INTRODUCTION:

Duckweeds are small, monocotyledonous flowering plants, which occur in standing and slowly flowing waters almost all over the world (Landolt, 1986). Living organisms need variable amounts of heavy metals (Lane and Morel, 2000). Heavy metals are considered a hazardous threat to the environment due to three key criteria, i.e. persistence, bioaccumulation and toxicity. (Mandakini et al, .2016). Aquatic plants grow in or near waters that can be emergent, submerged or free floating.

They are an important constituent of aquatic communities due to their roles in oxygen production, nutrient cycling, water quality control, sediment stabilization, to provide habitat and housing for aquatic life, and also for being considered efficient heavy metal accumulators (Vardanyan and Ingole, 2006). Due to these features these plants have been successfully used as biological monitors and remediation of environments polluted with heavy metals. Use of plants in metal removal recently (phytoremediation) has seemed as a capable alternative in the removal of heavy metal excess from soil and water (Mulligan et al,.2001, Glass et al,. 2000, Chaney et al,.1997). Phytoremediation can be categorized based on the pollutant fate or the mechanisms involved (EPA, 2000).

Heavy metals are widespread pollutants of great environmental concern as they are non-degradable, toxic and persistent with serious ecological ramifications and passes through food chains to humans (Chopra et al,. 2009). Heavy metals enter the plant tissues mainly through the roots and foliage, of which root uptake was the dominant pathspace. Metals can be moved from soil pore water into the plants though the roots in the form of dissolved ions (e.g., Cd²⁺) (McLaughlin et al. 2011). Onaindia et al. (1996) pointed out to possibility using of aquatic plants to infer to the water quality and provide the types and abundance of aquatic plants a good indicator of "changes in water sources and a" life "evidence of nutritional status (Arts, 2002).

The major micronutrients in plants is cobalt involved in the growth and metabolism Palit et al.(1994) uptake and distribution mechanism of Co²⁺in plants is species dependent and differently regulated (Bakkaus et al,.2005). Sree et al (2015) was investigated toxic effect of cobalt on Lemna minor L.

The phytoremediation potential of Lemna minor for the uptake of Cr (VI) at the optimum nutrient strength for Cr (VI) uptake was investigated by (Thayaparan et al., 2015). Capacity assessment for chromium absorption by Lemna minor was carried out for 7 days at different levels of chromium concentrations. The aim of research the possibility of using Lemnagibba to remove some heavy elements from aquatic environment and study of the response of Lemnagibba when exposed to heavy elements through the measurement of total chlorophyll and chlorophyll a and chlorophyll b, protein and enzymes.

MATERIALS AND METHODS:

The study was conducted in department of ecology lab since January to June 2017. The free floating macrophytes Lemnagibba was collected from Euphrates River. Plants were washed several times with tap water then distilled water in order to remove any small invertebrate and algae (Lytle and Smith, 1995). Plant acclimatized for day 7 in tap water .After acclimatization, plant was exposeto the chosen concentrationat 0.5, 1, 3, 6ppm for cadmium and (0.5, 1, 2.5, 5) ppm for cobalt and chromium at a time interval of 1, 7, 15 days. Triplicate batch tests were conducted in plastic container of 15 liter capacity. Desired heavy metal concentration was added in each container from prepared stock solution. About 100 gm. of plant was kept in each container marked the water level. All containerswere exposed enough to light for detention time of 15 days. Everyday tap water was added to maintain the same level in each container. The aquatic plant was exposed separately to the individual metal ion solutions of cadmium (CdCl₂), cobalt (CoCl₂) and chromium (K₂Cr₂O₇) were used as a source of Cr⁺⁶. After each time interval the plant was collected and washed with deionized water to remove any metal adhering to its surface. The washed plant sample was carefully dried. Sample wasdried for 48 h in an oven at 70ºC. After drying, the sample wasground and digestedaccording to Orson et al.(1992). Chlorophyll, chlorophyll a and b were measured according to Aminot and Rey,(2000) and calculatedby the following equation:

Total chlorophyll (mg/gm) =

{A645(20.2)+A663(8.02)}v/w x 1000

Chl.a(mg/gm) =12.7(A663)- 2.69(A645) v/w x 1000

Chl.b(mg/gm) =22.9(A645)- 4.68(A663) v/w x 1000

Total protein was measured according to (Pak, 2010). Superoxide dismutase was measured according to (Marklund and Marklund,1974) and enzyme activity was calculate by equation of (Frary et al; 2010).

