Phytoremediation-Pot Studies

Transcription

1 4.1 Introduction The development of industrialization anthropogenic activities led to an increased accumulation of heavy metals, which results in contamination of soil [1]. Generally heavy metals are non degradable, therefore can sustain for longer time in environmental segments. Due to this bioaccumulative nature heavy metals may alter the food chain and cause various toxic effects on plants, humans and animals. In environment the common hazardous heavy metals are Cd, Pb, Sn, Zn, Cr and Cu [2]. Cr normally exists in two oxidation states in soils; Cr (VI) is highly toxic to plants and animals in high doses and is responsible for inducing cancer and teratism, resulting liver and kidney damage. From the environment cadmium can be easily uptake by organisms [3]. Cadmium and chromium were highly transportable in soil-plant system than Cr and other heavy metals [4]. Therefore remediation of lands polluted with toxic heavy metals is very essential for environmental conservation. Remediation could be done by many different technologies however their procedure is laborious and costly [5]. Attention was given to phytoremediation by which plant is applied to absorb, transform and detoxify heavy metals. The phytoremediation method was simple, efficient, cost effective and environmental friendly [6]. The concept of using plants to clean up contaminated environment is not new. About 300 years ago plants were proposed for use in the treatment of waste waters. Some plant species are endemic to metallic-ferrous soil and can tolerate greater than the used amount of heavy metal or other compounds [7]. Plants that accumulate metals to high concentrations are sometimes referred to as hyper accumulators [8]. Hence, for the progress of phytoremediation tracing out of new hyper accumulators which can overcome limitations like slow growth rate, less resistance and poor competitiveness with native plants and survive in unfavorable conditions is required [9]. There are many phytoremediation technologies like phytoextraction, phytofiltration, phytostabilization, phytovolatilization and phytodegradation are 99

2 adopted [10]. Phytoextraction is found to be best method for the accumulation and tanslocation of heavy metals from root to shoot and remediate soil without damaging the soil composition and fertility [11]. Phytoextraction could benefit in many ways like restoring soil fertility and minimizing leaching. Most of the research on phytoremediation was based on pot experiments and hydroponic culture, and very few studies evaluated the phytoextraction potential of hyper accumulators under field trials [12]. This research was designed with the objectives to analyse the Cadmium and Chromium accumulation and translocation from the contaminated soils. The aim of this research was (i) to analyse the growth performance of plant, (ii) to evaluate the potential of selected plant species for the accumulation of heavy metal in different concentrations of metal amended soils along with the physico chemical parameters of soils (iv) to study the accumulation of cadmium and chromium in roots, shoots of the plant (V) to compare the bioaccumulation and translocation factor of cadmium and chromium in selected plant species. 4.2 Materials and Methods The present study was designed to investigate the phytoextraction of three selected heavy metals cadmium, chromium and lead from polluted soil by Mirabilis jalapa and Datura inoxia.the criteria used for selecting plants for phytoremediation were high metal tolerance, high bioaccumulation factor, short life cycle, high propagation rate, wide distribution and large shoot biomass. (a) (b) Figure 4.1. Schematic diagram of Datura inoxia(a) and Mirabilis jalapa(b) 100

3 4.2.1 Collection and characterization of soil The soil samples were collected form uncontaminated sites and air dried, mixed and sieved (2 mm) prior to determine the physicochemical characteristics. The physical parameters of the soils analysed are ph, EC, moisture and soil texture are mentioned as follow pH The ph of soil was determined by the method of Sims and Heckendorn (1991) in 1:2 soil-water and sediment- water slurry using a digital ph meter (Systronics). From air dried soil, 25g soil was taken in a beaker, added 50ml of double distilled water and stirred until the homogenous slurry was achieved. It was allowed to settle for 15 minutes, and ph of the supernatant liquid was measured using a digital ph meter Electrical Conductivity (EC) The EC of the soil (ms/sm) was determined in 1:2 soil water (Jackson, 1973) using a direct reading digital conductivity meter (Elico CM180), with a cell constant of Moisture From fresh soil sample, 10gm of soil was weighed and placed in an aircirculation oven at C to dry to a constant weight. It was cooled in desiccators and weighed to calculate the percentage fresh moisture from loss in weight. ( ) ( ) Lossin wt.on drying gm Moisture (%) = 100 Initialsample wt. gm Soil Texture Soil texture was determined using Mechanical Analysis of USDA System (United States Agricultural Department). It expressed the proportions of the various sixes of particles present in soil. The fractionation system is described here, which is a widely used International scale. 101

