PHYTOREMEDIATION OF SYNTHETIC WASTEWATER CONTAINING COPPER BY USING NATIVE PLANT

This study was aimed to assess the efficiency of N.oleander to remove heavy metals such as Copper (Cu) from wastewater. A toxicity test was conducted outdoor for 65-day to estimate the ability of N.oleander to tolerate Cu in synthetic wastewater. Based on a previous rangefinding test, five concentrations were used in this test (0, 50, 100, 300, 510 mg/l). The results showed that maximum values of removal efficiency was found 99.9% on day-49 for the treatment 50 mg/l. Minimum removal efficiency was 94% day-65 for the treatment of 510 mg/l. Water concentration was within the permissible limits of river conservation and were 0.164 at day-35 for the 50 mg/l treatment, decreased thereafter until the end of the observation, and 0.12 at day-65 for the treatment 100 mg/l. the concentrations of water samples exceeded the permissible limits for 300 and 510 mg/l throughout the observation. Bioaccumulation factor (BAF) for N.oleaner was found to be greater than one for all the treatments. Higher translocation factor (TF) were 1.65, 1.73, 2.61 and 2.34 mg/l for 50, 100, 300 and 510 mg/l, respectively. This study revealed that N.oleander can tolerate and treat Cu concentration in wastewater.


INTRODUCTION
Heavy metal contamination of water is one of the most important environmental problems throughout the world. These elements arises from natural or man-made activities through industrial and agricultural practices without proper control for their emission and discharge to the surrounding which have resulted in contamination of water and soil around the world (8). These elements are known to be carcinogenic and cause serious health issues to humans and noxious effects on animals, microorganisms, and plants because they are non-biodegradable and highly toxic even at low concentrations (6). Heavy metals are categorized as either non-essential or essential to biological activities. Non-essential ones are those elements that have an unknown biological role for all organisms such as Cd, Pb, Cr, As and Hg (11). Essential metals are needed in trace amounts by living organisms and have an important role in many physiological and biochemical functions such as Fe, Mn, Cu, Ni and Zn. several methods have been used to treat heavy metals from water such as solvent extraction; membrane filtration; precipitation by chemical reagents; coagulation; adsorption and ion exchange (1). However, these methods are economically unfeasible due to their high cost and generate additional waste to get rid of. Phytoremediation is inexpensive and ecofriendly treatment strategy which involves using plants to remove various organic and inorganic contaminants from water taking advantage of their abilities to absorb pollutants by their roots and transport it to their aerial parts (12). Cu is an essential element for living organisms and plays a significant role in various physiological processes, though it can cause detrimental health effect when exceeds allowable limits (1). In humans, severe health concerns are related to excessive Cu intake such as kidney damage, vomiting, intestinal irritation cramps, or even death for long term exposure (18). In plants, the exposure to high Cu concentrations may cause leaf chlorosis, stunting, growth retardation, photosynthesis disruption and oxidative stress (4). Plants with fibrous root and higher biomass are thought to be more suitable for phytoremediation of heavy metals from water. Terrestrial plants usually produce long fibrous root systems with large surface area for metal sorption (14). The aim of this study was to determine the ability of N. oleander to tolerate and uptake Cu from wastewater.

MATERIALS AND METHODS Experimental setup
The phytotoxicity experiment was conducted outdoor, in order to accommodate field conditions, and continued for 65 days from October 2018 to January 2019. Nine glass pots used in the test, each one with dimensions of 35cm x35cm x30cm (LxWxD), equipped with a bottom tap for sampling purposes. Each pot was filled with 6 cm layer of gravel ≥4 mm size placed in the bottom, intermediate layer of 7cm fine gravel with a size (2-4 mm), while the upper layer where 7 cm of sand with a size (0.1-2 mm) with an overall weight of 12 kg, (Figure 1). The characteristics of the sand used in the test are as follows: 17% CaSO4, 31% CaCO3, 0.8% organic matter, and pH value of 7.3 with a loamy sand texture. Five pots were planted (each of which transplanted randomly with eight healthy plants) and designated as: T PC ; T P1 ; T P2 ; T P3 ; T P4 corresponding to Cu treatments of 0, 50, 100, 300, 510 mg/l concentrations, respectively. Four pots were kept unplanted for comparison and designated as: T 1 , T 2 , T 3 and T 4 to represent Cu treatments of 50,100, 300, 510 mg/l concentrations, respectively ( Figure 2). Tpc control pot was used in order to compare the results of plants in contaminated planted pots to observe the effect of Cu toxicity. The concentrations used in this experiment were based on the result of the visual observation of the physical status of plant growth during a preliminary test conducted prior to the toxicity test.

