Use of Phytoremediation in Salt Pollution. A case of Indah Water Konsortium (IWK)

Introduction

Background

Contaminations of soil especially from Indah Water Konsortium (IWK) by heavy metals are one of the results of human activities. It is one of the serious environmental issues in the world. The heavy metals usually comes from mining activities, smelting of metallic ferrous, electroplating, pesticides and fertilizer applications, municipal waste generation and also the sludge dumping (Ogundiran and Osibanjo, 2008; Krishna and Govil, 2005). In Malaysia, the management of contaminated soil and sludge are very complex because of the amount of the waste that has been generated by human from day to day.

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Soil pollution can be defined as the build-up in soils of persistent toxic compounds, chemicals, salts, radioactive materials, or disease causing agents, which have adverse effects on the plant growth and animal health. Soil pollution has been controlled by putting regulations on the use of DDT and introduction of alternatives to it. Mining cannot be stopped because we are in constant need for mineral ores for different applications (Gupta et al., 1982).

Phytoremediation is a method of using the living green plants for in situ risk reduction and / or removal of contaminants from contaminated soil, water, sediments, and air. Risk reduction can be through a process of removal, degradation of, or containment of a contaminant or a combination of any of these factors. During phytoremediation, plants may improve soil aeration via their root system, which enhances rhizosphere microbial activity and contaminant degradation. Microbial activity is stimulated by root exudates that serve as sources of carbon and energy for microorganisms that oxidize and/or degrade organic contaminants (Alkorta and Garbisu, 2001). Phytoremediation is energy efficient, aesthetically pleasing method of remediating sites with low to moderate levels of contamination and it can be used in conjunction with other more traditional remedial methods as a finishing step to the remedial process. Phytoremediation is the use of plants to partially or substantially remediate selected contaminants in contaminated soil, sludge, sediment, ground water, surface water, and waste water. It utilizes a variety of plant biological processes and the physical characteristics of plants to aid in site remediation. Phytoremediation has also been called green remediation, botano-remediation, agroremediation, and vegetative remediation. Phytoremediation is a continuum of processes, with the different processes occurring to differing degrees for different conditions, media, contaminants, and plants. A variety of terms have been used in the literature to refer to these various processes.

Phytoextraction is one of the mechanisms that can be applied while using phytoremediation concept. Phytoextraction is the use of plants to remove contaminants from the environment and concentrate them in above-ground plant tissue Phytoextraction was primarily employed to recover heavy metals from soils; however, this technology is now applicable to other materials in different media. Greenhouse-based hydroponic systems using plants with high contaminant root uptake and poor translocation to the shoots are currently being researched for removal of heavy metals and radionuclide’s from water. These plants also are referred to as hyper accumulators (Kumar et al., 1995).

The term heavy metal is refers to any metallic chemical that has a relatively high density and at low concentration, it will become toxic or poisonous. A few examples of heavy metals include mercury (Hg), arsenic (As), chromium (Cr), thallium (Tl), lead (Pb), and cadmium (Cd). Heavy metals are naturally occurred in the Earth’s crust. They cannot be destroyed. In the small amount, they enter our bodies through air, drinking water and also food. As the functions of trace elements, some of the heavy metals such as copper and zinc are needed to maintain the metabolism of human body (Duffus, 2002). But, with the higher concentrations, they can contribute to poisoning.

Heavy metal analysis was conducted by using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Inductively Coupled Plasma Mass Spectrometry or ICP-MS is an analytical technique used for elemental determinations. The technique was commercially introduced in 1983 and has gained general acceptance in many types of laboratories.

Geochemical analysis labs were early adopters of ICP-MS technology because of its superior detection capabilities, particularly for the rare-earth elements (REEs). ICP-MS has many advantages over other elemental analysis techniques such as atomic absorption and optical emission spectrometry, including ICP Atomic Emission Spectroscopy (ICP-AES) (USGS).

Physical changes occur when objects undergo a change that does not change their chemical nature. A physical change involves a change in physical properties. Physical properties can be observed without changing the type of matter. Examples of physical properties include: texture, shape, size, color, odor, volume, mass, weight, and density.

Problem Statement

As we know, the Acacia mangium is a wild plant and can grow very fast. The heavy metals content in the IWK sludge need to be clean. The Acacia mangium is used to act as a phytoremediator to reduce the contaminats which are contain in the soil and sludge. Wild plant, such as Acacia mangium is one of the species that can absorb the heavy metals that contain in the soil. There are lots of heavy metals that are abundant in the contaminated soil (sludge). Acacia mangium is use to obtain the possibility of the Acacia mangium to absorb the heavy metals. Acacia mangium can function to clean the contaminated soil (sludge) that caused by human activities such as mining, municipal solid waste dumping and electroplating. There are few technologies that can undergo under this phytoremediation. Examples of the technologies are phytoextraction, phyrovolatilization and rhizofiltration.

Objectives

a.To determine the amount of heavy metals that can be accumulate in the roots of Acacia mangium by using the contaminated soil from Indah Water Konsortium (IWK).
b.To study and determine the effect of heavy metals uptake towards the physical changes and the growth performance.

Significant Study

Nowadays, there are lots of pollution occurred in our country and also all over the world. Soil pollution is one of the example of pollutions occur. This study was conducted is just about to reduce the cost for cleaning the sludge and also the contaminated soil. In this research, Acacia mangium have been chosen for being a phytoremediator. This phytoremediator functions to clean the contamination in the soil. The contamination from the soil and sludge also may react as the fertilizer.

Literature Review

Contamination of Soil

Human activities such as mining, smelters, production of energy, municipal solid waste dumping and fuel production are examples of the activities that can contribute to the contamination of the soil (Chehreganli, 2008). The contamination of the soil especially the agricultural soil will contain the heavy metals that may affect the plants productivity and the safety of the plant to be eaten (Zheljazkov, 2008). The heavy metals content in the soil is due to the excessive fertilizer that is given planting and also from mining activities.

