INTRODUCTION:

Heavy metals present in the soil causes soil pollution and disturb the environmental cycle. When present in excess amount, heavy metals possess severe impact on the fertility of soil and thus affect the crop productivity and yield (Yang et al., 2005). Heavy metals are present in the soil in the form of free radicals or exchangeable ions or complexes^2,3. Metals, which are present in the form of ions, show movement along the upper part of the plant⁴. Phytoremediation technique is used to overcome these problems.

Many weed plants are used for phytoremediation process as these are able to remove all the contamination from the soil. Chenopodium species are widely also used for this purpose. However C. album shows more collection of toxic metals such as chromium, mercury, lead and cadmium. Higher content of chemicals were absorbed in the leaves followed by stem and roots.

The plant (Chenopodium album) which is used as a phytoremediator should have two main features. Firstly, it should be an accumulator of high amounts of metal and for this it should produce large biomass. Secondly, the plant which is a metal accumulator should be responsive to the agricultural processes. Thus, the metal accumulation in shoots is more preferred as compared to roots because shoots can be cut out easily⁵.

Plants that are willing to decontaminate the soil heavy metal pollution should also possess one or more of the following properties;

(i) The roots of the plants should absorb the pollutants from soil either in the solid form or in the liquid form,

(ii) The contaminating material should bind with the root tissue by physical means or by chemical means

(iii) Plants should translocate the absorbed metals from their root region to the shoot part so that their presence in root will not harm the soil.

The process in which plants, plant parts or plant extract is used to remove the heavy metal contamination from soil is called phytoremediation. This plant based process used for the removal of metals proves to be an emerging concept in which metals are accumulated and translocated in the plant cells⁶ (Long et al., 2002). The other aspect which influences the phytoremediation process is the mobility of metal in soil and plant. Thus the metal should be mobile. Lead present in soluble form can be easily accumulated by the plant and leads to remove it from the soil.

Phytoremediation technique might be a time consuming process because the process is directly proportional to the growth rate however ability of plant to decontaminate soil is related with the biomass produced by plant. It is generally a technique in which remediation of heavy metals is done by using plants or wild weeds⁷. It is composed of five main processes namely:

(i) Rhizofiltration:

In this process, the part of plant used to detoxify soil is the roots. In this process the root and the microorganisms present within and around the root region are used. The highly toxic metals such as Ar, Zn, Pb, Cr, Cu, Ni, and Hg can be removed by using this process⁸. Plants such as Sunflower, rye, spinach, corn, mustard were studied as they are potent source to remove lead from the soil,

(ii) Phytostabilization:

This is another technique of phytoremediation in which mobility of toxic metals in the soil is reduced. As a result, it limits the bio magnification which takes place due to leaching and soil erosion. Phytostabilization takes place through various processes such as sorption, precipitation, complexation or metal valence reduction. Plants having dense root system are more effective for this process. When roots are dense and strong they can stabilize and immobilize large amount of heavy metals⁷,

(iii) Phytoextraction:

It is the best method which is used to clean contaminants majorly from the soil. Soil structure and its fertility are not affected by this process. It is also known as phytoaccumulation,

(iv) Phytovolatilisation:

In this technique, the contaminants present in the soil are absorbed by the plants and are transformed into a volatile form and finally the volatile form is transpired into the atmosphere. The plant must take up water and other organic and inorganic contaminants which in turn lead to Phytovolatilisation. The process is majorly used to decrease the concentration of mercury present in the soil. The mercuric ions are converted into lesser toxic form that is the elemental form of mercury⁹

(v) Phytodegradation:

The process which involves the reduction of contaminants by transformation, breakdown, stabilization and evaporation of toxic substances from the soil and the polluted water present in the soil, it is called phytodegradation. It is generally the formation of simpler molecules from complex and organic taken up by the plants¹⁰.