Plant selection
Nursery grown Nerium oleander of the same age and average height of 25 cm has been used in the study. It is an ornamental, evergreen plant found in most parts of Iraq and can be found along the streets and highways of Baghdad city, (Figure 3).

Preparation of synthetic wastewater
The experiments were conducted in a single exposure system using Cu as the contaminant at various concentrations. The Cu levels used in this study were 0, 50, 100, 300 and 510 mg/L, prepared using Copper nitrate Cu (NO3)2.3H2O from (BDH Chemicals Ltd, UK) diluted in tap water. Five liters of the synthetic wastewater was added to each pot, and the level of water in all pots was maintained constant at 2 cm below the soil surface to simulate a subsurface flow system (SSF) similar to that used in a constructed wetland. Water losses were compensated by adding (≈0.5 L) tap water for approximately 1-2 time/week to avoid variation in Cu concentrations due to evaporation and evapotranspiration.

Cu analysis in water, plant, and soil
The toxicity experiments continued for 65 days. Physical observation was conducted to observe the effect of Cu contamination level on the plant compared to the control. The water samples were taken after (7,14,21,28,35, 49 and 65 days) and analyzed using Atomic Absorption Spectrometry (ASS). One plant was harvested on each sampling day (7, 14, 28, 49, 65 days) separated into (roots, stems, leaves). Cu extraction from the plants was performed using wet digestion method 6HNO 3 : 2H 2 O 2 adopted by (3). Soil samples were analyzed at 7, 14, 21, 28, 35, 49 and 65 days, total concentration of Cu in soil samples was determined using aqua regia digestion procedure (7). The Cu concentrations in plant and sand samples were also analyzed using AAS.

Determination of wet weight and dry weight
The overall health of plant growing in heavy metals contaminated medium can be estimated through plant biomass (5). Plant biomass could be obtained from the measurement of plant wet and dry weight. Plant samples were harvested at the end of the observation. For dry weight (DW) measurements, Plant samples were placed in a drying oven (BINDER, Hotline International, Germany) set at 105 • C until reaching constant weight for a maximum of 2 hours (19), and weighted using electrical balance (Sartorius ENTRIS 64i-1S, Germany).

Calculation of removal efficiency, BAF, and TF
The efficiency of N.oleander to remove Cu from water was calculated using eq.1 (13): Where 0 = initial Cu concentration in wastewater (mg/l), = Cu concentration with time (mg/l). The potential of plants in accumulating a given metal from contaminated substrate can be evaluated using BAF, it is estimated by using eq.2 (13): The amount of Cu transferred from roots to aerial parts is estimated using eq.3 (15)≔ =