4 The range of particle size in this scale is as follows. Grade Particle Diameter (mm) Coarse sand Fine sand Silt Clay <0.002 One hundred grams of oven dried soil was taken, and sieved with the sieves of pore sizes 2.0 mm, 0.2 mm, 0.02 mm and the soil left in each sieve were weighed as W1, W2, and W3 respectively, W1 represented % of gravel, W2 represents % Course sand, whereas W3 represented the % fine sand. The sieved down soil represented % of the silt and clay and clay content was determined. The chemical parameters of the soils analysed are organic matter, N, P, K, and metals are mentioned briefly as follow Organic carbon This was determined by Walkley Black method and expressed as percentage. From air dried and sieved soil, 1.5 g of soil was taken in 250 ml Erlenmeyer flask. 25 ml of 0.4N K 2 Cr 2 O 7 and 20 ml of concentrated H 2 SO 4 was added to the flask and swirled gently to mix the reagent with the soil. The contents of the flask were allowed to stay for 30 minutes after which 100ml of DDW and 5 drops of Ferroin Indicator were added to it. The solution was titrated against 0.4 N FeSO4.7H2O to get the colour change from greenish brown to dark green and from blue (turquoise) to red (maroon). A blank was also run in the same manner. Soil organic carbon is calculated as % organic carbon = (B-T)XF Where B = ml of FeSO4 for blank titration T = ml of FeSO4 for blank titration F = (3/7XN/W) N = Normality of FeSO4.7H2O W = Weight of sample (g) 102

5 Soil organic matter Percentage of organic matter was obtained by multiplying the percentage organic carbon with % organic matter = % organic carbon X The factor is the ration of organic matter to organic carbon based on assumption that soil organic matter contains 58% carbon Available nitrogen (nitrate nitrogen) Available nitrogen in soil was determined as nitrate nitrogen. 5gms of airdried sieved soil was taken in a polythene container. To this 50 ml of DDW was added and the mixture was stirred for 10 minutes and then filtered. Nitrate nitrogen was determined in the filtrate using phenol disulphonic acid method by colour measurement using UV Visible spectro photometer. A suitable aliquot of the soil extract was taken into an evaporating basin and brought to dryness on a steam bath. After cooling the residue, 2ml of phenol disulphonic acid was added to dissolve the residue. To this 20 ml of DDW and 18 ml of ammonium hydroxide were added and the volume was made up to 50 ml and optical density was measured at 410 nm against a blank solution (distilled water). Nitrated nitrogen was determined from the calibration curve using following formula: ( ) ( ) ( ) mg C extractant volume ml Nitrate nitrogen = g aliquot ml sample wt g Where C = mg of Nitrate nitrogen obtained from the graph ( ) ( ) ( ) mg C extractant volume ml Nitrate nitrogen = g 10 aliquot ml sample wt g Where C = mg of Nitrate nitrogen obtained from the graph. 103

6 Available Phosphorus This was determined by molybdenum blue method. Five grams of air-dried sieved soil sample was extracted with 50 ml of 2.5% acetic acid and extract was filtered and the filtrate was used for the determination of available phosphorus. Suitable aliquots of soil sample extract were taken in 50ml volumetric flasks. To this 2ml of ammonium molybdate and 5 drops of stannous chloride solutions were added. The optical density of blue colour was measured at 690 nm against a blank using UV-visible spectrophotometer (Systronic 108). Readings on the spectrophotometer were recorded in between minutes after the addition of the last reagent. The concentration of phosphate was computed from a standard curve, using the following formula; ( ) ( ) ( ) C extractant volume ml Phosporus (%) = aliquot ml sample wt g Where c = Mg of phosphate obtained from the graph Phosphorus (mg/100gm) = Phosphorus (%) X Potassium available Potassium content in the soil was determined. 25gm of air dried, sieved soil was taken in 500ml of Erlenmeyer flask. To this 125 ml of 1 M ammonium acetate (NH4AC) solution (ph 7.0) was added with shaking to completely wash the soil and the content was allowed to stand overnight. The extract was filtered through a Whatman filter paper no.42 through Bucher funnel under light suction using multiple washings. The leachate was transferred to 250ml volumetric flask, brought to volume with 1 M ammonium acetate and homogenized Estimation of plant available elemental fractions in the soils Plant available metal and other nutrients were estimated by the standard methods. From the sieved soil, 5 g sample was weighed and transferred into Erlenmeyer flask followed by the addition of 50 ml of 2.5% acetic acid to each 104

7 flask. Samples were stirred and allowed to stand for 24 hours, filtered through Whatman No. 41 and stored in polythene containers. Plant available metals were analyzed by Atomic Absorption Spectrophotometer and the metal concentration (M) was quantified using the following formula. Metal (Mg/g) = C X Ss/Sw Where C = Concentration in mg/g given by AAS Ss = Sample solution or final soil sample. Sw = Weight of the soil sample taken Total Cd, Cr and Pb metals Total metal concentration in the soil samples was determined by Mixed Acid Digestion Procedure. Soil samples were dried at 500C and sieved through <2 mm size using a nylon sieve. The sieve samples were round using a mortar and pestle and 0.5g was accurately weighed and transferred into Kjeldahl flask and digested slowly on moderate heat and increasing later. The digestion was continued for minutes after the appearance of white fumes, cooled diluted with DDW and filtered using Whatman filter paper N0.44 and made up to 50 ml with DDW. A blank was prepared in similar manner. Later metal in the solution have been quantified by Atomic Absorption Spectrophotometer. Total metal concentration in soil was determined by the following computation. Metal (mg/g) = C X Ss/Sw Where C = Concentration in mg/g given by AAS Ss = Sample solution or final soil solution. Sw = Weight of the soil sample taken. 105

