Heavy metal remediation in copper (Cu) contaminated soil of Andosol and Latosol using spinach (Amaranthus sp.) as bioaccumulator

Concentration of heavy metal copper (Cu) which often present as pollutant in the environment can be minimized by one of remediation techniques called phytoremediation. It is a relatively inexpensive, simple, effective technique and showed minimal effect to the environment. This study aims to investigate the effectiveness of spinach (Amaranthus sp.) in reducing Cu concentration in Andosol and Latosol soils. Three (3) different types of planting media namely Andosol, Latosol, and sandy soils with control treatment (without Cu) and Cu treatment (as CuSO4.5H2O) at a dose of 100 mg/kg or (1.5 gr per 5 kg of soil) were used in this study. The results showed that spinach was able to accumulate heavy metals both in the roots and leaves. However, heavy metal absorption was found to be higher in root tissue compared to other organs observed. The treatment of Latosol soil + compost with spinach plant showed the highest accumulation of Cu compared to other treatments. While in Andosol soils, Cu metal commonly found in the form of residues. Thus, it can be concluded that phytoremediation of Cu using spinach in Andosol and Latosol soil showed promising results.


Introduction
Soil contamination is caused by the presence of xenobiotic (man-made) chemicals or other changes in the soil's natural environment.This type of contamination usually arises from bursting underground storage tanks, application of pesticides, contamination of sewage from landfills or direct discharge of industrial waste [1].The occurrence of this phenomenon correlates with the level of development of industrialization as the more industrial waste is generated.
The level of heavy metal pollution that is exposed to the environment causes serious problems in nature because these metals cannot be decomposed naturally.According to Singh et al. [2] and Sankhla et al. [3] there are at least 20 metals classified as toxic waste which pose a major risk to human health.Heavy metals that are commonly found in the environment such as Cd, Cu, Pb, Co, Zn and Cr have phytotoxic properties at both low and very high concentrations which are detected in waste [4].The heavy metal Cu is classified as an essential heavy metal in very low concentrations.If it is in an accessible form, it will be absorbed by plants so that Cu will become toxic to living organisms.The effect of Cu's toxicity will be seen if this metal enters the organism's body in large quantities or exceeds the tolerance value of the organisms.Heavy metals can accumulate in the media and are quickly absorbed by plants; and although at very low concentrations, heavy metals can be toxic [5].High concentrations of heavy metals in the media can enter the food chain and adversely affect organisms.
Up until now, treatment of Cu-containing waste uses physicochemical methods such as chemical precipitation, ion exchange and reverse osmosis [6].The weakness of this treatment is that it requires expensive monitoring equipment and systems.The concept of biological waste treatment using growing media is known as phytoremediation.Phytoremediation relies on the role of plants to absorb, degrade, transform and immobilize heavy metal pollutants [7] as the plants have the ability to accumulate heavy metals which are essential for growth and development.Phytoremediation only works effectively to the depth of the plant roots, and plants that have absorbed contaminants run the risk of heavy metals entering the food chain.Therefore, it is necessary to add organic matter.The presence of organic matter in the soil besides being utilized by microorganisms as a source of energy, can also react with heavy metals to form complex compounds (organo-metallic complexes), thereby reducing the reactivity and toxic effect of metals [8,9].
Amaranthus spinosus and Amaranthus viridis are species that are able to thrive in polluted environments and have relatively fast life cycles.So that this plant has the potential to accumulate pollution in large quantities without showing any signs of the external damage.Based on this, it is necessary to conduct research on the extent of the ability of spinach plants to accumulate heavy metals, especially Cu.