Soil contamination is either solid or liquid hazardous substances mixed with the naturally occurring soil. Usually, contaminants in the soil are physically or chemically attached to soil particles, or, if they are not attached, are trapped in the small spaces between soil particles (U.S EPA). Soil contamination is the occurrence of pollutants in soil above a certain level causing a deterioration or loss of one or more soil functions. Also, Soil Contamination can be considered as the presence of man-made chemicals or other alteration in the natural soil environment. This type of contamination typically arises from the rupture of underground storage tanks, application of pesticides, and percolation of contaminated surface water to subsurface strata, leaching of wastes from landfills or direct discharge of industrial wastes to the soil. The most common chemicals involved are petroleum hydrocarbons, solvents, pesticides, lead and other heavy metals. The occurrence of this phenomenon is correlated with the degree of industrialization and intensity of chemical usage. Figure 2.1 shows the example of soil pollution occurred by human activities.

Soil heavy metal pollution is a widespread global problem. Soil is one of the essential components in agro ecosystems. Also, it is the final sink of many pollutants. Anthropogenic activities including metal smelting, compost and fertilizers may bring heavy metals into agricultural soil (Alloway, 2004). Studies have shown that heavy metals were potentially toxic to crops, animals and humans when contaminated soils were used for crop production (Xian, 1989; Costa, 2000). Copper and Zn are essential micronutrients and are phyto toxic at very high concentrations from plant standpoints (Gupta and Kalra, 2006). Although Pb and Cd are not essential elements for plants, plants take up Cd and Pb from soils and accumulate them in their edible parts. The consumption of the edible plant parts by human is an important pathway for soil Pb and Cd to affect human health (Wang et al., 2006).

Sludge from Indah Water Konsortium (IWK)

Normally, all of the sewerage systems from individual septic tanks (from house) to the most sophisticated mechanical plants which can produce sludge. Sludge is an active organic compound which can rapidly turn septic if left untreated. For untreated sludge is a significant environmental and public health hazard. However, treated stabilized sludge is inert, stable and safe to use. It can be utilized to condition soil or as landfill (www.iwk.com.my).

By the year 2005, Malaysia will be producing 4.3 million cubic meters of domestic sludge annually. As a result, many new sludge treatment and disposal facilities will be needed to manage the large volume. Malaysia produces 3.2 million cubic meters of domestic sludge yearly. However, facilities to treat and dispose of this sludge are limited. Currently, sewage treatment plants with excess capacity are being used to treat septic tank sludge. One viable solution is to construct sludge lagoons that will serve as sludge holding and treatment facilities. However, for long-term use in urban areas, sludge settling tanks and digestors are required, such as at the Pantai Sewage Treatment Plant in Kuala Lumpur. As an immediate solution, Indah Water is proposing to use existing sewage treatment plants with excess capacity. For a short-term strategy of between two to five years, Indah Water proposes the construction of sludge lagoons, while the long-term strategy would be to construct sludge digestion and mechanical dewatering facilities (www.iwk.com.my).

Sludge Treatment

Normally, sludge that produce from wastewater treatment process is high. The disposal of the excessive sludge will be forbidden in the near future, thus will increased attention has been turned to look into potential technology to reduce sludge (Garg, 2009). There are few options for sludge treatment. They are stabilization, thickening, dewatering, and also drying. The sewage sludge is stabilized in order to reduce pathogens, to eliminate offensive odors and to inhibit, reduce or eliminate the potential for putrefaction to happen. Otherwise, stabilization is used to reduce volume, the production of usable gas such as methane, and also improve the dewater ability of the sludge. For the other technology such as thickening, dewatering and drying, they are used to remove water from the sewage sludge. But, there are several techniques in dewatering devices that can be use to remove moisture. The most close techniques to nature and very effective is dewatering in drying beds. The advantages of drying beds are low in costs; infrequent attention is required and high solid content in the dried products, especially during arid climates. There are also some disadvantages while using drying beds. They are large space is required, the effect of climatic changes on drying characteristics, labor intensive sludge removal, insects and potential to produce odors (Pabsch, 2003).

Phytoremediation

There are some advantages from phytoremediation. They are the phytoremediation cost are much less than traditional in situ and ex situ processes, the plants can be easily monitored to ensure the proper growth, the valuable metals can be reclaimed and reused through this processes and also preserves the nature of the environment. But, they are also having limitations while doing this process. The limitations are phytoremediation is confined to the area that covered by the depths of the roots, leeching of contaminants into groundwater cannot be fully prevented by plant based remediation systems and the plant survival is at risk due to the toxicity of the contaminated soil as well as general soil condition.

Phytoremediation Technologies

There were few technologies that can be applied as phytoremediation process.

Phytoextraction

Phytoextraction is one of the technologies that use plant to remove the contaminants. Generally, phytoextraction is the uptake of contaminants such as heavy metals by roots of the plant and translocation within that plant. The contaminants are normally removed by harvesting process by that plant. The concentration technology leaves a much smaller mass to dispose of compare to excavate of the soil or other media. This technology is most often applied to heavy metal contaminated soil, as shown in figure 2.1. Phytoextraction is used to treat the soil, sediments and also treat sludge. It can be used to a lesser extent for treatment of contaminated water. The phytoextraction studies were conduct by using hydroponically grown plants with the added contaminant in solution. It may not reflect actual conditions and results the occurred in the soil. The coefficient of phytoextraction measured under field conditions were likely seems to be less than those determined while doing laboratory analysis (Nanda Kumar et al.,1995).

Rhizofiltration

The other technology is rhizofiltration. Rhizofiltration is the adsorption process or precipitation onto plant roots, or an absorption into the roots of contaminants that are in solution which is surrounding the root zone area, due to biotic or abiotic processes. Plant uptake, concentration, and translocation might occur, depending on the contaminant. Exudates from the plant roots might cause precipitation of some metals. Rhizofiltration first results in contaminant containment, in which the contaminants are immobilized or accumulated on or within the plant. Contaminants are then removed by physically removing the plant. Extracted groundwater, surface water and waste water can be treated by applied this technology. Generally, rhizofiltration is applicable to this two conditions; low concentration and high water content. But, for soil, sludge or sediments, this technology does not really work well because the contaminant needs to be in solution in order to be sorbed towards plant system. This technology contributes few advantages. The advantages are either terrestrial or aquatic plants can be used. Although terrestrial plants require support, such as a floating platform, they generally remove more contaminants than aquatic plants. For this system, it can be either in situ (floating rafts on ponds) or ex situ (an engineered tank system). An ex situ system can be placed anywhere because the treatment does not have to be at the original location