8 Exchangeable and organically bound trace metals were found by some workers to represent plant available forms Preparation of cadmium chloride, potassium dichromate and lead nitrate solution The aqueous stock solutions of cadmium, chromium and lead were prepared by dissolving 1.3 g of Lead Nitrate from Fisher scientific (Ls6200) was dissolved in deionised water to give 1000 ml of the solution. The mixture was stirred by the stirring bar until all the lead was completely dissolved. The stock solutions were used to contaminate the soil to attain the desired concentrations of heavy metals, i.e., 0, 25, 50, 75 and 100 mg/kg Sample collection The seeds of Mirabilis jalapa and Datura inoxia were collected form uncontaminated sites and sterilized for 30 minutes, washed with tap water and then sowed directly in to prepared soils 5 seedlings each plant species with similar size selected for phytoextraction experiment Pot study experiment A green house study was carried out in an artificially amended sandy soil with cadmium, chromium and lead using different concentrations (0, 25, 50, 75 and 100ppm). Pot culture experiments were conducted using garden soil. Air dried soil of 5kg/each pot sieved through a 4 mm sieve and then placed into each pot after mixing with CdCl 2.2.5H 2 O and K 2 Cr 2 O 7. The known concentrations of salts were mixed with air dried soil filled pots and left for stabilization for 2 days. Three replicates of each treatment were considered. Plants were grown under natural light in order to keep all plants under similar conditions. Artificial fertilizers or amendments were not added to enhance growth or metal uptake during the course of experiment Plant growth & Physiological study For growth studies individual plants were grown under similar conditions and 9 plants for each concentration were harvested a time interval of 15, 30 and

9 days without damaging the roots. Plant height (cm) was measured from root leaves to the top the plant from each pot. The collected plant samples were separated into roots and shoots (stem and leaves). These are air dried first for two days, and then dried in hot air oven at 65 for 72 hours. Shoot and root length (cm) and dry biomass (g) of the plant parts (shoot and root) were taken for each treatment and interval. Leaf pigments chlorophyll a, chlorophyll b and carotenoids of the leaves in the test species were measured on all the harvest days. 0.5 gm of fresh material was weighed accurately ground to a fine pulp using a mortar and pestle with the addition of 80% acetone, centrifuged and the clear supernatant was transferred to a 50ml of volumetric flask. This process was repeated until the residue turns to colorless and the entire solution was made up to the mark with 80% acetone to read the values in UV-VS Spectrophotometer [13] Metal analysis 0.5gm of ground plant materials was accurately weighed and digested for the determination of metals using Kjeldahl flask and digested slowly using mixed acid digestion procedure [13] with nitric acid (HNO 3 ) and perchloric acid (HClO 4 ) mixture (2:1) in a a closed system. The digest was heated on a hot plate until it became clear and then it was allowed to cool and filtered through a whattman filter paper. The filtrate was collected in a 50ml volumetric flask and diluted up to the mark with distilled water. Then the filtrate was used for the analysis of metals (Cd, Cr and Pb) in sample solutions were quantified using Atomic Absorption Spectrophotometer [13]. As mentioned previously, each experiment was run in triplicate. Results were shown as mean standard error. Total Cd, Cr and Pb content of soil was determined by mixed acid digestion procedure and the available metal i.e., Acetic acid (2.5% m 1:1) extractable metal were determined by Atomic Absorption Spectrophotometer Statistical Analysis Bioaccumulation factor (BCF) and translocation factor (TF) were used to evaluate plant phytoextraction efficiency. The significance of the present work has 107

10 been analyzed by statistical validation in terms of standard deviation (SD) using SPSS 13.0 statistical software Bioconcetration factor Bioconcentration factor provides an index of the ability of the plant to accumulate a particular metal with respect to its concentration in the soil substrate. It is a ratio of the heavy metal concentration in the plant tissue (root, shoot) to that in soil. It is calculated as follows [14] Translocation factor BCF = ( Metal) plant tissue Metal soil Translocation factor indicates the ability of the plant to translocate the metals from root to shoot at different concentrations. It is a ratio of concentration of the heavy metal in shots to that in its roots. It is calculated as follows [15]. TF = ( ) ( ) Metal shoot Metal root The translocation property describe that the content of heavy metals accumulated in shoots of a plants should be higher than those in its roots i.e., TF>1 to be considered as a accumulator plant where as the ratios are invariably <1 is non accumulators [16] Results and Discussion Physiochemical parameters of soil The soil used for the experiments was sandy with 70% of sand, 20%of slit and 7% of clay contents. According to Table 4.1 some selected soil characteristics as follows: PH :8.02, and it was determined by using a digital PH meter(systonics) [13], EC:0.116ms and it was determined in 1;2 soil and sediment water slurry using a digital conductivity meter(elico cm 180) [14], Organic matter: 1.1% and it was determined by Walkley Black method [13], Available nitrogen: 0.05(mg/100g) [15], available phosphorous 0.6(mg/100gm) and it was determined by molybdenum 108