There are number of plants or weeds can be utilized for phytoremediation, here in the research work we utilized Chenopodium album. It is an annual herb which belongs to the family chenopodiaceae. It is also known as pigweed in English and bathua in Hindi. It is considered as a wild weed which grows everywhere. It is a plant which occurs throughout the world. Phytochemical analysis revealed the presence of alkaloid compounds^11,12, Apo carotenoids¹³, flavonoids, phytoecdysteroids¹⁴ and an unusual xyloside in the plants. The main antioxidants present in Chenopodium album are ascorbic acid and glutathione. C.album is a weed plant which faces a wide array of temperature during its developmental stages (up to 5 – 45°C) in north India. The most heat stable enzyme present in C.album is the Superoxide Dismutase (SOD) as the weed itself is heat adaptive. It was analysed that Chenopodium album shows phytostabilization of lead and little translocation of Pb take place from roots to shoots. It was analyzed that some Chenopodium species for their antioxidative and cytotoxic activity. Four species of the plant were evaluated which were C. album, C. hybridum, C. rubrum and C.urbicum. Herb extract of C.album and seeds of C. urbicum contains maximum amount of polyphenol compounds. Extracts of C. rubrum and C. urbicum has the best antioxidant effect.

The current study aims to study the remediation of metals (Cr, Hg) from soil utilizing Chenopodium album as the phytoremediator plant. The antioxidant properties of the plants were analyzed during metal stress conditions and the biochemical parameters of the Chenopodium album were studied during metal stress condition.

MATERIAL AND METHODS:

POT EXPERIMENTATION:

Experiments were carried out to analyze the stress of heavy metals (Cr, Hg) and phytoremediation capability of the plant Chenopodium album. Seeds of similar size were taken and were sterilized with sodium hypochlorite and then washed with double distilled water. Pots of similar size were taken. Soil was taken at a depth of 30cm from the surface of the nearby field and was dried and homogenized. Seeds were sown in the pots followed by watering. Pots were grown in replicates of four. After the emergence of plant seedlings (15 days old), different concentrations of mercury and chromium were given. It was followed by further exposure of 2 times with a time gap of 15 days. Both the metals were given in the concentrations of 50mg/kg, 100mg/kg and 150mg/kg of soil respectively. The metal salts were provided to the plant while watering in aqueous form. The chromium treated plants with these concentrations show rapid absorption of metal which results in complete damage of the plant. Therefore the chromium stress was given in 10mg/kg, 15mg/kg and 20mg/kg to the new plant seedlings and various parameters were studied.

Biochemical tests:

Chlorophyll content of Chenopodium album:

Chlorophyll is the main pigment present in plants which imparts green colour to the plants. The total amount of chlorophyll present in Chenopodium album is measured by using Arnon’s method (1949)¹⁵. Chlorophyll a was detected at 660nm, chlorophyll b at 645 and the carotenoids at 470nm.

Formula to calculate chlorophyll content:

To calculate the total amount of chlorophyll a and chlorophyll b and total chlorophyll content in the plant, Arnon’s method was used in number of protocols¹⁵. The formula for calculating the total chlorophyll content was estimated by the formula given below¹⁶:

ml acetone

Chl a(mg kgˉ)=[(12.7 ×A663)-(2.69 ×A645)]× ––––––––––––––––

mg leaf tissue

ml acetone

Chl b(mg kgˉ)=[(22.9 ×A645)-(4.68 ×A663)]× ––––––––––––––––

mg leaf tissue

Total chlorophyll = chl a +chl b

Carotenoid content of Chenopodium album:

Total carotenoids were measured by the method of Cyanotech Corporation (2001) with slight modifications¹⁷. 0.5g of sample was macerated with 25ml of acetone containing dimethyl sulphoxide. Homogenate was filtered through Whatman filter paper no. 1. The solution was washed with same amount of acetone and DMSO. Absorbance was taken at 471nm. Total carotenoids content was calculated by the formula¹⁸.

abs max. sample vol.

mg of carotenoids per ml of sample = –––––––––––––– × –––––––––

250 ×100ml acetone Df

Nitrate activity test:

The activity of nitrate reductase enzyme was measured by using the method¹⁹ for nitrate determination. After analysis, pink color was observed and absorbance was taken at 540nm, followed by the formula:

Formula to calculate Nitrate Reductase Activity:

µmol NO₂ˉgˉ FW at 10 minutes = Abs.at 540 ×0.081 ×25.8 × 1/0.2

µmol NO₂ˉ FW at 40 minutes = Abs.at 540 ×0.081 ×25.8 ×1/0.2

Total NR activity =

2[Nitrate at 40 min.-Nitrate at 10 min.]µmol NO₂ˉgˉ FW

Protein estimation:

The total amounts of proteins present in Chenopodium album were determined by the method²⁰of protein estimation. BSA stock (1mg in 100ml distilled water) was prepared as a standard and comparison with the standard was carried out. Analytical reagent A, B, C and Reagent D i.e. Folin’s reagent was taken in equal concentration (1:1v/v). For the determination of proteins, equal amount of plant extract was taken in different test tubes and then the reagents were added. Standard curve was prepared to calculate total protein content.