Phytostabilization

Phytostabilization can be defined as; (1) immobilization of a contaminant in soil through absorption and have be accumulated by roots, adsorption onto roots, or precipitation within the root zone of plants, and (2) the use of plants and plant roots to prevent contaminant migration via wind and water erosion, leaching, and soil dispersion. Phytostabilization can change metal solubility and mobility or impact the dissociation of organic compounds. The plant affected soil environment can convert metals from a soluble to an insoluble oxidation state (Salt et al., 1995). Phytostabilization can occur through sorption, precipitation, complexation, or metal valence reduction (EPA 1997a). Plants can also be used to reduce the erosion of metal contaminated soil. The term phyto lignification has been used to refer to a form of phytostabilization in which organic compounds are incorporated into plant lignin (Cunningham et al. 1995b). Compounds can also be incorporated into humic material in soils in a process likely related to phytostabilization in its use of plant material. The advantages by using this technology are the unnecessary of soil removal; the cost is low and less disruptive than other more vigorous soil remedial technologies and disposal of hazardous materials is not required.

Rhizodegradation

Rhizodegradation is the breakdown of an organic contaminant in soil through microbial activity that is enhanced by the presence of the root zone. Rhizodegradation is also known as plant-assisted degradation, plant-assisted bioremediation, plant-aided in situ biodegradation, and enhanced rhizosphere biodegradation. Root-zone biodegradation is the mechanism for implementing rhizodegradation. Root exudates are compounds produced by plants and released from plant roots. They include sugars, amino acids, organic acids, fatty acids, sterols, growth factors, nucleotides, flavanones, enzymes, and other compounds (Shimp et al., 1993; Schnoor et al., 1995a). The microbial populations and activity in the rhizosphere can be increased due to the presence of these exudates, and can result in increased organic contaminant biodegradation in the soil. Additionally, the rhizosphere substantially increases the surface area where active microbial degradation can be stimulated. Degradation of the exudates can lead to co-metabolism of contaminants in the rhizosphere.

Plant roots can affect soil conditions by increasing soil aeration and moderating soil moisture content, thereby creating conditions more favorable for biodegradation by indigenous microorganisms. Thus, increased biodegradation could occur even in the absence of root exudates. One study raised the possibility that transpiration due to alfalfa plants drew methane from a saturated methanogenic zone up into the vadose zone where the methane was used by methanotrophs that co metabolically degraded TCE (Narayanan et al., 1995). The chemical and physical effects of the exudates and any associated increase in microbial populations might change the soil pH or affect the contaminants in other ways.

Rhizodegradation has the advantages. The advantages are contaminant destruction occurs in situ; translocation of the compound to the plant or atmosphere is less likely than with other phytoremediation technologies since degradation occurs at the source of the contamination and mineralization of the contaminant can occur.

Phytodegradation

Phytodegradation (also known as phytotransformation) is the breakdown of contaminants taken up by plants through metabolic processes within the plant, or the breakdown of contaminants external to the plant through the effect of compounds (such as enzymes) produced by the plants. The main mechanism is plant uptake and metabolism (US EPA, February 2000). Additionally, degradation may occur outside the plant, due to the release of compounds that cause transformation. Any degradation caused by microorganisms associated with or affected by the plant root is considered rhizodegradation. Phytodegradation is used in the treatment of soil, sediments, sludges, and groundwater. Surface water can also be remediated using phytodegradation. The advantages for this technology are contaminant degradation due to enzymes produced by a plant can occur in an environment free of microorganism (for example, an environment in which the microorganisms have been killed by high contaminant levels). Plants are able to grow in sterile soil and also in soil that has concentration levels that are toxic to microorganisms. Thus, phytodegradation potentially could occur in soils where biodegradation cannot.

Phytovolatilization

Phytovolatilization is the uptake and transpiration of a contaminant by a plant, with release of the contaminant or a modified form of the contaminant to the atmosphere from the plant through contaminant uptake, plant metabolism, and plant transpiration. Phytodegradation is a related phytoremediation process that can occur along with phytovolatilization (US EPA, February 2000). Phytovolatilization has mainly been applied to groundwater, but it can be applied to soil, sediments, and sludges. Phytovolatilization has the advantages.

The advantages are contaminants could be transformed to less-toxic forms, such as elemental mercury and dimethyl selenite gas and contaminants or metabolites released to the atmosphere might be subject to more effective or rapid natural degradation processes such as photodegradation.

Heavy Metals Contamination

Generally, ‘heavy metals’ must have a specific weight (mass) which is higher than 8 g/cm? (Fulcrum Health Limited, 2007). From other sources, ‘heavy metals’ can be determined based on their density of the elemental form of each metal with the densities above than 7 g/cm? (Bjerrum, 1936). The term ‘heavy metal’ has been taken to mean that all metals and also metalloids exclude the alkali and alkaline earth elements (Bryan, 1976).

Heavy metals are one of the toxic metals. They are individual metals and metal compounds that negatively affect people’s health. Some toxic, semi-metallic elements, including arsenic and selenium, are discussed in this page. It is often used as a group name for metals and semimetals (metalloids) that have been associated with contamination and potential toxicity or eco toxicity. In very small amounts, many of these metals are necessary to support life. However, in larger amounts, they become toxic (Duffus, 2002).

Heavy metals become dangerous because they tend to bioaccumulate. Bioaccumulation is the increasing in the concentration of the chemical in the organism over the time, compared to chemical’s concentration in the environment. The compounds accumulate in living things at any time they are taken up and stored faster than they broken down (metabolized). These heavy metals can enter a water supply by industrial and also consumer waste, or even from acid rain which break down the soil. Then, the heavy metals were released into streams, lakes, rivers and to groundwater.

Heavy Metals Characteristics

Copper

Copper is one of the elements that occurred in a small quantity in the environment and needed for the normal growth and also function in metabolism system for all living organism (Schroeder et.al, 1966; Carbonell and Tarazona, 1994). Copper is most widely scattered in the nature ores that contain sulfides, arsenic, chloride and also carbonate. Generally, as we all know, in plant, copper is needed especially in seed production, disease resistance and regulation of water.