11 blue method [16 ] and potassium 0.06% and it was determined by flame photometric method [16] and total Cd,Cr and Pb concentrations 2.29 mg/kg, 2.5 mg/kg and 4.0 mg/kg respectively, available Cd, Cr and Pb concentrations 0.57mg/kg & 0.6 mg/kg and 1.0 mg/kg respectively. Table 4.1. Physio-chemical properties of soil. Physio-chemical characteristics Colour Reddish Brown Gravel (%) 2 Sand (%) 70 Silt (%) 20 Clay 7 texture Sandy PH(1:2:5) 8.2 EC(mS/cm) Chemical characteristics Organic matter (%) 1.1 N(mg/100g) 0.05 P(mg/100g) 0.6 K (%) 0.06 Heavy metals Available cadmium (mg/kg) 0.57 Total Cadmium(mg/kg) 2.29 Available Chromium(mg/kg) 0.6 Total chromium(mg/kg) 2.5 Available Lead(mg/kg) 1.0 Total Lead(mg/kg)

12 Effect of cadmium stress on plant growth and physiology in Mirabilis jalapa andddatura inoxia Growth studies of Mirabils jalapa with cadmium amendments During the period of observation, no toxic symptoms were observed in any test concentrations, shoot length and shoot dry matter showed no significant reduction relative to control. The mean values have increased showing no inhibitory effect with increases soil Cd on all harvest days (Table and Fig a, b). The maximum shoot length and shoot dry matter were 37.5cm and 3 mg/kg in TC4 soils on day 45 respectively. However the root length and root dry matter were significantly reduced in TC4 soils. The maximum reduction was observed on day 45. The mean values have decreased shown that inhibitory effect on root length and root dry matter with increase in soil Cd which is proved in the earlier reports. 110

13 Table Shoot length, Root length, Shoot biomass and Root biomass in different test concentrations on different harvest days in Mirabilis jalapa Shoot length Root Length Shoot dry matter Root dry matter Days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±5.41 Day ±2.35 ±2.65 ±5.62 ±4.23 ±2.54 ±1.25 ±2.65 ±3.21 ±3.69 ±2.78 ±2.05 ±2.14 ±2.36 ±2.65 ±4.56 ±2.08 ±2.69 ±2.17 ±2.84 ±2.37 Day ±3.54 ±2.54 ±3.21 ±2.98 ±5.36 ±2.65 ±2.75 ±2.15 ±3.45 ±2.65 ±4.23 ±1.25 ±1.56 ±1.98 ±1.75 ±2.34 ±2.98 ±5.36 ±2.87 ±2.87 Results are means ±SD (n=5) 111

14 Figures showing variations in root length, shoot length, root dry matter and shoot dry matter in different concentrations and harvest days of Mirabilis jalapa with cadmium amendments a b Leaf pigments of Mirabils jalapa with cadmium amendments As shown in Table & Fig a-c same trend observed in physiological parameters. The mean values of chlorophyll a, chlorophyll b and carotenoids were decreased with the increase of soil Cd which was similar to previous studies [17]. The maximum reduction in chlorophyll a, chlorophyll b and carotenoids were 0.54mg/g, 0.35mg/g and 0.23mg/g on day 45 in TC4soils respectively 112

15 Table Chlorophyll a, Chlorophyll b and Carotenoids in different test concentrations on different harvest days in Mirabilis jalapa Chl a (mg/g) Chl b (mg/g) Carotenoids (mg/g) Days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± Day ± ± Day ± ± ± Results are means ±SD(n=5) 113

16 Figures 4.22a-c Showing variations in Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests Cd contaminated soils 4.2.2a 4.2.2b 4.2.2c 114

17 Growth studies of Datura inoxia with cadmium amendment. During the period of observation, no toxic symptoms were observed in any test concentrations, shoot length and shoot dry matter showed no significant reduction relative to control. The mean values have increased showing no inhibitory effect with increases soil cd on all harvest days (Tab & Fig. 4.23a-b). The maximum shoot length and shoot dry matter were 28.5cm and 3.5 mg/kg in TC4 soils on day 45 respectively. However the root length and root dry matter were significantly reduced in TC4 soils. The maximum reduction was observed on day 45. The mean values have decreased shown that inhibitory effect on root length and root dry matter with increase in soil Cd which is proved in the earlier reports. 115

18 Table Plant length and dry matter in different concentrations on different harvest days in Cd contaminated soils in Datura inoxia Shoot length Root Length Shoot dry matter Root dry matter Days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± ± ±2.0 2 ± ± ± ± ± ± ± ± ± ± ± ±2.54 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.85 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.21 Results are means ±SD(n=5) 116

19 Table Chlorophyll a, Chlorophyll b and carotenoids in different test concentrations on different harvest days in Mirabilis jalapa Chl a (mg/g) Chl b (mg/g) Carotenoids (mg/g) Days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± Day ± ± Day ± ± ± ± ± ± ± ±0.01 Results are means ±SD(n=5) 117

