Abstract
Our environment is polluted from heavy metal using industries. It is due to anthropogenic activities. The heavy metals are in excess is toxic to animals, plants and microorganisms. The excess of copper can affect the entire plant systems. Copper is one of the major source pollutants in ocean and terrestrial. To remove the excess copper pollution by biosorption is an easiest technique by using Ulva lactuca. In this present study, Cow pea Vigna unguiculata (L) seedlings were treated with 6 mM copper chloride, water control and Ulva lactuca bio sorbent (2 g/L, 4 g/L, 6 g/L and 8 g/L), its influence on the biochemical, enzymatic and morphometric characteristics were studied. The morphometric parameters such as fresh weight, leaf area, shoot length, dry weight and root length is increased than that of 6 mM treated control. The pigments such as chlorophyll a, chlorophyll b, total chlorophyll, carotenoids are steadily increased and anthocyanin content was decreased than the control 6 mM copper chloride. Biochemical characters such as protein and soluble sugar were increased with 6 mM of copper chloride. On the opposing the contents of proline, free amino acid, total phenol and leaf nitrate were decreased in the 6 mM of copper chloride. The enzyme activities such as peroxidase, catalase are decreased and the nitrate reductase is increased. Atomic Absorption Spectroscopy (AAS) was employed to conclude the copper in the treated and control plants and 6 mM Cu2+ reveals that vigorously affected the Vigna unguiculata (L) (cow pea) plants and The green algae Ulva lactuca is effectively biosorbed the copper heavy metal and get rid from soil pollution and improve crop yield.
Keywords
Cow pea; Copper toxicity; Bio sorption; Atomic absorption spectroscopy.
Introduction
Heavy metal pollution is common in India. Mining and agriculture areas are the documented cases of metal toxicity. Heavy metal toxicity is the primary concern and bioavailability of the heavy metals plays many roles in the soil. Organic matter, pH, clays, redox conditions and hydrous oxides and total metal concentrations these factors affect the bioavailability. Heavy metal toxic due to human influences such as metal releasing industries [1]. Copper ion as an essential micronutrient and is important in maintaining cellular homeostasis [2]. However, Excess of copper is highly toxic to growth of plants, damage the plant internal structure and completely inhibit the plant growth.
The heavy metal copper at high concentrations to the plants show the blue to brown or yellow in colour and growth is stunted. Excess copper levels decrease plant vigour, iron intake and seed germination. Various problems arise during the neutralizing copper in soil. The copper toxicity is due to continuous use of fungicides. The persistence of copper in soil is many years and has solubility is low.
Copper is not regularly present at higher concentration in soil, but when it’s present at above the permissible concentration then it effects the environment [3]. Copper toxicity is a problem of both agricultural and environmental significance. Sources of copper contamination by agrochemicals, agricultural wastes, urban, smelting, mining and industries. The indicator of bioavailability of copper is Cu2+ [4]. Macro algae are among the most complex and reliable organisms in studies of heavy metal pollution due to their rapid rate of metal accumulation from aqueous solutions [5].
Biological monitoring of marine water primarily focuses on the red algae [6], brown algae [7] and most of the studies says that Ulva lactuca accumulate high heavy metals and only concentrations of these elements in the organisms, and not of the sample site (water, sediments) have been characterized.
Various organisms are used to analyse the heavy metal concentrations in natural environment on the extent of contamination and shall reveal possible applications of using Ulva lactuca as bioindicator [8]. Most studies reported the Ulva lactuca do accumulate and bio indicate the heavy metal ions. Most of the Ulva lactuca algae are grown in marine and brackish water [9].
Materials and Methods
The Experimental and control plants seeds were allowed to grow in 1:1:1 ratio of uniformly mixed red, black and sandy soil and allowed to grow in the seeds. After twelve days, the seedling of (cow pea) Vigna unguiculata (L) were treated with various concentrations of copper chloride with Ulva lactuca was collected from uvari coastal area Tamil Nadu, shade dried and finally powdered by milling (Set I).
The optimum concentration of 6 mM of copper level based on LSD analysis [10] was mixed with various concentrations (2 g/L, 4 g/L, 6 g/L and 8 g/L) of Ulva lactuca seaweed dry powder, constantly shaken in shaker for 12 h, filtered using filter paper and the plants of another set was treated with the filtrate (Set II).
Twelve days later plants of Set I and Set II, the growth parameters such as root length and shoot length are measured by scale and represented in cm2. The morphology of leaf area was measured by conventional graphical method by drawing the outline of the leaf in a graph sheet and counting the small and big squares with in it and expressed in cm2. The dry weight and fresh weight of the seedlings were obtained using an electronic weighing balance.
To quantify the chlorophyll from leaves, fresh leaves were deveined and cut into small bits, from the pool leaf bits, a sample of 100-200 mg was weighed. The leaf bits were homogenized in 100% acetone liquid using mortar and pestle. The homogenate was centrifuged at 4000 rpm (revolution per minute) for 5 min at room temperature (27ºC). Extraction with 100% acetone liquid was repeated till the pellet becomes white/pale yellow in colour. The supernatant was used for the estimation of photosynthetic pigments.