The toxicity level of copper occur naturally in some soil whereas others may contain high levels of copper as a result of anthropogenic release of the heavy metals to the environment through mining, smelting, open dumping and manufacturing. Average content of copper in plant is 10 mg/kg (Baker and Senef, 1995). At concentration above the requirement for optimal growth, copper can inhibit growth and interfere in an important process such as photosynthesis and respiration (Prasad and Strzalka, 1999). Both humans and animals need some amounts of copper in their diets, but very high concentrations of copper can be toxic causing adverse effects. The most common symptoms of copper toxicity to human are injury to red blood cells, injury to lungs, as well as damage to liver and pancreatic functions.

Chromium

Chromium is the first transition series element with atomic number 24, atomic mass 52 g/mol and density 7.2 g/cm3. The common oxidation states are +2, +3 and +6. Chromium is the seventh most abundant metal in the earth’s crust (Katz and Salem, 1994) and an important environment contaminant released into the atmosphere due to its huge industrial use (Nriagu and Nieboer, 1988). Source of chromium in the environment are waste chromate from paint, corrosion inhibitors from water cooled heat exchange system, waste solution from dyeing and leaching from sanitary landfills.

In nature, chromium exists in two different stable oxidation states; Cr (III) and Cr (VI). Cr (VI) is found to be more toxic than Cr (III) (Panda and Patra, 1997). Naturally occurring in soil, Cr range from 10 to 50 mg/kg and in plant is about0.006-18 mg/kg. Chromium is toxic to plants and does not play any role in plant metabolism (Dixit et al., 2002). Accumulation of chromium by plant can reduce growth, induce chlorosis in young leaves, reduce pigment content, alter enzymatic function, damage root cells and cause ultrastructural modifications of chloroplast and cell membrane (McGrath, 1985; Panda and Patra, 1997; Panda and Dash, 1999; Choudhury and Panda, 2004).

Iron

Iron is a metallic chemical element of atomic number 26. Its symbol is Fe, atomic weight is 55.847, specific gravity is 7.874, melting point is 2,795°F (1,535°C), and boiling point is 4,982°F (2,750°C). Iron is one of the transition metals, occurring in group 8 of the periodic table. Iron is the fourth most abundant element in the earth’s crust and the second most abundant metal, after aluminum. Iron is essential for plants and plays critical roles in important processes such as photosynthesis and respiration( Jeong and Connoly, 2009).

Zinc

Zinc is one of the metallic elements. And, it has the symbol of Zn and atomic number 30. In the group 12 of periodic table, zinc is the first element in the group (Habashi, 2008). Zinc is chemically similar to magnesium (Mg) because of its ion and the oxidation state of +2. Zinc is an essential mineral for biological processes and also in concern of public health (Hambidge and Krebs, 2007). Zinc deficiency affects about two billion people in this world and performed with many diseases. For children, it will cause growth retardation, delayed sexual maturation, infection susceptibility, and diarrhea. Zinc contributes to the death of 800,000 children in the world per year. Enzymes with zinc atom in reactive center are spread widely in chemistry, such as alcohol dehydrogenase in humans.

Cadmium

Cadmium, with symbol of Cd and atomic number 48 is one of the chemical elements. Similar to zinc, it prefers the oxidation state +2 in the most of its compounds and similar to mercury (Hg) because it shows low melting point compared to transition metals (Jennings and Thomas, 2005). Average concentration of cadmium in the earth’s crust is between 0.1 and o.5 parts per million (ppm). It was discovered by Stromeyer and Hermann, both in Germany. Cadmium occurs as a minor component in most of zinc ores and the byproduct is zinc.

Cadmium was used as a pigment and also for corrosion resistant plate on steel (Miller and Mullin, 1991). Cadmium compounds used to stabilize the plastic. With the exception of its use in nickel-cadmium batteries and cadmium telluride solar panels, the use of cadmium is generally decreasing in its other applications.

Essential Plant Nutrients

Generally, plants require about 13 mineral nutrient elements to grow. The elements needed for the plants to complete their life cycle. These elements were called essential plant nutrients. For macronutrients such as nitrogen, phosphorus, calcium, potassium, sulphur and magnesium needed for plants in the large amount. Micronutrients such as iron, copper, chlorine, boron, zinc, manganese and molybdenum required in small amount (Tucker, 1999). The nutrient elements were differ in the form of they were absorbed by the plant, by their functions in that plant, their mobility in the plant and by plant deficiency or symptoms of toxicity. There were five types of deficiency or toxicity symptoms. They were chlorosis, necrosis, lack of new growth, accumulation of anthocynanin resulting in purple or reddish colour, and stunting or reduced growth. Table 2.2 shows the essential plant nutrients: their relative amounts in plants, functions and classification. Then, table 2.3 shows generalized symptoms of plant nutrient deficiency or excess.

Acacia Mangium

There are roughly 1300 species of Acacia existing in the world but the discovery for Acacia sp. in Malaysia is known as Acacia mangium. Acacia is a genus of shrubs and trees belonging to the subfamily Mimosoideae of the family Fabaceae, first described in Africa. Acacia mangium is native to Australia, Indonesia, Papua and New Guinea. It is a low-elevation species associated with rain forest margins and disturbed, well-drained acid soils (pH 4.5-6.5). It is usually found in the humid, tropical lowland climatic zone characterized by a short dry season.

Acacia recover wastelands like ex-mining area, returning nutrients to poor soils and providing shade for other plants to take hold. Its ability to grow well on infertile soils, especially those low in phosphorus, make it a favorite for rehabilitation of mine spoils and eroded sites. The species are characterized by polymorphism of vegetative characters where bi-pinnate leaves are replaced by a type of foliar organ called phyllode. Phyllodes are morphologically distinct from ordinary foliage leaf. In this research, Acacia mangium will be use as a sample to determine the uptake of heavy metals in the sewage sludge that we take from Indah Water Konsortium (IWK) Juasseh.