20 Figures 4.2.3a-b showing variations in root length, shoot length, root dry matter and shoot dry matter in different concentrations and harvest days of Datura inoxia with cadmium amendments 4.2.3a 4.2.3b Leaf pigments of Datura inoxia with cadmium amendments The mean values of chlorophyll a, chlorophyll b and carotenoids were decreased with the increase of soil Cd which was similar to previous studies [17]. As shown in Table & Fig a-c same trend observed in physiological parameters. The maximum reduction in chlorophyll a, chlorophyll b and carotenoids were 0.76mg/g, 0.54mg/g and 0.34mg/g on day 45 in TC1, TC4 and TC2soils respectively 118

21 Figures 4.2.4a-c showing variations in Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests Cd contaminated soils 4.2.4a 4.2.4b 4.2.4c 119

22 Effect of Chromium stress on plant growth and physiology Growth studies Mirabilis jalapa with chromium amendments Root system of the plant shown significant difference relative to control. Root length and root dry matter were decreased as the dosages of Cr increased from 25 to 100 ppm. The tallest plants were found in control pots (Table 4.3.1& Fig a-b). The maximum reduction in root length and root dry matter observed in TC4 soils on day 45. Same trend observed in shoot system of the plant.whereas in shoot system, Shoot length and shoot dry matter were increased as the dosages of Cr increased in test concentrationstc1 to TC4. The mean values have increased shown no inhibitory effect on shoot system with increase in soil Cr. Figures 4.3.1a-b showing variations in root length, shoot length, root dry matter and shoot dry matter in different concentrations and harvest days of Mirabilis jalapa with chromium amendments 4.3.1a 4.3.1b 120

23 Table Plant length and dry matter in different concentrations on different harvest days in Cr contaminated soils in Mirabilis jalapa Shoot length Root Length Shoot dry matter Root dry matter Days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.65 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.03 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.23 Results are means ±SD(n=5) 121

24 Table Chlorophyll a, Chlorophyll b and. Carotenoids content in different concentrations at different harvests (mg/g) in Cr contaminated soils Mirabilis jalapa Harvest Days Chl a (mg/g) Chl b (mg/g) Carotenoids (mg/g) C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± Day ± ± ± Day ± ± ± Results are means ±SD(n=5) 122

25 Leaf pigments in Mirabilis jalapa with chromium amendments As shown in Table.4.3.2& Fig 4.3.2a-c total chlorophyll and carotenoid values were affected, which was similar to some previous studies [18]. The maximum reduction in chlorophyll a, chlorophyll b and carotenoids were 0.4mg/g, 0.28mg/g and 0.12mg/g on day 45 in TC4soils respectively. The mean values have decreased shown that inhibitory effect on physiological parameters of test concentrations. Figures 4.3.2a-c Showing variations in Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests Cd contaminated soils 4.3.2a 4.3.2b 4.3.2c 123

26 Growth studies in Datura inoxia with chromium amendments Shoot system of the plant shown significant difference relative to control. Shoot length and Shoot dry matter were decreased as the dosages of Cr increases in soil. The tallest plants were found in control pots. The maximum reduction in shoot length and shoot dry matter observed were 12.5cm and 1.5mg/kg in TC4 soils on day 30. Whereas, in root system, root length and root dry matter were increased as the dosages of Cr increased in test concentrationstc1 to TC4. The maximum values in root length and root dry matter observed were 6.3cm and 3.5mg/kg in TC1 soils on day 30 and day 45. The mean values have increased from control to TC1 and thereafter reduced in TC2 to TC4 soils which shown inhibitory effect on root system with increase in the dosage of Cr concentrations in soil. Figures 4.3.3a-b showing variations in root length, shoot length, root dry matter and shoot dry matter in different concentrations and harvest days of Datura inoxia with chromium amendments 4.3.3a 4.3.3b 124

27 Table Plant length and dry matter in different concentrations on different harvest days in Cr contaminated soils in Datura inoxia Shoot length Root Length Shoot dry matter Root dry matter C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ±1.0 3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.5 Day ± ± ± ± ± ± ± ± ± ±0.7 3 ± ± ± ± ± ± ± ± ± ±1.36 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.99 Results are means ±SD(n=5) 125

28 Table Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests (mg/g) in Cr contaminated soils in Datura inoxia Chl a (mg/g) Chl b (mg/g) Carotenoids (mg/g) C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± Day ± ± ± ± ± ± ± Day ± ± ± ± ± ± ± ±0.12 Results are means ±SD(n=5) 126

29 Leaf pigments in Datura inoxia with chromium amendments As shown in Table & Fig a-c Total chlorophyll (chlorophyll a and chlorophyll b) and carotenoid values were affected with the increasing the dosage of chromium concentration in soils. The maximum reduction in chlorophyll a, chlorophyll b and carotenoids were 0.61mg/g, 0.25mg/g and 0.13mg/g on day 45 in TC4soils respectively. The mean values have decreased shown that inhibitory effect on physiological parameters of test concentrations. The control value is more than all test concentrations showing the effect of chromium. Figures 4.3.4a-c Showing variations in Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests Cr contaminated soils 4.3.4a 4.3.4b 127