% Inhibition of pyragallol reduction/50 % X reaction volume

SOD Activity (units)=-----------------------------------------------------

Total test period

Catalase enzyme was measured according to Aebimethod (1983), and enzyme activity was calculateddepending on equation (Frary et al; 2010).

Catalase Activity (unit /g)

= (Δabs/min X reaction volume)/0.001

The bio concentration factor (BCF) is a useful parameter and it provides the ability index of a plant to accumulate metals with respect to metal concentration in the medium and it was calculated on a dry weight basis (Zayed, et al., 1998).

BCF = Trace elements concentration in plant tissue (µg.g^-1)/ Initial concentrationof the element in the external nutrientsolution (mg.l^-1)

Statistical analysis:

Two-space ANOVA test was used for further statistical analyses. The value P<0.05 was considered statistically significant. All statistics were done using the excel computer 2007.

RESULTS AND DISCUSSION:

1- Bio Concentration Factor (BCF):

The bio-concentration factor (BCF) indicating the ability of the plant to accumulate metals in their tissue as shown in table 1. The BCF values at different cadmium concentrations (0.5, 1, 3 and 6) mg/L were estimated onday 1, 7 and 15. The BCF high value was 59231 at 1 mg/L on day 15 and low value was 756 at 0.5 mg/L on day 1, while at cobalt concentration (0.5, 1, 2.5 and 5) mg/L BCF was estimated highas 61492 at 1 mg/L on day 15 and low value was 870 at 0.5 mg/L on day 1, and for chromium concentrations (0.5, 1, 2.5 and 5) mg/L BCF was recorded as the highest value 5362 at 0.5 mg/L on day 1 and the lowest value as105 at 2.5 mg/L on day 1. Lemnagibba has the ability to accumulate cadmium and cobalt in the tissues at low concentrations which is generally classified as a good accumulator. Cadmium is absorbed rapidly by the roots and can accumulate in plants (Silvane et al,. 2011). The ambient metal concentration in aquatic media is one of the critical factors that influence the metal uptake efficiency in aquatic plants (Gupta et al, .1995). The physiological need of metals in plant and uptake kinetics directly or indirectly affects the accumulative process for certain species of metals (Rashmi and Surindra, 2015). Lemna sp. tolerates large amounts of metal without adverse impact on its growth and development; it is linked to one specific location, so it is the real representative of the area; it is easily available for collection, identification and handling; and the plant specimens are similar in size and age, making it easy to select a representative sample. Its disadvantage is that it does not have a long lifetime enough to fully exhibit the phenomenon of bioaccumulation (Vlatko et al, .2015).

2- Removal ratio:

The current study recorded high removal ratio of Cd at 1 mg/L on day 15 as 78 % while it had high removal ratio of Coat 1 mg/L on day 15 as 79 % but Cr⁺⁶ recorded 60% (at 2.5 mg/L on day 15 as shown in Table 2) This is due to L.gibba plant ability to absorb the Co and Cd in low concentration and bioaccumulation inside tissues. The results of Cd removal are in agreement with that studies by earlier authors (Sen and Mondal,1990 ; Tkalec et al,.2008; Rashmi and Surindra, 2015 ). Few past researchers have also described similar result that duckweed can be a possible material to remove heavy metals from water and in this process the pH of water plays an important role in removal process (Xie et al,. 2013; Sobrino et al, .2010). The pH affects the solution chemistry of the metals, the activity of the functional groups in the biomass and the competition of metallic ions (Khellaf and Zerdaoui, 2013).

3- Physiological state of plant:

Results of metal analysis in Lemnagibba confirmed the accumulation of cadmium, cobalt and chromium within the plant and a corresponding decrease of metals in the water. Accumulation showed that the Cd metal increase gradually with day 7 and day 15 and higher values at 3 ppm onday 15, while concentration of Cd in medium decrease gradually throughout the experiment period without the production of any toxicity or reduction in growth (figure 1 a,b). This is due to tolerance of Lemnagibba of little amount of concentration of cadmium ion and accumulate in tissues.