Materials and methods
The soils used in this study were Andosol soil from Sukamantri Village and Latosol soil from Darmaga area, which were taken at a depth of 0-30 cm, as well as 30 x 50 cm pots, CuSO 4 .5H 2 O, seeds of A. spinosus.and A. viridis, chemicals for soil and plant analysis and laboratory equipments (AAS, shaker, centrifuge, tools glass, funnel, and filter paper).
The preparation of the planting medium was as follows: the soil was air-dried and sieved (< 5 mm sieved), weighed 5 kg each and put into respected pots.The treatments were A0 (control); A1 (Andosol + A. spinosus.);A2 (Latosol + A. spinosus); A3 (Andosol + A. viridis); A4 (Latosol + A. viridis); A5 (Andosol+ A. viridis + compost); A6 (Latosol + A. viridis + compost).Cu is given in the form of CuSO 4 .5H 2 O at a dose of 1.5 g of CuSO 4 .5H 2 O per 5 kg of soil.Cu treatment is done by mixing CuSO 4 .5H 2 O with a little soil in a container that has a lid and stirring until homogenous.Then the mixture was added to the soil and stir once again until homogenous.
Planting of A. spinosus and A. viridis were done after the plants are one week old.Plants are maintained until they are 5 WAP (Week After Planting).Analysis of total heavy metals by drying of plant organs (roots and leaves) at the end of the study.The former planting soil is composited according to the treatment and soil type and then analyzed to determine Cu content.

Cu accumulation in spinach plants
Absorption and accumulation of heavy metals by plants can be divided into three continuous processes, namely absorption of metals by roots, translocation of metals from roots to other plant parts, and localization of metals in certain cell parts to prevent inhibiting the plant's metabolism.The levels of heavy metals in the soil and their absorption by plant species are influenced by soil conditions and the ability of plants to absorb metals in the soil.The result showed a decrease in heavy metal (Cu) levels in the soil after 5 WAP treatment (Figure 1).In Figure 1 it can be seen that A. spinosus can absorb metals higher in leaf, root, and stem tissues compared to A. viridis.Plants usually perform general mechanisms in maintaining homeostasis under high concentrations of heavy metals ions [10].Plants use complex processes to adapt in metabolism to environmental changes.This process includes adaptation to extreme conditions including resistance and tolerance mechanisms, then involves metal immobilization in roots and cell walls [11].Tolerance is related to the internal absorption of toxic elements [12].The seeds of the A. spinosus.plant come from landfills, so it is assumed that these plants have adapted to the high heavy metal concentration in the soil.The high content of Cu in the plant tissue of A. spinosus indicates that the plant is able to absorb Cu more efficient compared to A. viridis.Thus, A. spinosus originating from landfills or other polluted soils can be used as phytoremediation to reduce heavy metal Cu contamination in soil.
The ability of spinach plants to absorb metals in Andosol and Latosol soils which accumulated in the root, stem and leaf tissues can be seen in Table 1.The results showed that in the two types of plant used, heavy metal was found in the roots, stems, and leaves.The highest metal absorption was found in plant biomass, especially roots, in treatment A2 and the lowest in treatment A3.The highest levels of heavy metals are found in the roots of plants.This is presumably because the roots are the first organs that come into contact with toxic compounds and are able to accumulate more than shoots [13].Roots can be used as an important index in heavy metal tolerance.Leaf tissue has a protein with a high NH 2 active group.The NH 2 group on the leaves is a compound that can bind metals, causing Cu to be adsorbed in the leaves [14].
Moreover, the addition of compost in treatments A5 and A6 did not increase the accumulation of heavy metal absorption in spinach plants.Compost will interact with polluted soil through the chelation mechanism, because compost is able to increase organic acids with high molecular weight [15].So that the soil that is given compost contains heavy metals into metal forms that are not available to plants.Based on this, the application of compost is able to withstand the absorption of heavy metals by plants so that it has low accumulation in plants.The presence of heavy metal ions is very dynamic in the plant nutrient transport system.The high concentration of accumulated heavy metal ions in the planting medium without the addition of organic matter indicates that compost as an organic matter added to the soil can positively prevent the movement of heavy metal ions into the plant tissue system, compared to soil without organic matter.At the same time at the presence of the compost can withstand the flow rate of metal ions in high amounts only up to the root tissue.So that the residue of heavy metals in the roots becomes high.