30 4.3.4c Effect of lead stress on plant growth and physiology Growth studies of Mirabilis jalapa with lead amendments During the period of observation, no toxic symptoms were observed in any test concentrations, shoot length and shoot dry matter showed no significant reduction relative to control. The mean values have increased showing no inhibitory effect with increased dosage of lead on all harvest days (Table &Fig 4.4.1ab). The maximum shoot length and shoot dry matter were 17.5cm and 1.4 mg/kg in TC2 soils on day 45 respectively. However the root length and root dry matter were increased from 15 to 30 days and thereafter a gradual decrease from 30 days to 45 days observed. The maximum reduction was observed were 3.0cm and 0.2mg/kg on day 45, the mean values have decreased shown that inhibitory effect on root length and root dry matter with increased dosage of chromium in soils. 128

31 Table Plant length and dry matter in different concentrations on different harvest days in Pb contaminated soils in Mirabilis jalapa Shoot length Root Length Shoot dry matter Root dry matter C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ±0.32 ±0.56 ±0.98 ±0.85 ±0.98 ±0.85 ±0.98 ±0.85 ±0.95 ±0.96 ±0.14 Day ±0.85 ±0.35 ±0.91 ±0.84 ±0.52 ±0.42 ±0.87 ±0.96 ±0.65 ±0.41 ±0.96 ±0.63 ±0.21 ±0.61 ±0.66 ±0.41 ±0.14 ±0.56 Day ±0.96 ±0.65 ±0.42 ±0.28 ±0.95 ±0.41 ±0.21 ±0.24 ±0.85 ±0.85 ±0.54 ±0.21 ±0.5 ±0.56 ±0.35 ±0.65 ±0.24 ±0.12 ±0.75 Results are means ±SD(n=5) 129

32 Figures 4.4.1a-b Showing variations in root length, shoot length, root dry matter and shoot dry matter in different concentrations and harvest days of Mirabilis jalapa with lead amendments 4.4.1a 4.4.1b Leaf pigments of Mirabilis jalapa with lead amendments Chlorophyll a, chlorophyll b and carotenoids of mirabilis jalapa for different treatments in pot study experiment shown in Table 4.4.2& Fig 4.4.2a-c leaf pigments such as chlorophyll a, chlorophyll b and carotenoids were effected which was in agreement with previous studies(p das ).. In Mirabilis jalapa the maximum reduction was observed in chlorophyll a, chlorophyll b and carotenoids were 0.38, 0.28 and 0.17 respectively and shown inhibitory effect with the increased dosage of metal. 130

33 Table Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests (mg/g) in Pb contaminated soils in Mirabilis jalapa Chl a (mg/g) Chl b (mg/g) Carotenoids (mg/g) C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± ± ± ± ± ± Day ± ± ± ± ± ± ± ± ± ± ± ± ±0.14 Day ± ± ± ± ± ± ± ± ± ± ± ± ±0.52 Results are means ±SD(n=5) 131

34 Figures 4.4.2a-c Showing variations in Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests Cr contaminated soils 4.4.2a 4.4.2b 4.4.2c Growth studies in Datura inoxia with lead amendments As shown in the Table & Fig a-b, in Datura inoxia the maximum shoot length and root length were observed as 16.6cm in TC3 soils and 18cm in TC3 soils respectively and shown no inhibitory effect. Maximum shoot dry matter and root dry matter were observed as 1.03mg/kg and 0.14 mg/kg in TC1 soils. 132

35 Table Plant length and dry matter in different concentrations on different harvest days in Pb contaminated soils in Datura inoxia Shoot length Shoot dry matter Root Length Root dry matter C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Day ± ± ± ± ± ± 0.78 ± ± ± ± ± ± ± ± Day ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.42 Results are means ±SD(n=5) 133

36 Figures 4.4.3a-b Showing variations in root length, shoot length, root dry matter and shoot dry matter in different concentrations and harvest days of Datura inoxia with lead amendments 4.4.3a 4.4.3b Leaf pigments of Datura inoxia with lead amendments Chlorophyll a, chlorophyll b and carotenoids of Datura inoxia for different treatments in pot study experiment shown in Table & Fig a-c the maximum reduction observed in chlorophyll a, chlorophyll b and carotenoids were 0.4, 0.41 and 0.22 in TC4 soils respectively and shown inhibitory effect on plant with increased dosage of lead concentration in soils. 134

37 Table Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests (mg/g) in Pb contaminated soils in Datura inoxia Chl a (mg/g) Chl b (mg/g) Carotenoids (mg/g) C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.12 Day ± ± ± ± ± ± ± ± ± ± ± ± ±0.14 Day ± ± ± ± ± ± ± ± ± ± ± Results are means ±SD(n=5) 135

38 Figures 4.4.4a-c Showing variations in Chlorophyll a, Chlorophyll b and Carotenoids content in different concentrations at different harvests pb contaminated soils 4.4.4a 4.4.4b 4.4.4c 136