RESULTS AND DISCUSSION Cu concentration in water medium
The results of Cu concentration for all treatments in planed and unplanted pots are represented in (Figure 4a, b).   (Figure 5). The concentration increased up to day-28 for T P1 and T P2 and continues to decrease till the end of the observation. For T P3 and T P4 , Cu concentration in plant tissues increased up to day-14 and continues to decrease to the end of the observation. The highest Cu concentrations in plant were 337.6 mg/kg, 354 mg/kg, 433 mg/kg and 484 mg/kg for T P1 , T P2 , T P3 and T P4 respectively. From these results, one can conclude that the highest plant uptake of N.oleander reached at 28-day to remediated Cu polluted water up to 100 mg/l, while for higher concentrations up to 510 mg/l Cu polluted water reaches highest uptake at half of this period 14-days. (Figure 6a) shows the images of N.oleander at the beginning of the phytotoxicity test. Plants images at the first week of observation are shown in (Figure 6 b). Leaves chlorosis was observed at T P2 from the first week of observation. At higher concentrations of T P3 , T p4 plant withering symptoms were started from day-7 observation. Figure 6 c depicts the images of plants for the day-28. Effect of toxicity increased gradually until the end of the observation (Figure 6 d). Many researchers (9,10) reported that Copper toxicity may cause leaf chlorosis; oxidative stress; reactive oxygen species (ROS) formation and Growth retardation.  Figure 6 (a-d). N.oleander during phytotoxicity test Cu concentration in soil medium Cu levels in soil for both planted and unplanted pots are depicted in (Figure 7). The concentration of heavy metals in soil medium increased with increasing the added doses for all aquaria with and without plants. This agrees with the findings of Tangahu et al., (20). The results of Cu concentration in planted pots generally increased for day-14 of observation for most exposure doses and were 17.2, 35.8, 102.8 and 200 mg/kg for 50, 100, 300 and 510 mg/l, respectively and decreased until the end of the experiment. This phenomenon could be related to plant uptake mechanism (20). For unplanted pots, the concentration of Cu in soil was almost stable for each treatment and no significant variation noticed for all pots throughout until the end of the observation. This stability is probably due to soil water reaction and equilibration without interference of plant. The highest Cu concentrations recorded for soil in unplanted pots were 18.8 and 37.5, 108.7 and 187 mg/l for 50, 100, 300 and 510 mg/l, respectively. In general, high Cu concentration in the soil medium could be attributed to high Cu affinity to soil medium, also soil characteristics play a significant role, (16).  Figure 7(a-d). continue Figure 7. (a-d). Cu concentration in soil medium Effect of Cu on wet weight and dry weight Plant wet weight and dry weight is an important parameter used to estimate their ability to tolerate heavy metals (22). The wet weight and dry weight measured at the end of observation decreased along with increasing Cu concentration in water, a decrease in dry weight of 11, 37, 57 and 85% compared with the control was found when Cu concentrations were 50, 100, 300 and 510 mg/l, (Figure 8).  (Table 1). The BAF values for all treatments were higher than one which indicates that N.oleander has the capacity to adsorb Cu from the water medium, translocate and store it in the above ground parts (2). The initial concentration effect on Cu uptake by plants in each treatment resulted in different trends of BAF values over the different treatments. Most of the TF values of Cu by N.oleander, as shown in (Table 2), are greater than one especially with higher concentrations TP3 and TP4, this may relate to Cu availability in water at higher concentrations (21). In addition, the variation of TF values could be caused by the difference of Cu concentration in different plant parts that depends on the biochemical and physiological factors that participate in heavy metal accumulation and distribution in the morphological parts of the plant (17). The results of the experiments performed with oleander grown in synthetic wastewater with increasing concentrations of Cu revealed that Cu removal by N.oleander decreased with increasing initial concentration added. The results of Cu concentrations in water were within acceptable river discharge criteria for both T P1 and T P2 . Higher removal efficiency obtained at T P1 , so this concentration will be applied in the future for horizontal subsurface flow constructed wetlands experiments. The plant developed no visible toxicity symptoms for Tp1, while chlorosis appeared at T P2 , and some plants in T P3 and T P4 showed withering symptoms. Plant biomass is an important parameter used to assess their tolerance to heavy metals. The plants exposed to single Cu concentration in water caused some reduction in the growth of N.oleander. BAF values were higher than one for all the treatments. TF values fluctuated during the observation, the main Cu accumulation was in the plant shoot, particularly for higher concentrations. Nerium oleander is obviously suitable for phytoremediation applications of water polluted with Cu.