content is often measured to assess the effect of HM stress on plant growth and physiology. Many studies reported reduced chlorophyll in different species under HM pollution (Liu et al., 2014, Iqbal et al., 2017). Decreased Chl content connected with HM pollution might be a result

of inhibition of some enzymes involved in Chl biosynthesis. Heavy metals were reported to affect Chl formation and inhibited the synthesis of Chl precursor, aminolevulinic acid (ALA) (Gill, 2014). Previous studies found that HM stimulated chlorophyll degradation, and reduction in Chl content was observed in plants grown in HM polluted soils (Sharma and Dubey, 2005). Even moderate amounts of some HMs such as Cd affected the content of photosynthetic pigments, which could lead to a reduction in photosynthetic potential and a disruption of plant growth processes (de Souza et al., 2012). In another study, Iqbal et al. (2013) assumed that lead inhibits chlorophyll biosynthesis by impairing the uptake of essential photosynthetic pigment elements, such as magnesium, potassium, calcium and iron. While Malar et al. (2014) related the reduced accumulation of photosynthetic pigment linked with HM treatments to the peroxidation of chloroplast membranes as levels of ROS generation increased under such conditions.

Fig (3): Effects of Pb, Zn and Cd on total chlorophyll of T. nilotica and C. erectus trees

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Heavy metal concentrations : The obtained results indicated that Pb, Zn and Cd accumulated in shoots and roots of C. erectus and T. nilotica as plants became older to reach their maximum accumulation at the end of the experiments (Figs 4 and 5). After 6 months of the highest HM treatments the Pb or Zn ratio of shoots/roots was about 1.9 and 1.4 in C. erectus and T. nilotica, respectively, while Cd ratio was about 1.7 and 1.2, respectively. At any sampling time the amounts of HM accumulated in plant roots were significantly higher than those accumulated in plant shoots. The results showed also that HMs increased in plant tissues with increasing these metals in the growth media and reached the highest levels at the highest treatments.

Maximum accumulation of Pb, Zn and Cd in C. erectus was about 465, 478 and 48 mg.kg

-1 dwt,

respectively, in roots, and about 241, 252 and 26 mg.kg

-1dwt, respectively, in shoots. In T. nilotica the

maximum values were 410, 401 and 40 mg.kg-1

dwt, respectively, in roots, and 301, 215 and 30 mg.kg

-

1dwt, respectively, in shoots. Therefore, the

translocation factor value for Pb, Zn and Cd was 0.52, 0.53 and 0.54 in C. erectus and 0.73, 0.54 and 0.75 in T. nilotica, These values are less than 1 for both species. Therefore HMs were largely stored in plant roots as compared to those accumulated in plant shoots. These results support those obtained by Gratao et al. (2005) who found a positive relationship between the accumulation of HMs and the ability of plants to tolerate these metals. Thus, some species have a ability to tolerate HM toxicity via prevention of metal translocation from roots to shoots as reported by El-Gamal and Tantawy (2010) who found that plants differ in their ability to accumulate the heavy metals from the growth media.

Although higher concentrations of the studied metals were found in roots of C. erectus and T. nolitica, significantly high amounts were also measured in the above-ground tissues and together with the fact that there was no significant reduction in the biomass suggested that both species could be a possible candidates for Pb, Zn and Cd accumulators. An efficient HM accumulation mechanism in seedling roots could represent a new and interesting phenomenon for establishment of phytoremediation strategies, in which higher levels of the contaminant remain tightly attached to plant tissues (Brunet et al., 2008). The ability of the plant to accumulate and tolerate HM ions indicates that this plant species may have an efficient accumulating mechanism for

removal of HM ions from polluted sites and water bodies (Malar et al., 2014).

Fig (3): Effects of Pb, Zn and Cd on heavy metal content in T. nilotica and C. erectus shoots.

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Fig (3): Effects of Pb, Zn and Cd on heavy metal content in T. nilotica and C. erectus Roots.

Proline content: The recorded results indicated that proline concentration increased with increasing levels of heavy metals in the soil to reach its maximum value at 400 ppm of Pb or Zn and 20 ppm of Cd. After which proline concentration started to decrease significantly as compared with control plants. This increase in proline with increasing the level of HM pollution was more obvious during the second sampling time (120 days after treatments). Apparently both tree species accumulate proline against HM accumulation. At the second sampling date proline increased by about 28.1%, 21,8% and 37.5% in C. erectus seedlings treated with 400 ppm of Pb, 400 ppm of Zn and 20 ppm of Cd, respectively as compared with control treatment. The corresponding increase in T nilotica were about 26.5%, 29.4% and 12,2% respectively, comparing with control treatment.

Early studies showed that proline accumulated in plant tissues as a response of exposing plants to environmental stress (Sheetal et al., 2016). It is well known that abiotic stresses, including heavy metal stress, result in producing high amounts of reactive oxygen species (ROS) in plant cell leading to plant toxicity (Malar, 2014). Many workers reported that ROS is one the initial responses of plants to stress (Acosta-Motos et al., 2017; Tchounwou et al., 2012). Production of free radicals and reactive oxygen species is enhanced in the presence of metals, and this can seriously disrupt normal metabolism through oxidative damage to cellular components (Ang et al., 2010). To repair the damage initiated by ROS, plants have developed a complex antioxidant system. Antioxidants like systeine, proline, ascorbic acid to chelate the toxic metal ions (Emamverdian et al., 2015).