39 The yellowish colour of the leaves and the reduction in leaf pigments as shown in fig are indication of lead accumulation which has caused chlorophyll degradation (Prasad ). Summarizing the growth characteristics, it can be observed that root length and root dry matter and leaf pigments were greatly influenced by heavy metal stress. Lead is non essential heavy metal which is not known to have any metabolic function in plants and is toxic to plants at higher concentrations. It is in agreement with earlier studies Cadmium uptake In mirabilis jalapa As shown in table and figure 4.5.1a-c the maximum accumulation by roots and shoots were 36.2mg/kg & mg/kg in TC4 soils on day 45. The mean values have increased with increased soil Cd on all harvest days and shown no significant reduction, relative to control. Accumulation of Cd was found higher in shoots rather than in shoot which indicates that Cd is effectively translocated to shoot 137

40 Table Cadmium accumulations in soils, shoot and root, Bioconcentration factor (BCF) and Translocation factor (TF) in different test concentrations on different harvest days. Cadmium Accumulation Harvest Days 15 days 30 days 45 days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Total cadmium (µg/g) in Soils ± ± ± ± Available cadmium in soil (µg/g) ± ± ± ±0.63 Shoot (µg/g) ± ± ± ± ± ± ± ± ± ±0.52 Root (µg/g) ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.96 BCF of Shoot BCF of Root TF Results are means ±SD(n=5) 138

41 Figure a-c. Cd accumulations of shoot and root in different concentrations on day in Mirabilis jalapa a b c 139

42 In Datura inoxia The maximum accumulation by roots and shoots were 30 mg/kg & 28.5mg/kg in TC4 soils on day 45. The mean values have increased with increased soil Cd on all harvest days and shown no significant reduction, relative to control. Accumulation of Cd was found higher in roots rather than in shoot (Table & 4.52a-c). Figure a-c. Cd accumulations of shoot and root in different concentrations on day in Datura inoxia 4.5.2a 4.5.2b 4.5.2c 140

43 Table Cadmium accumulations in soils, shoot and root, Bioconcentration factor (BCF) and Translocation factor (TF) in different test concentrations on different harvest days Cadmium Accumulation Harvest Days 15 days 30 days 45 days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Total cadmium (µg/g) in Soils 2.29 ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.84 Aailable cadmium in soil (µg/g) ± ± ± Shoot (µg/g) ± Root (µg/g) ± ± ± ± ± ± ± BCF of Shoot BCF of Root TF Results are means ±SD(n=5) 141

44 4.7. Chromium uptake In Mirabilis jalapa The maximum accumulation by roots and shoots were 34.8 mg/kg & mg/kg in TC4 soils on day 45 as shown in Table4.5.3 and figure 4.5.3a-c. Accumulation of Cr was found higher in root rather than in shoot. The accumulation difference of Cr among the root and shoot was marginal. The possible reason might be that there were some other physiological reasons responsible for this phenomenon. The mean values have increased with increased soil Cr on all harvest days and shown no significant reduction, relative to control. Figure a-c. Cd accumulations of shoot and root in different concentrations on day in Mirabilis jalapa 4.5.3a 4.5.3b 4.5.3c 142

45 Table Chromium accumulations in soils, shoot and root, Bioconcentration factor (BCF) and Translocation factor (TF) in different test concentrations on different harvest days Chromium Accumulation Harvest Days 15 days 30 days 45 days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Total Chromium( µg/g) 2.5 ± ± ± ± ± ± ± ± ± ± ± ± ±0.37 Available chromium in soil (µg/g) 0.62 ± ± ± ± ± ± ± ± ± ±0.20 Shoot (µg/g) ± ± ± ± ± ± ± ± ± ± ± ± ±0.33 Root (µg/g) 0.87 ± ± ± ± ± ± ± ± ± ± ± ±0.20 BCF of Shoot BCF of ROOT TF Results are means ±SD(n=5) 143

46 Table Chromium accumulations in soils, shoot and root, Bioconcentration factor (BCF) and Translocation factor (TF) in different test concentrations on different harvest days Chromium Accumulation Total Chromium(µg/g) Harvest Days 15 days 30 days 45 days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC ± ± Available chromium in soil (µg/g) ± Shoot (µg/g) 0.6 ± ± ±0.09 Root (µg/g) ± BCF of Shoot BCF of Root TF Results are means ±SD(n=5)

47 4.7.2 In Datura inoxia The maximum accumulation by roots and shoots were 19.3 mg/kg & 22.9 mg/kg in TC4 soils on day 45 as shown in Table and figure 4.5.4a-c). Accumulation of Cr was found higher in shoot rather than in root. The possible reason might be that there were some other physiological reasons responsible for this phenomenon. The mean values have increased with increased soil Cr on all harvest days and shown no significant reduction, relative to control. Figure a-c. Cd accumulations of shoot and root in different concentrations on day in Datura inoxia 4.5.4a 4.5.4b 145

48 4.5.4c 4.8. Lead uptake In Mirabilis jalapa In Mirabilis jalapa as shown in the Table & Fig a-b the maximum uptake of lead observed in roots in TC4 soils and in shoots in TC4 soils respectively. Lead content was higher in roots as compared that in shoot parts 146