Fractionation of Cu in soil.
Fractionation analysis by extraction method was carried out with the aim of indirectly assessing the potential mobility and availability of Cu metal in the soil.One of the negative impacts of heavy metal pollution on plant growth is determined by its levels and physicochemical forms in the soil [16].Sequential extraction results for Cu metal in two types of soil can be seen in Figure 2.
In Figure 2 it can be seen that Cu in the form of dissolved fraction in both soil types, both control and treated soils, has very low values ranging from 1.89 ppm to 2.65 ppm.The concentration of Cu found in the form of exchangeable fractions in both soil types, both in the control and in the compost addition treatment, was relatively low.The dissolved and exchangeable forms are the forms of Cu available to plants.As stated by Dube et al. [17] that metal cations in soil are found in various physicochemical forms namely (1) simple or complex ions in soil solution, (2) easily exchangeable ions, (3) bound to organic matter, (4) occluded or co-precipitated in the crystal structure of oxide, carbonate or phosphate minerals and other secondary minerals, or (5) ions in the crystal lattice of silicate minerals and primary minerals.These forms can balance each other, are considered easily absorbed by plants and are known as active fractions which respectively show a decrease in the degree of absorption by plants [18].
The residual fraction consists of the fraction bound to the oxides, the fraction bound to the organic matter, and the residue fraction itself.In Andosol and Latosol soils, Cu in the form of the residual fraction was found in the dominant amount.The presence of exchangeable Cu in the experimental soil will be difficult to degrade into bioavailable Cu metal has a high/strong affinity for organic matter contained in the soil [19], this makes it difficult for Cu metal to be released into its free form or available to living things.The residual fraction in Andosol soils have a high organic matter content, so Cu is more commonly found in the form of fractions bound to organic matter.According to Andersson [20] soil organic matter prefers Cu compared to transition metal divalent cations.The result of Darmawan and Wada [21] showed that Cu in soils containing Al-humic would be dominated by the form of the fraction bound to the oxides and the fraction bound to the oxides and the fraction bound to organic matter.This is also in line with the addition of compost to the Andosol soil medium, the amount of metal in the form of residues is increasing.These heavy metals are strongly bound in the soil solution system to form complexes.It is believed that the presence of compost in the soil can absorb/bind heavy metal ions to form complex bonds.Metals that are strongly bound to minerals or silicates have the most stable bonds and are difficult to detach to become available.In Latosol soils which are dominated by 1:1 type clay mineral (kaolinite), Cu is more commonly found in the form of fractions bound to the oxides.This is different from the results of a study by Darmawan and Wada [21] where Cu in soils dominated by clay minerals 1:1 (kaolinite) is more commonly found in the form of exchangeable fractions.This is because the Latosol soil used as Latosol Horizon B. According to Schaefer et al. [22], Latosol is a soil that contains lots of Fe and Al oxides.Where in Latosol soil, the lower (deeper) the content of and Al oxides is higher, so the Fe and Al oxide content in the B horizon (subsoil) is higher than the A horizon (topsoil).As a result, Cu metal binds a lot with Fe and Al oxides.Hsu [23] stated that Latosol is a mineral soil rich in sesquioxides as a result of silicate leaching.Non-crystalline iron oxides and aluminium silicates have high selectivity for Cu The results of the analysis of fractionation of heavy metal forms in the soil are also consistent with the accumulation of heavy metal content in plant tissues, that heavy metals in treatment A5 and A6 have low levels of absorption in plant tissues.This is because the addition of compost to the planting medium makes heavy metals dominant in the form of residues, i.e. forms that are not available to plants.However, based on the analysis of Cu concentration in plant tissues, A. spinosus grown in Latosol soil absorbed the highest Cu, which was 52.78 ppm in the roots and 18.23 in the leaves and stems.In contrast, spinach plants grown on pure Andosol soil without the addition of compost, only absorbed 28.49 ppm Cu in the roots and 11.85 ppm in the stem and leaves.

Conclusion
Heavy metal absorption by plants was found to be higher in root tissue; and A. spinosus.was able to accumulate heavy metal Cu higher than A. viridis.Meanwhile, Latosol soil+compost (A.spinosus) was able to increase the absorption of the most accumulated Cu heavy metal content.Whereas in Andosol soils, Cu is more commonly found in the form of residues, which is a combination of fractions bound to organic matter and bound to oxides.

Table 1 .
Average absorption of heavy metals in the biomass (roots, stems, and leaves) of spinach plants for 5 WAP.