In spectrophotometric measurements the blanks were used for the subtraction of all the readings. The absorbance was measured at 645 nm, 470 nm and 662 nm, for chlorophyll a, chlorophyll b and carotenoids respectively using a lab systronics spectrophotometer model no: 106. The amount of chlorophyll b, total chlorophyll and chlorophyll a was calculated using the formulae of Welburn and Lichtenthaler [11].
Chlorophyll a (mg/L)=11.75 × A662-2.35 × A645
Chlorophyll b (mg/L)=18.61 × A645-3.96 × A662
Total chlorophyll=chlorophyll a+chlorophyll b
The carotenoid content was calculated using the following formula:
Xanthophyll+Carotenes=1000 × A470-2.27 Ca-8.14 Cb/227
Ca=Total chlorophyll a
Cb=Total chlorophyll b
The anthocyanin content was estimated by inoculating 200 mg of leaf samples inoculating in 200 ml of extraction medium that consists of methanol, diluted water and HCl acid in the ratio of 50:20:1, respectively. The incubation was extended to 48 h at 4ºC in dark condition with agitation. After 48 h, the solution was collected and the optical density is measured at 630 nm and 657nm. The absorbance value of anthocyanin content was estimated by Mancinelli et al. method [12].
Absorbance of anthocyanin=A530–0.3 × A657
The total soluble sugar or glucose was measured by anthrone solvent method likewise the total amino acid content was measured by ninhydrin indicator method [13]. The protein content was measured, alkaline copper mixture with folin phenol [14]. The proline was estimated using glacial acetic acid and sulphosalicylic acid [15]. The biochemicals were estimated using standard value. The in vivo nitrate reductase (NR) enzyme activity was assayed according to Jaworski et al. [16] method with modification. 100 mg fresh leaf material chopped and it was incubated in scintillation vials containing 5 mL of incubation medium composed of 200 mM KNO3, 100 mM KH2PO4- KOH buffer pH 7.5, 1% (v/v) n-propanol and 0.1% Triton X 100(v/v). Incubation was carried out in dark for 1 h at room temperature (27ºC) with occasional shaking. Aliquots of 0.5 ml was taken from the vials and analysed for nitrite after 1 h incubation. To 0.5 ml distilled water (H2O), 1 of 3% sulphanilamide and one ml of N-1-N (Naphthyl ethylene-diamine dihydrochloride) were added in quick succession. 15 min were allowed for colour formation and absorbance was measured at 540 nm. The nitrite was estimated with the help of a standard nitrite curve. The activities of peroxidase and catalase enzyme extracts of the leaves of experimental plants were extracted by grinding per gram of leaf in five mL of 100 mM phosphate buffer (pH 6.0) and filtered through a three layered cheese cloth and run at 3000 rpm for 30 min. The supernatant obtained was served as the source for crude enzymes such as peroxidise and catalase. To assay peroxidise enzyme activity, the enzyme extract was added to pyrogallol which gets oxidized to a coloured derivative in the presence of hydrogen peroxide (H2O2) (1% v/v).
The amount of pupurogallin formed during the reaction was assayed spectroscopically [17]. To 2 mL of pyrogallol phosphate buffer (0.058 M pyrogallol dissolved in 0.1 M phosphate buffer pH 6.0), an aliqot of 0.1 ml of enzyme extract was added. Then, absorbance was set to zero at 420 nm. To this, 0.5 mL of hydrogen peroxide (H2O2) (1% v/v) was added. Then, the content was thoroughly mixed and the absorbance was measured using systronics model no. 106 spectrophotometer. The difference in the absorbance at an interval of 20 s for a period of 3 min was measured. The peroxidise activity was expressed as moles of hydrogen peroxide (H2O2) reduced per unit enzyme per unit time.
To assay the catalase enzyme activity, three ml of phosphate buffer was added to one mL of H2O2 and one ml of enzyme extract [18]. The reaction mixture was incubated at 25ºC for 1 min. The reaction was terminated by the addition of 1 ml of sulphuric acid (H2SO4). The end point was the persistence of pink colour at least for 15 s. The catalase activity was expressed in micromoles hydrogen peroxide (H2O2) catalysed per unit time per mg protein. The heavy metal accumulation of the experimental plants was analysed at the end of the plant life. Heavy metal copper concentrations in plants were analysed using Baker et al. method [19,20].
The plant sample as a whole was washed, dried in oven at 160ºC for 40 min and digested in a mixture of (HNO3) and perchloric acid (10:1). Then the solution was centrifuged at 5000 rpm (revolution per minute) for 5 min and double filtered with What mann filter paper no: 4 and the filtrate was analysed for copper concentration by Atomic Absorption Spectrometry (Shimadzu Model AA–6300), available in the Science Instrumentation Centre of Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi, Tamil Nadu.
Statistical Analysis
For the growth parameters the average value of ten independent samples and for the enzymatic readings, pigmental parameters and biochemical parameters the average value of five were considered. The data were reported as mean ± SE (standard error) and the percent activity was represented in the parentheses. Statistical analysis - One way ANOVA – Turkey test was done using the statistical package, Origin – version 7.0.