49 Table Lead accumulations in soils, shoot and root, Bioconcentration factor (BCF) and Translocation factor (TF) in different test concentrations on different harvest days Lead Accumulation Harvest Days 15 days 30 days 45 days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Total cadmium (µg/g) in Soils ± ± ± Available cadmium in soil (µg/g) ± ± Root (µg/g) ± ± ± ±0.07 shoot ± ± ± ± ± ± BCF of Shoot BCF of Root TF Results are means ±SD(n=5) 147

50 Figure a-c. Cd accumulations of shoot and root in different concentrations on day in Datura inoxia 4.5.5a 4.5.5b In Datura inoxia 4.5.5c In Datura inoxia as shown in Table and Fig a-c maximum uptake of lead observed in roots as mgkg-1 in TC3 soils, where as the maximum uptake of shoot was 31.72mgkg-1in TC3 soils respectively. Lead content was higher in shoots as compared that in areal parts. 148

51 Table Lead accumulations in soils, shoot and root, Bioconcentration factor (BCF) and Translocation factor (TF) in different test concentrations on different harvest days Harvest Days Lead Accumulation 15 DAYS 30 days 45 days C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 C TC1 TC2 TC3 TC4 Total cadmium (µg/g) in Soils ± ± ± ± Available cadmium in soil (µg/g) ± ± ± ± Shoot (µg/g) 1.2 ± ± ± ± ± ± ±0.09 Root (µg/g) ± ± BCF of Shoot BCF of Root TF Results are means ±SD(n=5) 149

52 Figure a-c. Cd accumulations of shoot and root in different concentrations on day in Datura inoxia 4.5.6a 4.5.6b 4.5.6c 150

53 Based on the results it is evident that the Datura inoxia was a more tolerant and higher accumulation of lead compared to Mirabilis jalapa and also proved that with the increase in concentration of the heavy metal exposure to the plant bioaccumulation of the plant increased. In both the plants lead accumulation by the plants with increase in all test concentrations Biological concentration factor & Translocation factor in Cadmium, Chromium & Lead In Mirabilis jalapa In Cd As shown in table (3) in Cd, the maximum BCF of shoot and root were 2.15 and 1.60 in TC4 on day 45 respectively. The BCF values in experiment increased with increased soil Cd concentrations and all of them higher than 1.0 suggesting that Mirabilis jalapa has stable feature of Cd accumulation. The BCF of root and shoot has increased with time showing that the plant is extracting more metal. The increase in BCF with time was positive and it is proved in earlier reports [19]. Translocation values in the experiment were respectively. Plants must have the ability to translocation Cd from root to shoot in order to continue absorption of Cd from soil. Translocation to shoots is one of the mechanisms of resistance high Cd levels. The overall results shown that the maximum TF observed in TC4 on day 45. All of them higher than 1.0 in different test concentrations on different harvest days indicate the ability of Cd translocation from roots to shoots. In Cr As shown in table (3) in Cr, the maximum BCF of shoot and root were 2.08 & 1.68 observed in TC4 on day45 respectively. All of them higher than 1.0 suggesting that Mirabilis jalapa has stable feature of Cr accumulation. The BCF of root and shoot has increased with time showing that the plant is extracting more metal. The increase in BCF with time was positive and it is proved in earlier reports [19]. Translocation values in the experiment were The maximum TF observed in TC4 on day 45. All of them lower than 1.0 in different test 151

54 concentrations on different harvest days indicates the poor ability of Cr translocation from roots to shoots In Pb to evaluate the efficiency of lead phytoextraction in plants, BCF and TF were calculated. Results indicate based on the table (3) the bioconcentration factor of Mirabilis jalapa in shoot and root ranged in between , respectively. The maximum biocentration factor value observed in TC3 soils in both root and shoot. The average Translocation factor values were found to be in order of , in Mirabilis jalapa the maximum value found in control. As the TF values were lower than 1.0, they indicate the limited ability of lead to translocte from roots and shoots In Datura inoxia In Cd As shown in table (3) in Cd, the maximum BCF of shoot and root were 2.15 and 1.60 in TC4 on day 45 respectively. The BCF values in experiment increased with increased soil Cd concentrations and all of them higher than 1.0. Translocation values in the experiment were respectively. Plants must have the ability to translocation. In Cr As shown in table (3) in Cr, the maximum BCF of shoot and root were 1.45 & 1.14 observed in TC4 on day45 respectively. Maximum translocation values in the experiment were 1.19 observed in TC4 on day 45. In Pb higher bioconcentration values in shoot and root were , respectively. In both of the plant species the maximum bioconcentration factor value observed in TC3 soils. The average Translocation factor values of respectively. In Datura inoxia maximum translocation factor values observed in TC4 soils and all the TF values were higher than 1.0 which indicates strong potential to remedy lead contaminate site The BCF values in both the plants were higher than 1.0 under different test concentrations, suggesting that both plants have a stable feature of lead accumulation 152