Below is the taxonomy for the Acacia mangium (Starr and Loope, 2003 ):

• Family : Fabaceae (Pea family) (Wagner et al., 1999).
• Latin name: Acacia mangium Willd.
• Common names : Mangium, Mangium wattle, mange, forest mangrove
• Taxonomic notes: The genus Acacia is made up of about 1,200 species that are widespread but with a large number in Australia (Wagner et al., 1999). Acacia mangium hybridizes naturally with Acacia auriculiformis, producing hybrids which grow faster than either parent, but tend to retain the poor form of A. auriculiformis.
• Nomenclature : The genus name is derived from akakia, the Greek name for Acacia arabica (Lam.) Willd., which is derived from akis, a Greek word meaning sharp point, in reference to the thorns of the plant (Wagner et al., 1999).

Acid Digestion

Acid digestion is one of the methods that necessary to dissolve metal samples for analysis. Most lab chemistry involves dissolving a salt with water or organic material with solvent to obtain a known volume solution for chemical analysis. Metals will not dissolve in water or organic solvents; therefore acid digestion is a method of dissolving the metal into solution, which can then be analyzed by laboratory methods to determine the amount of metal or element present (Edgell, 1988 and Kimbrough and Wakakuwa, 1989).

Inductively Coupled Plasma Mass Spectroscopy (ICP-MS)

ICP technology was built upon the same principles used in atomic emission spectrometry. Samples are decomposed to neutral elements in high temperature argon plasma and analyzed based on their mass to charge ratios (Wolf, 2005). An ICP-MS can be thought of as four main processes, including sample introduction and aerosol generation, ionization by an argon plasma source, mass discrimination, and the detection system. The schematic below illustrates this sequence of processes.

Because atomization or ionization occurs at atmospheric pressure, the interface between the ICP and MS components becomes crucial in creating a vacuum environment for the MS system. Ions flow through a small orifice, approximately 1 millimeter in diameter, into a pumped vacuum system. Here a supersonic jet forms and the sample ions are passed into the MS system at high speeds, expanding in the vacuum system (Jarvis et al., 1992). The entire mass spectrometer must be kept in a vacuum so that the ions are free to move without collisions with air molecules.

Since the ICP is maintained at atmospheric pressure, a pumping system is needed to continuously pull a vacuum inside the spectrometer. In order to most efficiently reduce the pressure several pumps are typically used to gradually reduce pressure to 10-5 mbar before the ion stream reaches the quadruple. If only one pump were used, its size would be excessive to reduce the pressure immediately upon entering the mass spectrometer.

One of the advantages to ICP-MS is the extremely low detection limits for a wide variety of elements. Some elements can be measured down to part per quadrillion ranges while most can be detected at part per trillion levels. The table 2.below shows some common detection limits by element.

Statistical Analysis

Statistical analysis is an experiment usually results in some means or proportion affected of different groups such as control and treated animals. Means will differ because each animal is different. Proportions affected could differ by chance. Means and proportions may also differ as a result of the treatment (Rossiter, 2006). The aim of the statistical analysis is to calculate the probability that differences as great as or greater than those observed could be due to chance.

If this probability is high, then chance may be the explanation, if it is low then a treatment effect may be the explanation. These days the actual calculations are almost always done using a computer.

Most measurement data where the aim is to compare means can be analyzed using an analysis of variance (ANOVA), a t-test or a non-parametric method. Scores and proportions often use a chi-squared test, while dose-response relationships use regression analysis. Other methods may be needed when there are multiple outcomes. The methods described in the sub-pages give a brief introduction.

Materials and Methods

Plant Samples

Acacia recover wastelands like ex-mining area, returning nutrients to poor soils and providing shade for other plants to take hold. Its ability to grow well on infertile soils, especially those low in phosphorus, make it a favorite for rehabilitation of mine spoils and eroded sites. The species are characterized by polymorphism of vegetative characters where bi-pinnate leaves are replaced by a type of foliar organ called phyllode. Phyllodes are morphologically distinct from ordinary foliage leaf. In this research, Acacia mangium will be use as a sample to determine the uptake of heavy metals in the sewage sludge that we take from Indah Water Konsortium (IWK) Juasseh. The criteria of the plants that being chosen were same height, same diameter of stem and also have the same amount of leaves. Figure 3.1 shows the Acacia mangium plant.

Chemicals Used

For acid digestion, the chemicals that being used were nitric acid and hydrochloric acid. For nitric acid, the chemicals that been used was Nitric acid-65%. The brand for this chemical is SYSTEM. The CAS no. for this chemical is [7697-37-2]. The amount of the chemicals used is about 2.5 L. The brand for hydrochloric acid that being used is Fischer Scientific. The chemical is Hydrochloric acid-37%. The CAS no. for this hydrochloric acid is [7647-01-0]. For ICP-MS analysis of heavy metals, the chemical that been used is 29-Multi element ICP-MS calibration. This standard consist of 5% HNO3 . The element that can be detected by using this standard are Al, As, Ba, Be , Bi, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, In, K, Li, Mg, Mn, Ni, Pb, Rb, Se, Na, Ag, Sr, Tl, V, U and Zn.

Chemicals

• Nitric acid
• Hydrochloric acid
• Deionized water (MiliPore type 1)
• 29-Multi element ICP-MS calibration

Experimental Design

Firstly, the different medium for each treatment were prepared. The treatment prepared by mixing the soil and sludge from Indah Water Konsortium (IWK). The treatments were 100% of soil, 30% sludge+70% soil, 50% sludge+50% soil and 70% sludge+30% soil. Then, the Acacia mangium were planted in the soil. The initial reading for height, diameter measured. Then, after 30 days, the height and diameter were measure again. The measurements were taken for the period of 30 days, 60 days and 90 days. After that, the harvesting process began. After harvesting, the plant was cut into three parts. The three parts were roots, stems and leaves. The harvesting processes were continued for the period of 60 days and 90 days. Then, the plant dried into the oven for about 2 days. After two days, the biomasses of each part of the plant were measured. After measure the biomass, the plants were grind until got the small size. After that, the sample of roots, leaves and stem were digested by using nitric acid and hydrochloric acid. The ratio for this mixture was 3:1, which was 300 ml of hydrochloric acid with 100 ml of nitric acid. The standard solution for ICP-MS analysis was prepared. The dilution that needed was 100 ppb, 10 ppb, 20 ppb and 30 ppb. Finally, the analysis for heavy metals were conducted by using ICP-MS. Figure 3.2 showed the diagram for experimental design that occurs in this project.