Determination of antioxidants activity:

Estimation of Malondialdehyde:

Malondialdehyde is estimated by the method proposed by Heath and Packer (1968)²¹. 1gm of leaves were taken, and crushed in 5ml of 0.1% of TCA (Trichloroacetic acid). The mixture formed was centrifuged at 5000rpm for 20 minutes. To the supernatant present in the test tube, 6ml of 20% TCA containing 0.5% of TBA (2-Thiobarbituric acid) was added. Test tubes were heated at 95°C for 30 minutes. Absorbance was taken at 532nm.

Formula to calculate total MDA content:

Absorbance ×1000

µmol MDA gˉ FW = –––––––––––––––––––––

155

Proline estimation:

Proline content was determined by using the method²². A standard of proline was also prepared. 0.1gm of proline was dissolved in 100ml of distilled water. Different concentrations of proline were taken. The standard curve was plotted and then compared with the sample.

Formula to calculate total proline content:

(µgmlˉproline ×ml toulene) 5

µmoles per gˉ tissue FW = –––––––––––––––––––––– × –––––

115.5 µgµmolˉ 0.5

SOD activity:

The activity of SOD enzyme was calculated by the method²³. The test was done in both light and dark conditions. Light reaction test tubes were utilized and the dark reactions test tubes were covered with foil and kept in dark conditions. After 10 minutes the absorbance was taken at 560nm.

Formula to calculate SOD enzyme activity:

blank OD-Treatment OD

total SOD content=––––––––––––––––––––––– × 100

Blank OD

RESULTS AND DISCUSSION:

POT EXPERIMENTATION:

Figure 1: Pot experimentation of Chenopodium album before and after treatment of metals in different concentration of mercury and chromium metals

Figure 2: Representation of changes in plants morphology, when treatments of 50mg/kg Hg, 100mg/kg Hg, 150mg/kg Hg, 10mg/kg Cr, 15mg/kg Cr, and 20mgkg Cr was given and the control was given with DW.

Chlorophyll content test:

When Chenopodium album, was treated with mercury and chromium stress than there occurs various changes in the photosynthetic pigments. The chlorophyll a and b of Chenopodium album decreases with increase in metal concentration. The control have higher amount of chlorophyll content and as the stress was given the chlorophyll content decreases. Mercury stress significantly decreases the chlorophyll content but maximum decrease was seen in chromium treated plants. Carotenoids also decreases with the increase in metal stress. The carotenoids were also present in higher amount in the control and it decreases with the increase in metal stress.

Figure 3: Graph showing the total chlorophyll and carotenoids content in C. album, when mercury and chromium were given.

The chlorophyll and carotenoids were present in plants which imparts colour to the plant. Chlorophyll a and b provides green colour to the plant while carotenoids are responsible for the red and yellow colour. This experiment shows a gradual decrease in the chlorophyll and carotenoids pigments. The decrease may be due to the heavy metals stress which stops the activity of various enzymes. The activity of certain enzymes which are responsible for the production of photosynthetic pigments may be blocked. Major reasons responsible for the decrease in photosynthetic pigments are: The activity of enzyme protochlorophyll reductase is affected due to heavy metal stress which was responsible for the production of chlorophyll²⁴. It also occurs due to inhibition of δ- aminolevulinic acid dehyratase (ALA- dehydratase).

Nitrate Reductase activity test:

The enzyme responsible for the assimilation of nitrate from soil into the plants is nitrate reductase. The NR activity was highly affected when treatment of heavy metals were given to the plant C. album. The NR activity gradually decreases with the increase in heavy metal stress of mercury and chromium. In case of chromium the nitrate activity was highly reduced due to stressed conditions as compared to mercury. NR activity was stopped by the heavy metal stress²⁵.

Figure 4: Graph showing decrease in Nitrate Reductase activity due to metal stress of mercury and chromium

The amount of nitrate enzyme activity may be decreased due to many reasons. It may be reduced because of decrease in photosynthetic pigments that is decrease in chlorophyll content. As nitrate enzyme uses NADH for the assimilation of nitrate to nitrite and that NADH is produced by chlorophyll pigments. Nitrate enzyme also requires energy produced from photosynthesis. Thus, due to decreased chlorophyll content, the nitrate enzyme activity is decreased.

