1 Introduction

In an ecological study,any metal or metalloid that causes environmental problems which cannot be biologically degraded should be considered as a heavy metal. Heavy metals are natural components of the Earth's crust,but in many ecosystems the concentration of several heavy metals has reached toxic levels as a consequence of anthropogenic activities. Many industries,such as textiles,metal production,electroplating,battery and cable manufacturing,mining,tanneries,steel production, and automotive manufacturing,release heavy metals such as cadmium,copper,chromium,nickel, and lead in waste waters(Srinivas et al., 2013). Fifty-three elements are categorized as heavy metals,defined as the group of elements whose densities are greater than 5 g/cm³ and recognized as ubiquitous environmental contaminants in industrialized societies(Padmavathiamma and Loretta, 2007). The most common heavy metals at hazardous waste sites are cadmium(Cd),chromium(Cr),copper(Cu),lead(Pb),mercury(Hg),nickel(Ni), and zinc(Zn)(USEPA,1997).

Lead(Pb),with atomic number 82,atomic weight of 207.19, and a specific gravity of 11.34,is a bluish-grey metal that occurs naturally in minute amounts within the Earth's crust. It has also been referred to as plumbum,lead metal, and pigment metal. Lead contamination is common in the environment because of its wide applications(Jeanna,2000); industrial processes that involve the use of Pb include mining,smelting,manufacture of pesticides and fertilizers,dumping of municipal sewage, and the burning of fossil fuels that contain Pb additives. Many commercial products and materials also contain Pb,including paints,ceramic glazes,television glass,batteries,medical equipment(e.g.,X-ray shields,fetal monitors), and electrical equipment. Presently,the use of Pb in the production of ammunition has also increased tremendously.

One of the greatest risks to human health is Pb contamination(Lasat,2000). Pb is a major natural as well as anthropogenic pollutant and has accumulated in many different terrestrial and aquatic ecosystems(Verma and Dubey, 2003). In 2000,U.S. Environmental Protection Agency listed Pb as a potential carcinogenic element in nature. Inhalation and ingestion are the two major routes of human exposure, and the effects from both are the same(Jeanna,2000). Pb accumulates in the body organs,which may lead to poisoning or even death. The gastrointestinal tract,kidneys, and central nervous system are also affected by the presence of Pb. Children exposed to Pb are at risk of impaired development,lower IQ,shortened attention span,hyperactivity, and mental deterioration,with children under the age of six being at a more substantial risk(Jeanna,2000). Adults usually experience decreased reaction time,loss of memory,nausea,insomnia,anorexia, and weakness of joints when exposed to Pb.

Pb has limited solubility in soil and is generally not available for plant uptake due to complexation with organic matter,sorption on oxides and clays,or precipitation as carbonates,hydroxides, and phosphate. Hence,the two major limitations of Pb phytoremediation are low bioavailability in soil and poor translocation from root to shoot. Adding organic fertilizer could improve soil structure and then promote desorption of Pb out of the soil surface,thus enhancing its bioavailabilite. Zhang et al.(2009)found that continuously adding organic fertilizer inhibits the activity of Pb,Hg, and Cd. because the organic fertilizer increases the soil organic matter and then inhibits the activity of metals.

Heavy metals such as Cd and Pb are non-essential elements for plants. Accumulation of high concentrations of these elements in plants,usually affects the absorption and transport of essential elements,which disturbs the plant metabolism and impairs plant growth and reproduction(Xu and Shi, 2000). Thus plants,like all living organisms,have evolved a suite of mechanisms that control and respond to the uptake and accumulation of both essential and non-essential heavy metals. The two best-characterized heavy-metal-binding lig and s in plant cells are the phytochelatins(PCs) and metallothioneins(MTs). PCs and MTs are different classes of cysteine-rich,heavy-metal-binding protein molecules. PCs are enzymatically synthesized peptides,whereas MTs are gene-encoded polypeptides(Cobbett and Goldsbrough, 2002). Energy is also required for the translocation of metals from root to shoot as well as for their allocation to various tissues and cell types. Heavy metals affect the cell division of plants, and the effects are different and depend on the concentration.

The plants studied here were Sida acuta Burm. F. and Chromolaena odorata(L.)R. M. King & H. Robinson. Hence,this study was designed to determine the morphological variations that occur in each of the plant species grown in Pb polluted soil and their biomass accumulation.

2 Materials and methods 2.1 Study area

The study was carried out in a screen house in the Biological Gardens of Obafemi Awolowo University campus,Ile-Ife,Nigeria. The experiment was carried out in February 2014, and the average temperature in the screen house was 35.2 °C and the light intensity was 11,380 lux.