Treatment of The Plant

For the first step, four types of the treatment have been prepared. As shown in Table 3.1, the treatments were distributed to 4 different types. The first treatment is 100% soil have been used to plant the Acacia mangium. It is also known as control treatment. The second treatment is 70% soil with 30% sludge have been used to plant the tree. This treatment also known as treatment B. The third treatment is treatment C. It is contain 50% soil with 50% sludge from IWK. The last treatment is 30% soil with 70% sludge and assume as treatment D. For each treatment, about twenty of Acacia mangium were planted.

Sample Analysis

Before all the analysis will conduct, the Acasia mangium is planted by using IWK contaminated soil. Every month, we need to harvest the tree. For each harvest, a tree needs to be removed. The growth performance for the tree needs to be measure. The parameters are height, biomass and the diameter of the tree.

Acid Digestion

About 0.5 g of dried samples of leaves, roots and stems were first weighted and then placed in clean digestion tubes. Then, a mixture of concentrated hydrochloric acid, HCl and nitric acid, HNO3 with mixed ratio of 3:1 was then added to digest the samples. The start of the digestion is beginning with temperature at 40oC for an hour and then, the temperature was increased to 140oC for another 2 to 3 hours until digestion process finished.

The digestion process has finished when the sample solution was clear or pale yellow and no more brownish fume released. The same procedure repeated for soil sample that we used before we plant the tree.

After the solution cooled down, the solution samples were then diluted to make 100mL solution and filtered through no.42 filter paper into sample bottles. The blanks samples (normally distilled water) were carried out the same way as samples. These solutions then were analyzed with Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) for five different elements; iron (Fe), chromium (Cr), zinc (Zn), copper (Cu) and cadmium (Cd).

The main priority for analysis in the laboratory, glassware or containers for the samples have been acid washed by soaking the glassware overnight in 5% acid solution (HNO3 or HCl) and then rinsed thoroughly with distilled water.

Analysis of Heavy Metals

Heavy metal analyses were done using ICP-MS, an inorganic analytical instrument made by Perkin-Elmer TM. The characteristics of analysis by this instrument are listed in the table below. Blank determination was carried out for calibration of the ICP-MS. Calibration curves of heavy metals analysis was obtained from concentration of the given standard interval. The heavy metals that need to be analyzed are Fe, Cu, Cr, Zn and Cd. Standard solutions were prepared from 10000 ppb stock solution supplied by Perkin Elmer. The standard solution need to be run first. It is because we need to have the calibration curve before the sample analyzed. The calibration curve considered accepted if the r? value is greater than 0.995. The range for the calibration curve must be ± 15% from the actual concentration. For example, if concentration 100 ppb, the accepted value can be within 85 ppb to 115 ppb. Recovery test was done by using known concentration of a standard solution determined with ICP-MS as a sample to obtained 90% to 95% recovery throughout this study.

Standard Preparation

From the given standard solution which has the concentration about 10000 ppb, the solution need to be dilute until we get 4 different standard solution. From the prepared standard, the ICP-MS will test the standard. Table 3.2 shows the standard solutions that we need to be prepared.

Statistical Analysis

ANOVA Analysis

The tests we have learned up to this point allow us to test hypothesis that examine the difference between only two means. Analysis of Variance or ANOVA will allow us to test the difference between 2 or more means (Bower, 2000). ANOVA does this by examining the ratio of variability between two conditions and variability within each condition. A t-test would compare the likelihood of observing the difference in the mean number of words recalled for each group. An ANOVA test, on the other hand, would compare the variability that we observe between the two conditions to the variability observed within each condition.

Results and Discussion

Data analysis

Height

The graph below shows the result for height of Acacia mangium after plant it for about 3 months. On the y-axis, it represents the treatment. For number 1, it is 100% soil which is considered as control treatment, number 2 is mix of 30% of sludge and 70% of soil. For number 3 and 4, each of them represents 50% sludge and 50% soil, and the other is 70% sludge and 30% soil. From the graph, for the control treatment, the height of the plant increase when the period of time for the plant was long. Then, for the treatment 3, the height was higher than the other treatment. For the treatment 2 and treatment 4, the situation was same with the control treatment. Although the increasing was not really huge, but it still can be considered as the normal growth for this plant. The percentage of height changes of the plants increase, although in a small amount.

For the treatment 1, treatment 2 and treatment 4, the growth of the plant was normal. From time to time, the height increase with the period of time. The mixing of sludge and soil for this three treatment can be considered successfully act as fertilizer. For the normal plant, they need fertilizer to help them to live well. If the fertilizer supply was enough, hence the growth of the plant must be well. For example, height of the plant. Treatment 3 was the mixture of the sludge which is only 50% and soil which is about 50%. So, from this result, it shows that after have rapid growth for the whole period of 90 days, most of the nutrient that contain in the sludge were uptake by Acacia mangium to grow well. The plant can uptake most of the sludge that contain in the soil.

As shown in Appendixes A, the mean for the height is 57.044 cm. The value for R-square is 0.862. It is mean that the height of tree has strong correlation with the harvesting time and also the different treatment. Besides, from the Appendixes A, if comparing between the height and harvesting time, the p-value is 0.0323, which is greater than 0.05. The p-value indicates that there were no significant effect between the height and harvesting time. The height was not influenced with the harvesting time. For the different treatment with height, the p-value is less than 0.05. The different treatment, with the different mixture of soil and sludge, gave the different impact towards the growth of the plant.

From Figure 4.3, the biomass for leaves during first month was higher compared to the other two months. It is because, during the first month, there was much heavy metal that still contain in the soil. So, the plant has lot of chances to gain more nutrients for their growth. But, for control treatment, during the first harvest, the biomass was low compared to the other harvest. It is because, the plant was tried to adapt the new conditions. Otherwise, the control treatment only has soil, without sludge. Then, they did not get enough nutrients to grow well. For the other treatment, the sludge played a role as nutrient supply to the plant. From the result, we can see that the trend of biomass for leaves was quite good. For the period of 90 days, there were lots of leaves that fall onto the ground. Hence, the biomass of leaves decreased while the period of time was longer. This can be related with the amount of heavy metals uptake by this plant. According to figure 4.6, the uptake for copper became low along the period of 90 days. Copper was needed by this plant for photosynthesis process. Then, copper translocations were done until leaves because photosynthesis process occurred in leaves area.