Figure 2 a and bshow the value average of cobalt in Lemnagibba remaining in watershow that accumulation in plant increased during day 7 and day 15 and higher value at 2.5 ppm onday 15 while in the medium, it recorded gradual decreaseonday 15. There was significant difference (p<0.05) between control and treated plants and between four cobalt concentrations (0.5, 1, 2.5 and 5)mg/L. It was referred by (Ince et al, .1999) that higher concentrations of Co²⁺ inhibited growth in L. minor while lower Co²⁺concentration stimulated growth. Stimulating effect of cobalt on growth rate was also observed in algae (Osman et al., 2004; Horvati´c and Perˇsi´c, 2007) and sweetpepper (Gadand Hassan, 2013). While induced stimulation of growth was not observed in the study of (Begovi´c et al.,2016).

Chromium is known to be a toxic metal that can cause severe damage to plant and animal (Panda and Choudhury, 2005), and an important environmental contaminate released into the atmosphere due to huge industrial use (Nriagu and Nieboer,1988). The current study showed metal accumulation in plant increased gradually in day 7 and day 15 compared with control (figure 3 a), while in the water medium it decreased gradually during study period(figure 3 b), and recorded high value at 5 mg/L on day 7, showing correlation with chlorophyll content decreased (Table 4), due to the accumulation of Cr by plant which can reduce growth, reduce pigment content,damage root cell and cause ultrastructural modification of the chloroplast and cell membrane (Panda et al,. 2003; Hu et al,.2004).

Protein plays an important role in the metabolism and cell membrane, where it regulates the processes that overlap the external and internal membrane (Kharat et al., 2009). Dissolved protein content is an important indicator of the plant's physiological condition (Doganlar et al., 2010) The results of the study showed a decrease in protein content in plants during study period (table 3), due to plant stress resulting from ROS (Reactive Oxygen Species), which is an oxygen-containing chemical reaction molecule such as superoxide anion (O₂), hydrogen peroxide (H₂O₂) and hydroxyl (OH^-), leading to an oxidative stress that produces these compounds as products during metabolism that affect plant cells and lead to their death, as well as the breakdown of protein, fat, and DNA (Smirnoff, 2005).

Chlorophyll is a green pigment responsible for plants photosynthesis for energy production and is present in the plant cell of the plastids (Lefsrud and Kopsell, 2005). Table 3shows the total chlorophyll ,chlorophyll a and chlorophyll b concentrations respectively, The results of these in (Table 3) showed a decrease in the amount of chlorophyll onday 15 due to formation of ROS (Reactive Oxygen species) resulting in plant stress that directly or indirectly affects photosynthetic process (Liu et al 2009 ;Khataee et al., 2011).

The enzyme SOD (a group of metallic enzyme which antioxidants) is based on an element in the defense against natural and industrial pollutants. The most important function of this enzyme is to restore the vitality of the cells and reduce the speed of destruction and used to detect the harmful effects of pollutants in aquatic (Sahan et al., 2010. This enzyme exists in green leaves either with direct effectiveness to regulate the amount of (ROS) and affect the leavesmore than roots which is similar to the high efficiency of the enzyme in the leaves, leakage of electrons from the transmission of electrons series in photosynthesis to the molecule of oxygen (Liu et al; 2009) .The results in the current study showed higher effectiveness of the SOD enzyme during the period of stress of metals onday 7 and day 15 ( Table 4) probably because the plant used it as a means of defense against pollutants.

The enzyme catalase is spread in organisms. Its function is to stimulate hydrolysis of hydrogen peroxide into water and oxygen. This enzyme is characterized by the highest rate of inversion. One part can convert 83,000 parts of hydrogen peroxide into water and oxygen per second (Nadji et al., 2010). Most aerobic objects and organisms are anaerobic and there is a direct correlation between chlorophyll dye and catalase enzyme activity. When chlorophyll production rates decrease, catalase activity decreases. The results of the study showed adecrease in the effectiveness of catalase enzyme during the exposed period (Table 4).