2.2 Collection of materials

Samples of top soils were r and omly collected from depths of 0-20 cm at the back of the Botany Department,Obafemi Awolowo University. The soil samples were air-dried for a week and then sieved using 2-mm mesh gauze to remove debris and stones. Seven-liter plastic pots of 23-cm diameter were used for the experiment. In order to allow aeration,the plastic pots were perforated at the base using a soldering iron. Viable seeds of Sida acuta and Chromolaena odorata were obtained from the wild.

2.3 Experimental design

The experiment was a factorial combination of Pb at five levels of concentration(0,200,400,800 and 1,000 mg/kg)laid out in a completely r and omized design and replicated three times with two plants and two levels(0 g/kg and 9.4 g/kg)of organic fertilizer OBD-Plus at the rates of 0 kg and 40 kg per hectare.

2.4 Pre-planting operations 2.4.1 Physical and chemical analysis of soil

The collected soil samples were analyzed for particle size distribution using the hydrometer method(Bouyoucos,1962), and the soil pH was determined following the procedure outlined by Eckerts and Sims(1995). The soil organic matter was determined using the Walkley and Black(1934)chromic acid digestion procedure,while the total nitrogen(N)was determined by the modified micro-Kjeldahl method(Bremner and Mulvaney, 1982). The samples were digested with a concentration of H₂SO₄ in the presence of H₂O₂ at 350 °C and the nitrogen in each digested sample was determined using the steam distillation technique(Keeney and Bremner, 1966). Available phosphorus was extracted using Bray-1 extractant(Bray and Kurtz, 1945), and after the extraction the phosphorus in the solution was determined using a Spectronic 20 atomic absorption spectrophotometer(AAS)set at 600-nm wavelength. Exchangeable cations were extracted with 0.01 M NH₄OAc(ammonium acetate)solution at pH 7.0, and magnesium and calcium ions in the extract were determined using the Spectronic 20. Potassium and sodium ions in the solution were determined using a Gallenkamp flame photometer(Tel and Hargert, 1984).

2.4.2 Preparation of plant seedlings

A nursery bed of 2 m² containing 50 seedlings was prepared for the two plants species and the seeds were planted at a depth of 2 cm. The nursery bed was watered to field capacity.

2.4.3 Preparation of Pb solution and the fertilizer amendment

The heavy metal used in the experiment was Pb, and Pb(II)nitrate {Pb(NO₃)₂} salt served as the source of the Pb used. Known weights of Pb with respect to each concentration of 0,200,400,800 and 1,000 mg/kg weredispensed into 1-L volumetric flasks,allowed to dissolve, and shaken vigorously. The resultant solutions were dispensed in a sterile bottle,allow to settle, and were ready for use. Organic fertilizer OBD-Plus with a nitrogen(N)content of 0.95% was used to augment the soil at concentrations of 0 g/kg and 9.4 g/kg,at the rates of 0 kg and 40 kg,respectively,per hectare.

2.4.4 Pollution of soil

Each pot was labeled using a permanent maker with respect to the concentration of heavy metal and fertilizer applied. A plastic tray was placed under each pot for the collection of excess water to prevent loss of pollutants. After the application of the pollutant,the pots were left for one week to allow for equilibration. Thereafter,organic fertilizer was applied to augment the soil.

2.5 Transplanting to polluted soil

Two weeks after germination of the seeds in the nursery bed,the seedlings with good growth and uniform height were selected and transplanted to each experimental pot which were 60 pots in total at the rate of one seedling per pot and were well watered.

2.6 Post-planting operations 2.6.1 Biomonitoring of the growth parameters

A week after transplanting,the growth parameters of the selected medicinal plants were measured. These were plant height,number of leaves, and leaf breadth and length. Plant height,breadth and length were measured using a 1-m ruler; the number of leaves was counted at each pot. This was done once a week for seven weeks. The leaf area was calculated using the method of Osei-Yeboah et al.(1983). The leaf area was calculated as: Leaf area = leaf length × leaf breadth × correction factor. The correction factor of Sida acuta and Chromolaena odorata was 0.7. Using leaf area meter,correct leaf area of the plants were determined. Correct leaf area divided by leaf length × leaf breadth gives the correction factor.

2.6.2 Harvesting

At 10 weeks after treatment,all the plants were carefully harvested and each plant sample was rinsed with tap water, and water droplets were removed using blotting papers. The plants were separated into roots and shoots and weighed using an electronic compact scale.

2.7 Laboratory analysis 2.7.1 Assessment of the biomass of the medicinal plants

The fresh weights of the shoots and roots were determined using a beam balance. This was done immediately after harvesting to prevent loss of moisture. The shoots and roots samples were then dried in the oven at 70 °C until they were well dried. After drying,the weights of the dried shoots and roots samples were determined. The dry matter yield was calculated from the wet and dry weights of the plant samples.