As shown in Appendixes C they were the results for biomass of leaves. The average mean for biomass of leaves was 8.511 g. The R-square value is 0.742, which can be considered as strong correlation between the biomass of leaves with the harvesting times and also the treatment. If we compared between harvest with the biomass, there were not have significant different. It means that the harvesting times do not affect the biomass of leaves. Same goes to the treatment and biomass. The p-value is greater than 0.05. There were also not have significant different between them.

Stem

Figure 4.4 shows the result that I get from this research. The graph is about the results of biomass for stem of Acacia mangium against the 4 different treatments. From all of the result that shown in the graph, at second harvest, biomass of stem for 4 different treatments higher than the other two harvest. For treatment C which is contain 50% of sludge mix with 50% of soil, the biomass is the highest compare to the other treatment. And, for control treatment, which is 100% soil shows the lowest biomass among the others.

During the second harvest, which is among the period of 60 days, the biomass of the stem is the most highest. From this, during this period of time, the plant, which is Acacia mangium has been successfully absorb all the nutrients that provide in the mixture of soil and sludge. Overall, during the second harvest, the biomass for stem for each treatment was higher compared to treatment for first and third harvest. During the period of 30 days, the plant undergoes adaption process which they were tried to make themselves comfortable with the soil and the sludge. But, after 60 days, their biomass was slightly decreased. It was happened because of the reduction of nutrient supply in the soil.

During the second month, the plant absorbs as much nutrient as they could. Besides, there were not enough sufficient nutrients to support their growth. This trend was similar with roots part. The highest biomass measured during the second month. The biomass decreased after 90 days.

As shown in Appendixes D, there were the results for biomass of stem. The average mean for biomass of leaves was 10.198 g. The R-square value is 0.868, which can be considered as strong correlation between the biomass of stem with the harvesting times and also the different treatment. If we compared between harvest with the biomass, there were have significant different. It means that the harvesting times affected the biomass of stem. Same goes to the treatment and biomass. The p-value is less than 0.05. There were also have significant different between them.

Root

From Figure 4.5, the graph shows the result for biomass of the plant. The result consists of root part only. From the entire bar, we can see that the highest biomass for root was at the 2nd harvest and for the control treatment. For the lowest biomass, the biomass root for treatment B which is 30% sludge, during the 3rd harvest has been detected. Randomly, during the 2nd harvest, the biomass for root from each treatment was quite high compared to 1st and 3rd harvest. During the 3rd harvest, the biomass of root for each treatment slightly decreased.

From the graph, the biomass of the root quite high during the 2nd harvest due to a few reason. The first reason was the nutrient or the metals that contain in the soil and played a role as fertilizer for the plant to grow. During the 1st harvest, the plants try to uptake all the nutrients to help their growth. But, in the period of 60 days, the plant had already comfortable with the soil. So, they gained more nutrients in order to grow and stay in that soil. After 60 days, the content of nutrients in the soil decreased. Then, the biomass of the plant became decrease. The same situation happened to all the treatment.

As shown in Appendixes E, there were the results for biomass of root. The average mean for biomass of leaves was 3.844 g. The R-square value is 0.810, which have strong correlation between the biomass of root with the harvesting times and also the different treatment. If we compared between harvest with the biomass, there were have significant different. It means that the harvesting times affected the biomass of root because the p-value less than 0.05. But, there were difference between treatment and biomass. The p-value is greater than 0.05. There were also not have significant different between them.

Heavy Metal Analysis

Copper

Copper is one of the elements that needed by plant to growth. From the graph, we can see that the uptake of heavy metals, especially copper was high during the first harvest. Mostly, the uptake of heavy metals which is copper was high in root parts. Not all of the uptake copper may translocation successfully to the top. Randomly, stem have the lowest uptake among all of the parts. The heavy metals uptake for the first harvest was high.

The metals uptake was high during the first harvest. It was due to the capability of the plant to survive. The plants need more nutrients if they wanted to live well. If the plants did not fight to get the nutrient, they may die in a few days. The uptake for copper still high during the second harvest, because they need to maintain grow in the soil. But, after 90 days, the uptake decrease. The content of nutrient or heavy metals in the soil reduce because of the rapidly uptake during the first and second harvest. Copper was needed by plants, in order to well growth.

According to Tucker et al., copper is needed to be function as a catalyst in photosynthesis and respiration process. It is a constituent of several enzyme systems involved in building and converting the amino acids to proteins. Copper is also important in carbohydrate and protein metabolism. Copper also affects the flavor, the strong-ability and the sugar content of fruits.

As shown in Appendixes F, there were the results for copper uptake by the plant. The average mean for concentration of copper was 0.032 mg/l. The R-square value is 0.457, which have weak correlation between the concentration of copper with the different part and also the different treatment. If comparing between part and concentration for copper, there were significant difference. It means that the different part does affected the amount of copper uptake because the p-value less than 0.05. But, there were different result between treatment and concentration of copper uptake. The p-value is greater than 0.05. There were no significant different between them, which mean that different treatment, does not, affected the concentration of copper uptake.

Chromium

From the graph, we can see that the uptake of heavy metals, especially chromium was high during the first harvest. Mostly, the uptake of heavy metals which is chromium was high in stem and leaves. Not all of the uptake chromium may translocation successfully to the top. Randomly, stem have the highest uptake among all of the parts. The heavy metals uptake for the first harvest was high.

The uptake for chromium in stem during the first harvest for control treatment was the highest. This can be related to the biomass of the stem for that particular treatment. From figure 4.4, we can see that the biomass for control treatment of stem was high compared to the first and third harvest. The uptake of chromium was related with the biomass. If the uptake was high, the biomass for the stem also may be increase. The highest biomass was influenced by the highest concentration uptake. Chromium was not necessary element for plant to grow.

As shown in Appendixes G, there were the results for chromium uptake by the plant. The average mean for concentration of chromium was 0.018 mg/l. The r-square value is 0.092, which have very weak correlation between the concentration of chromium with the different part and also the different treatment. If comparing between treatment and concentration for chromium, there were no significant difference. It means that the treatment does not affected the amount of chromium uptake because the p-value greater than 0.05. The result between different part and concentration of chromium uptake were also no significant difference. The p-value was greater than 0.05, which means that different part, not affected the concentration of chromium uptake.