The 0.5 ppm treatment on day 15 the air space increased in number and decreased in area, mesophyll cell occupied leaf center and epidermis cell increased in thickness compare with control (Plate 1 – A,B) where the 1 ppm treatment on day 15.The air spaced is integrated some of walls and decreased in area, mesophyll cell occupied leaf center and epidermis cell increased in thickness layers were multiplied (Plate 1,2,3 – C), but in the (6,5) ppm heavy metals treatment on day 15 the showing in general the lack of thickness of leaf and tortuosity of the epidermis surface ,air spacedecreased in number and increased in the area ,disintegration of mesophyll cell and epidermis cell increased in thickness(Plate 1,2,3 – D).

Table 1: Bio concentration Factor for heavy metal in L. gibba

Metal	Concentration of heavy metal mg/L	1 day	7 day	15 day
Cd	0.5	756	3220	899
	1.0	912	4204	59231
	3.0	2095	1634	9480
	6.0	1481	3263	5149
Co	0.5	875	16730	40841
	1.0	1242	17098	61492
	2.5	1316	6396	39471
	5.0	870	4075	4700
Cr	0.5	5362	2842	2381
	1.0	338	1462	929
	2.5	105	1874	3578
	5.0	273	1192	1220

Table 2: Removal ratio % for L. gibba of heavy metal.

Metal	Concentration of heavy metal mg/L	1 day	7 day	15 day
Cd	0.5	17	27	41
	1.0	9	25	78
	3.0	10	22	44
	6.0	18	38	55
Co	0.5	19	44	68
	1.0	9	64	79
	2.5	12	35	72
	5.0	26	49	56
Cr	0.5	32	37	39
	1.0	10	22	22
	2.5	10	51	60
	5.0	16	29	32

Table 3: Mean total chlorophyll, chlorophyll a, and b in Lemnagibba during exposed period.mg/gm

Con. of metal		Total chl.mg/gm			Chl. amg/gm			Chl. bmg/gm
Con. of metal		1 day	7 day	15 day	1 day	7 day	15 day	1 day	7 day	15 day
Cd	0.5 ppm	0.131	0.132	0.05	0.037	0.034	0.013	0.097	0.094	0.04
	1 ppm	0.122	0.09	0.03	0.026	0.02	0.008	0.091	0.066	0.033
	3 ppm	0.09	0.05	0.01	0.017	0.016	0.005	0.077	0.039	0.006
	6 ppm	0.08	0.06	0.02	0.014	0.019	0.006	0.07	0.042	0.008
	Control	0.129	0.148	0.202	0.030	0.041	0.060	0.102	0.112	0.16
Co	0.5 ppm	0.16	0.11	0.090	0.036	0.025	0.025	0.120	0.08	0.07
	1 ppm	0.13	0.11	0.108	0.03	0.02	0.04	0.1	0.09	0.06
	2.5 ppm	0.08	0.09	0.08	0.017	0.017	0.03	0.06	0.08	0.05
	5 ppm	0.06	0.07	0.06	0.017	0.013	0.02	0.04	0.060	0.04
	Control	0.12	0.13	0.14	0.029	0.026	0.046	0.096	0.105	0.095
Cr	0.5 ppm	0.1	0.1	0.037	0.021	0.02	0.006	0.082	0.079	0.045
	1 ppm	0.09	0.08	0.037	0.007	0.004	0.008	0.081	0.075	0.041
	2.5 ppm	0.1	0.09	0.04	0.007	0.006	0.009	0.085	0.082	0.037
	5 ppm	0.07	0.06	0.02	0.001	0.002	0.005	0.065	0.054	0.029
	Control	0.11	0.11	0.09	0.023	0.019	0.024	0.09	0.094	0.082

Table 4: Mean total protein, Superoxide dismutase, and Catalase in Lemnagibba during exposed period.