2.8 Statistical analysis

We conducted separate analyses of variance for the different plants species at different treatments, and mean separation by Fisher's least significant difference(LSD)for shoot height,number of leaves,leaf area, and biomass accumulation. All analyses were carried out using SAS version 9.2.

3 Results 3.1 Physical and chemical properties of the soil used in the experiment

The physical and chemical characteristics of the soil used in the screen house study are presented in Table 1. The textural classification of the soil was loamy s and . The pH in 1:1 soil to water was 5.8 for the topsoil,indicating a slightly acidic soil condition. The organic carbon content of the soil was 40.10 g/kg,while nitrogen(N),phosphorus(P), and potassium(K)values were 3.30,10.4, and 2,605 mg/kg,respectively. The calcium(Ca) and magnesium(Mg)contents were 31.0 and 2,432.5 mg/kg,respectively,with a Pb concentration of 0.098 mg/kg.

3.2 Growth performance of Sida acuta 3.2.1 Shoot height

The shoot heights of Sida acuta plants grown in different levels of Pb concentration without and with fertilizer application are shown in Table 2. From the results,the shoot heights of the plants in all the treatments increased across the study period. At 200 mg/kg Pb without and with fertilizer application,there was no significant difference in the shoot height of Sida acuta,except week 7 under fertilizer application(50.87 cm),when compared with the control plants. At higher Pb concentration levels(800 and 1,000 mg/kg),there was a significant difference(p <0.05)in the shoot heights of Sida acuta when compared with the controls.

3.2.2 Number of leaves

Table 3 presents the number of leaves of Sida acuta without and with fertilizer application. Without fertilizer application,only those of weeks 1 to 3 at a low level of Pb concentration(200 mg/kg)showed no significant difference from the control. Other levels of Pb concentration showed significant differences from the control. At 200 mg/kg of Pb concentration under fertilizer application,there was no significant difference in the number of leaves of Sida acuta when compared with control. Also in weeks 1 and 3 with fertilizer application,there was no significant difference in the number of leaves across the weeks,except at 400 mg/kg Pb concentration(15.33 cm)which showed significant difference from the control. As the Pb concentration levels increased,the number of leaves of Sida acuta decreased significantly(p <0.05)from the control.

3.2.3 Leaf area

Table 4 shows the leaf areas of Sida acuta grown in different levels of Pb concentration without and with fertilizer. At the 200 mg/kg Pb concentration level both without and with fertilizer application,there was no significant difference in the leaf area of Sida acuta when compared with the control. Furthermore,the leaf area in week 1 without fertilizer application showed no significant difference at all the levels of Pb concentration. However,at high levels of Pb concentration(400-1,000 mg/kg),there were significant differences(p <0.05)in the leaf area of Sida acuta when compared with the control.

3.3 Growth parameters of Chromolaena odorata 3.3.1 Shoot height

The shoot heights of the Chromolaena odorata in the different Pb concentration levels without and with fertilizer application are shown in Table 5. At weeks 1,3,4, and 5 without fertilizer application,there was no significant difference at all the Pb concentration levels when compared with the control. Also,weeks 1,2,3, and 5 with fertilizer application showed no significant differences at all the levels of Pb concentration when compared with the control. Other treatments showed significant differences(p <0.05)when compared with the control.

3.3.2 Number of leaves

Table 6 shows the number of leaves of Chromolaena odorata at different levels of Pb concentration without and with fertilizer application. At weeks 1,2, and 3 without fertilizer application,there was no significant difference,except at 1,000 mg/kg of Pb at weeks 2 and 3,when compared with the control. Also,at weeks 1,2,3, and 4 with fertilizer application,there was no significant difference in the number of leaves of Chromolaena odorata,except at 1,000 mg/kg of Pb at weeks 3 and 4,when compared with the control. Other levels of treatment showed significant differences(p <0.05)in the number of leaves of Chromolaena odorata when compared with the control.

3.3.3 Leaf area

The leaf areas of the Chromolaena odorata at different levels of Pb concentration without and with fertilizer application are shown in Table 7. The leaf areas of Chromolaena odorata without fertilizer application at week 1 showed no significant differences at all the concentration levels when compared with the control. Also,Chromolaena odorata under fertilizer at weeks 1 and 2 showed no significant differences at all the levels of Pb concentrations when compared with the control. At 200 mg/kg of Pb,both without and with fertilizer application,there was no significant difference in the leaf area of Chromolaena odorata when compared with the control,except in week 5 without fertilizer application. As the levels Pb of concentrations increased,there were significant differences(p <0.05)in the leaf area of Chromolaena odorata when compared with the control.