Protein estimation test:

The total amount of proteins present in C. album shows a significant decrease with increase in the concentration of the metal stress. Proteins were present in higher concentration in the control plant and then decreases as the mercury and chromium stress was given. The least amount of proteins was present in the plant treated with 20mg of chromium.

Figure 5: Decrease in protein content in C. album due to metal stress.

The decrease in protein content may be related with the decrease in the NR activity or due to toxic metal stress. Some of the important enzymes responsible for the formation of proteins get degraded. Proteases may get degraded and thus become unable to produce proteins. Thus the experiment shows the decreases in the protein content with the increase in heavy metal stress in C. album.

Antioxidant enzyme activity:

Malondialdehyde (MDA) test:

MDA is the enzyme which is produced as a result of lipid peroxidation. The total MDA content of C. album increases with the treatment of different concentrations of mercury and chromium. Least amount of MDA was present in the control and the content increases with the increase in metal stress. Thus the MDA contents increases with increase in the metal stress and present in higher amount in the plant treated with maximum concentration of chromium. Many researchers show similar result of increasing MDA when metal stress was given. The graph depicted below shows the formation of MDA which increases with increase in metal stress concentration.

Figure 6: Increase in MDA content of C. album after giving treatment of Cr and Hg.

Proline test:

Due to mercury and chromium stress, the proline content increases rapidly. The proline content increases in plant on treatment with heavy metal stress. Many other experiments done by different researchers also depicted the similar results. Proline was considered an osmeoregulator and plays vital role in osmoregulation²⁶. It is generally produced when the plant is treated with some stressed conditions which may be environmental or heavy metal stress. During stressed conditions, proline is formed in large amounts to reduce the stress levels and to protect the plant from damage. The proline content in this study has been increased to protect the plant from stressed conditions. Also proline acts as a source of nitrogen and carbon and it also behaves as a scavenger of free radicals and any harmful substances from plant.

Figure 7: The graph showed an increase in total proline content when mercury and chromium metal stress was given.

SOD enzyme activity:

SOD is the enzyme which is produced during heavy metal stress to protect the plant from damage. The total SOD enzyme activity increases in C. album with the increase in the heavy metals concentration. The control plant shows the least amount of SOD production however it is produced in larger amounts in the plants which are treated with mercury and chromium.

Superoxide dismutases are the enzymes which are responsible for the dismutation of radicals produced by superoxide into hydrogen peroxide and oxygen.

Figure 8: Showing the increased rate of SOD enzyme in C. album when metals were given at different concentration

Reactive oxygen species (ROS) produced during metal stress leads to cause disruption of some important organelles in the plant cells²⁷. To reduce the activity of ROS produced during abiotic or biotic stress the SOD enzymes are produced in higher concentration. There are two types of SOD enzymes one resent in the mitochondria and peroxisomes called manganese SOD and the other present in the cytosol, peroxisomes and chloroplast called Zn/Cu SOD. Thus, from this experiment it was concluded that the total amount of SOD enzymes increases the plant is subjected to heavy metal stress.

CONCLUSION:

Phytoremediation is the technique which is used to detoxify all the heavy metals from the soil or water by using plant extracts or the plant parts. It is the most simple and cost effective method for remediation of heavy metals. Chenopodium album shows high potential for removal of toxicity from soil. Different metal concentrations were given by performing pot experimentation. It was analyzed that chlorophyll and carotenoid content decreased under stress conditions as compared to control plant, whereas shortening of shoot length and falling and yellowing of leaves occurs in metal stressed plants. Nitrate reductase enzyme activity decreases with increase in exposure to heavy metals. Plants will stimulate the formation of reactive oxygen species (ROS) under abiotic stress, which can harm the production of biomolecules such as lipids, proteins and nucleic acids. Usually, membrane lipid peroxidation in plants is detected by measuring malondialdehyde (MDA). It was observed that malondialdehyde content increases with increase in metal concentration. The proline test showed higher concentration, as it is required to protect the plant from stress conditions and acts as an osmeoregulator. Similar results are observed in SOD enzyme activity which increases in metal stressed plants as compared to the control. Overall studies showed different parameters to prove that phytoremediation by wild weed is an effective technique for the up-taking of heavy metals and to make the soil and water free from contaminants.

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Received on 26.04.2019 Modified on 30.05.2019

Research J. Pharm. and Tech. 2019; 12(10):4851-4856.

DOI: 10.5958/0974-360X.2019.00840.0