Metal concentration ppm		Total protein (mg/gm)			SOD (units/mg)			Catalase(units/mg)
Metal concentration ppm		1 day	7 day	15 day	1 day	7 day	15 day	1 day	7day	15 day
Cd	0.5	20.07	12.25	11.50	0.088	0.113	0.132	40.42	23.63	7.88
	1	18.25	13.25	12.67	0.091	0.130	0.131	42.52	38.32	5.29
	3	18.50	10.25	8.25	0.094	0.119	0.123	43.05	21.53	14.80
	6	19.67	9.00	1.75	0.095	0.130	0.138	51.97	7.42	2.63
	Control	21.00	20.25	19.15	0.055	0.067	0.066	7.88	7.45	7.36
Co	0.5	18.75	14.50	13.75	0.118	0.131	0.148	10.03	5.83	4.30
	1	17.00	14.25	12.25	0.129	0.136	0.148	41.48	7.91	3.19
	2.5	15.75	12.75	10.25	0.139	0.143	0.158	67.73	15.82	1.56
	5	15.50	6.50	4.00	0.143	0.150	0.140	75.13	21.56	3.69
	Control	20.25	18.75	18.25	0.115	0.114	0.116	7.5	7.48	7.89
Cr	0.5	15.25	11.50	8.25	0.046	0.047	0.039	3.15	5.83	0.54
	1	11.25	8.25	6.75	0.048	0.050	0.029	4.30	6.47	3.20
	2.5	10.25	7.25	6.25	0.050	0.053	0.031	5.83	6.87	4.25
	5	7.25	6.75	6.00	0.058	0.061	0.033	6.91	7.91	2.2
	Control	19.25	18.00	17.25	0.047	0.047	0.041	6.58	6.56	6.99

Plate 1: A-longitudinal section in leaf of Lemnagibba during on day1 (control plant), magnification force 400x , scale bare =60μm, B- longitudinal section in leaf of Lemnagibba during day 15 (0.5ppm of Cd⁺² metal treatment) C- during day15 (1 ppm of Cd⁺² metal treatment)D- during day15 (3 ppm of Cd⁺² metal treatment) E-during day15 (6 ppm of Cd⁺² metal treatment)A = Air space,Vb = Vascular bundles, Ep= Epidermis cell.

Plate 2: A-longitudinal section in leaf of Lemnagibba during on day1 (control plant), magnification force 400x , scale bare =60μm, B- longitudinal section in leaf of Lemnagibba during day 15 (1ppm of Co⁺² metal treatment) C- during day15 (2.5 ppm of Co⁺² metal treatment)D- during day15 (5 ppm of Cd⁺² metal treatment) A = Air space,Vb = Vascular bundles, Ep= Epidermis cell.

Plate 3: A-longitudinal section in leaf of Lemnagibba during on day1 (control plant), magnification force 400x , scale bare =60μm, B- longitudinal section in leaf of Lemnagibba during day 15 (1ppm of Cr⁺⁶ metal treatment) C- during day15 (2.5 ppm of Cr⁺⁶ metal treatment)D- during day15 (5 ppm of Cr⁺⁶ metal treatment) A = Air space,Vb = Vascular bundles, Ep= Epidermis cell.

CONCLUSION:

The current study conducted the BCF values recorded higher values for cadmium and cobalt at 1 mg/l on day 15 while for chromium recorded at 0.5 mg/l on day 1.The removal ratio recorded higher proportion for cadmium and cobalt at 1 mg/l on day 15 but for chromium recorded at 2.5 mg/l on day 15.Founding positive correlation between chlorophyll amount and catalase enzyme and through study period the SOD enzyme increase but decrease of protein content of plant. In high concentration of heavy metal causesgeneral the lack of thickness of leaf and tortuosity of the epidermis surface, airspacedecreased in number and increased in the area,disintegration of mesophyll cell andepidermis cell increased in thickness.

Figure (1a ): Mean concentration of Cadmium ion in Lemnagibba during study period.L.S.D_0.5=143.6

Figure (1b): Mean concentration of Cadmium ion in water during study period. L.S.D_0.5=0.09

Figure (2a ): Mean concentration of Cobalt ion in Lemnagibba during study period. L.S.D_0.5=996

Figure (2b): Mean concentration of Cobalt ion in water during study period. .L.S.D_0.5=0.02

Figure (3a): Mean concentration of Chromium ion in Lemnagibba during study period. .L.S.D_0.5=23.9

Figure (3-b): Mean concentration of Chromium ion in water during study period. .L.S.D_0.5=0.05

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Received on 05.02.2018 Modified on 26.03.2018

Research J. Pharm. and Tech 2018; 11(10): 4229-4236.

DOI: 10.5958/0974-360X.2018.00775.8