3.4 The plants' biomass accumulation

The biomasses of the two plant species grown in different levels of Pb concentration without and with fertilizer application are shown in Tables 8 and 9,respectively. The biomass in all the treatments decreased across the concentrations of Pb. The higher the levels of Pb concentrations,the lower the biomass accumulation of shoots and roots. There was no significant difference in the two medicinal plants grown in Pb concentrations of 0 and 200 mg/kg of without fertilizer and with fertilizer application,except that shoots of Sida acuta with fertilizer application(10.493 g and 8.443 g of biomass)were significantly different. Others showed significant differences(p <0.05)from the control.

4 Discussion and conclusions

From the experiment,plants treated with organic fertilizer under different levels of Pb concentration showed significantly improved growth parameters than the unfertilized plants under the same various levels Pb concentration. This was in line with the work of Sun et al.(2003) and Lin et al.(2010), and can be attributed to the role of bio-organic fertilization in plant physiology,which provides plants with essential elements. The available nutrients might have helped in enhancing the leaf area,shoot height,number of leaves, and fresh weight of the studied specimens. These results are supported by the findings of Yaduvanshi and Swarup(2000),Muhammad(2008), and Yadana et al.(2009). The increase in fresh weight was also reported by Sarwar et al.(2008). But using different plant species,according to Singh and Agarwal(2001),the increase in leaf numbers as well as size due to sufficient nutrition can be explained in terms of a possible increase in the nutrient absorption capacity of plants as a result of better root development and increased translocation of carbohydrates from the source to the growing points.

At different levels of Pb concentration both without and with fertilizer application,the growth parameters of the two plant species decreased,which may be attributed to excessive accumulation of Pb in the soil. The fresh and dried weights of both roots and shoots were also reduced at different levels of Pb concentrations. This agreed with the experimental observations by Abdul(2010),where an increase in concentration of Pb induced significant growth inhibition in two varieties of maize(Zea mays L.). In addition,Nicholls and Mal(2003)also observed complete and rapid death of the aboveground stems and leaves of Lythrum salicaria L. treated with Pb and Cu. In their experiment,the leaves were affected first,followed shortly by the stems. In our studied,none of the transplanted plants died,but chlorosis and necrosis were observed on some of the leaves,mostly those treated with the highest concentration of Pb. Scanty chlorosis was observed on the leaves of plants treated with organic fertilizer.

It has already been shown by Verma and Dubey(2003),Strubinskai and Hanaka(2011), and Miao et al.(2012)that Pb inhibits plant growth by affecting the biochemical and metabolic processes,which are linked with normal growth parameters and development of plants. A number of other reports(e.g.,Kopyra and Gwozdz, 2003; Atici et al., 2005)have shown that heavy metals,including Pb,diminished plant growth and development. When and how the roots were affected was not apparent until after harvest. There were reductions in the roots of the two plants grown under Pb polluted soil,with the highest Pb concentration showing significant reduction in root length. This was also observed by Faheed(2005) and Kabir et al.(2008). Ouzounidou et al.(1992)demonstrated that the cells of roots were affected more severely by exposure to Cu than other parts of the plant were.They stated that this was because the plant roots were the first contact point of metals; the resultant root length reduction was more prominent in plants under Pb stress.

Moreover,Sharma and Dubey(2005)showed that the physiological processes of plants are affected by fertilization and Pb application,Pb phytotoxicity leads to inhibition of enzyme activities, and distribution of mineral nutrition,water imbalance,change in hormonal status, and alteration in membrane permeability. The photosynthetic apparatus may be especially sensitive to damage from heavy metals(Kovacevic et al., 1999). Heavy metals affect many processes vital to the photosynthetic pathway. For example,excess heavy metals have been shown to negatively affect chlorophyll biosynthesis(Ouzounidou et al., 1992),to alter the action of ribulose 1,5-bisphosphate carboxylase oxygenase(Cook et al., 1997),to interfere with the dynamics of the thylakoid membrane(Szalontai et al., 1999), and to inhibit the electron transport system in both photosystem I and photosystem II(Ouzounidou et al., 1992).

We studied the effects of applied organic fertlilizer on the growth parameters and the biomass of two medicinal plants,Sida acuta and Chromolaena odorata,grown in Pb-polluted soil. The plants performed better under organic fertilizer application,but the growth parameters and the biomass of the plants decreased as the levels of Pb concentration increased.

Acknowledgments:

We sincerely appreciate the diligence,patience,advice and support of my industrious supervisor,Prof. A. A. Adelusi of the Department of Botany,Faculty of Science,Obafemi Awolowo University,Ile-Ife,Nigeria; for his guidance,constructive criticisms and encouragement which contributed immeasurably to the success of this study. We also appreciate Dr.(Mrs.)O. E. Dada,Department of Biological Sciences(Ecology and Environmental Studies Unit),College of Natural Sciences; Joseph Ayo Babalola University,Ikeji-Arakeji,Nigeria; and Mr. Okunlola Lanre,Biological Sciences Department,Osun State University,Nigeria,for their advice and moral support.