Ferum

If we compared with the others heavy metal uptake, ferum gave slightly different result. The rapidly ferum uptake was during 90 days period of times, which was the third month. For the first two months, the plant does not really need ferum as their nutrient supply or fertilizer. The highest uptake for this element was during the third month, which was for control treatment. Mostly, the lowest concentration uptake was for stem.

The highest uptake was during the third month. This was because, during the first two months, the plant, which is Acacia mangium used Cd, Cu, Cr and Zn as their fertilizer. Then, after two months, the uptake for those particular metals decreased due to the highest uptake during first two months. Then, ferum were uptake high during the third months. This element combines with other metals such as Zn and Cu to help plant to grow well. Because of the combines, the plant may sustain for a longer time. Ferum is needed for the plants to help in the chlorophyll development and function. It is also play a role in energy transfer within the plant. Ferum also functions in plant respiration and plant metabolism. Other than that, ferum is involved in nitrogen fixation.

Appendixes H show the results for ferum uptake by the plant. The average mean for concentration of ferum was 5.073 mg/l. The R-square value is 0.314, which have weak correlation between the concentration of ferum with the different part and also the different treatment. If comparing between treatment and concentration for ferum, there were no significant difference. It means that the treatment does not affected the amount of ferum uptake because the p-value greater than 0.05. But, there were different result between different part and concentration of ferum uptake. The p-value is less than 0.05. There was having significant difference between them, which means that different part, affected the concentration of ferum uptake.

Zinc

Zinc is one of the metals that needed by plant to growth. From the graph, the uptake of heavy metals, especially zinc was high during the first harvest. Mostly, the uptake of heavy metals which is copper was high in root and leaves parts. Not all of the uptake copper may translocation successfully to the top. Randomly, stem have the lowest uptake among all of the parts. The heavy metals uptake for the first harvest was high. The highest uptake for zinc was in leaves.

The uptake for zinc in leaves during the first harvest for treatment C, which is 50% sludge, was the highest. This can be related to the biomass of the leaves for that particular treatment. From figure 4.3, the biomass for 50% sludge mix with 50% soil treatment of leaves was high compared to the other parts and harvest. The uptake of zinc was related with the biomass. If the uptake was high, the biomass for the leaves also may be increase. The highest biomass was influenced by the highest concentration uptake. Zinc was needed by plants in order to activate their enzymes.

In Appendixes I, there were the results for zinc uptake by the plant. The average mean for concentration of zinc was 0.974 mg/l. The R-square value is 0.193, which have very weak correlation between the concentration of zinc with the different part and also the different treatment. There were no significant difference between treatment and concentration for zinc. It means that the different treatment does not affected the amount of zinc uptake because the p-value greater than 0.05. The result between different part and concentration of zinc uptake were also no significant difference. The p-value was greater than 0.05, which means that different part, not affected the concentration of zinc uptake.

Cadmium

Cadmium was not really needed by this plant to grow. The highest accumulation or uptake was during the first harvest. Most of the cadmium was highly uptake in the root parts. The lowest uptake was in the stem. Treatment C can be assuming as successfully fertilized the plant.

The highest uptake was in root parts. During the first harvest, the uptake of cadmium in the root for treatment C, which was 50% sludge, is highest. During the third harvest, the uptake for cadmium was low. The uptake of stem for treatment B shows the lowest uptake which is negative value (-ve).it was because; the amount or concentration of cadmium was little on the soil. The ICP-MS cannot detect the uptake for cadmium because they were too low in the soil. Cadmium was one of the toxic elements. If the plants absorb more cadmium, it might be harm towards the plant growth.

As shown in Appendixes J, there were results for cadmium uptake by the plant. The average mean for concentration of cadmium was 0.0013 mg/l. The R-square value is 0.370, which have weak correlation between the concentration of cadmium with the different part and also the different treatment. If comparing between treatment and concentration for cadmium, there were no significant difference. It means that the treatment does not affected the amount of cadmium uptake because the p-value greater than 0.05. But, there were different result between different part and concentration of cadmium uptake. The p-value is less than 0.05. There were have significant different between them, which means that different part, affected the concentration of cadmium uptake.

Conclusion

From this study, there are few conclusions that can be concluded. Based on the objectives, it can be conclude that the Acacia mangium can be a good phytoremediator in order to clean the contaminated soil that occurs because of human activities. This is because the Acacia mangium uptake lot amount of heavy metals that contain in that soil. From the results, the heavy metals uptake was high according to their different parts which are roots, stem and leaves. The roots uptake the highest amount of heavy metals, followed by leaves and finally stem. From all of the heavy metals that analyzed in this study, ferum gave the highest amount of metals that uptake by Acacia mangium. Ferum have already occurred lots in the soil. So, the plant will absorb more ferum. Then, followed by zinc, copper, chromium and the least one was cadmium. The uptake was rapidly during the first two months because of the capability of the plant to absorb more heavy metals. For the physical changes of Acacia mangium, it can be conclude that there were rapid growths for this plant during the period of 60 days. Then, after 60 days, the growth decrease slowly. It was happened due to the amount of heavy metals that contain in the soil. Because of the content of heavy metals decrease, hence the plants do not have sufficient supply for nutrient towards their growth. The height, diameter of the stem and biomass gave the same results. The increasing during the 60 days period of time, and slightly decrease on the third month. There are few factors that may contribute to this project. There is little human error while conducting this project. They are different amount of water while watering the plant, the mixture of soil did not mixing well, error while doing acid digestion process, and an error while preparing the standard solution and also during dilution process.

Recommendation

For further study, this study needs to be continued. Firstly, the study must consider and analyze the soil. It is because, the amount or concentration of heavy metal that contain in the soil influence the plant to absorb the heavy metals. By knowing the amount of heavy metals that contain in the soil, the exact amount of heavy metals uptake can be determined. Besides that, the study needs to know the stress for the plant. It is means that the ability and capability of this plant to absorb more heavy metals. This study also needs to know the nutrient efficiency for this plant.

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