Under stress conditions, proline considers one of the most important materials that protect plant cells against these ROS (Ghasemi et al., 2017). The present study showed that proline concentration increased significantly in C. erectus and T. nilotica in the presence of high levels of Pb, Zn and Cd. In this regard, Dinakar et al. (2008) found that proline levels increased in Arachis hypogaea L. seedlings after 25 days with increasing heavy metal concentrations in the growth media. Theriappan et al. (2011) reported that proline concentration increased under high levels of heavy metal to decrease the water potential under these unfavorable conditions, thus, proline may accumulate to regulate water balance inside plants.

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Table (1). Effects of Pb, Zn and Cd on proline content of C. erectus and T. nilotica leaves

Tamarix nilotica Conocarpus erectus

Ppm 60 Day 120 Day 180 Day 60 Day 120 Day 180 Day

Pb

00 2.8 3.2 3.4 3.1 3.4 3.6

100 3.4 3.6 3.8 3.3 3.6 4.0

200 3.7 3.9 3.9 3.6 3.8 3.6

400 3.9 4.1 3.4 4.1 4.3 3.2

800 3.2 3.0 2.8 3.4 3.2 3.0

LSD5% 0.91 0.88 0.84 0.82 0.78 0.72

Zn

00 2.8 3.2 3.4 3.1 3.4 3.6

100 3.0 3.4 3.5 3.4 3.7 4.1

200 3.2 3.7 3.6 3.8 4.4 3.3

400 3.7 3.9 3.2 4.2 3.5 3.1

800 3.3 3.1 2.9 3.2 3.1 2.9

LSD5% 0.75 0.62 0.58 0.80 0.98 0.91

Cd

00 2.8 3.2 3.4 3.1 3.4 3.6

10 3.2 3.4 3.6 3.8 4.6 3.4

20 3.4 3.6 4.6 4.3 3.5 3.2

40 4.1 4.4 3.1 3.6 3.2 2.9

80 3.1 2.9 2.8 3.2 2.9 2.8

LSD5% 0.97 1.21 1.56 0.89 1.45 0.67

Activity of antioxidant enzymes

Peroxidase: Registered results showed that the activity of antioxidant enzymes were elevated with increased Cd and Pb concentration. Data in Fig (4) showed that peroxidase activity increased as Pb, Zn or Cd concentrations increased in the soil up to

certain level then the activity of the enzyme starts to decrease with any further increase in the level of the heavy metals. In general, the highest level of peroxidase was recorded at 400 ppm of Pb or Zn and 40 ppm of Cd, particularly at the third sampling time (180 days after treatments) for C. erectus and at the second sampling time (120 days after treatments) for T. nilotica as compared with control treatments.

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Fig. (4). Effect of Pb, Zn and Cd on peroxydase activity in leaves of C. erectus and T. nilotica tree seedlings

Catalase : It was clear from data plotted in Fig (5) that catalase activity increased only at 200 and 400 ppm of Pb or Zn, and at 10, 20 and 40 ppm for Cd in both plant species. At higher concentrations than those levels catalase activity started to decrease. The increase in catalase activity under HM treatments was more obvious in C. erectus than that detected in T. nilotica.

Early studies indicated that heavy metal stress causes an increase in the antioxidant enzymes activities including peroxidase and catalase in many plant species (Heidari and Sarani, 2011). It is well known that heavy metals enhance the formation of reactive oxygen species (ROS) which cause an oxidative stress and cause a physiological damage to plant cells (Acosta-Motos et al., 2017). In this regard, the increased activity of the antioxidant enzymes might reflect a damage response to stress factors, thus this conclusion was in agreement with that reported by Al Hassan et al. (2017), who presumed that high lipid peroxidation and anti-oxidative ability were parts of a damage response to salinity in rice cultivars.

The oxidative stress developed in plants when exposed to heavy metal stress was ascribed to heavy metal induced imbalance between the generations of toxic oxygen radicals and their scavenging through the anti-oxidative defense mechanism (Shahid et al., 2014). Therefore, antioxidant enzymes often provide an efficient system for detoxification and scavenging of the toxic oxygen species through an adaptive mechanism involving regulation of anti-oxidative enzymes such as CAT, POX and SOD (Heidari and Sarani, 2011) and enhance the accumulation of cellular antioxidants. These reactions regulate the conversion of the super oxide ions to hydroxyl (OH) ions (Cheng et al., 2016).

In the present study, it seemd that plants tried to protect themselves through the activation of the anti-oxidant system including antioxidant enzymes such as POX and CAD (Figs 4 and 5). These enzymes used to convert ROS to water and oxygen useful for plants (Heidari and Sarani, 2011). With the increase in heavy metal concentration in the soil or with increasing the time of plant exposure to heavy metals, the activity of the antioxidant system decreased (Heidari and Sarani, 2011).

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Fig. (5). Effect of Pb, Zn and Cd on catalase activity in leaves of C. erectus and T. nilotica tree seedlings

CONCLUSION: The present work is considered one of the important studies focusing on the use of native tree species in phytoremediation of Pb, Zn and Cd contaminated soils. The results indicated that Conocarpus erectus and Tamarix nilotica trees are considered good accumulator candidates to Pb, Zn and Cd metals. Therefore, both tree species could involve in the phytoremediation technology program in order to reclaim the heavy metal polluted soils.

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