Removal of heavy metals from wastewater by natural growing plants on River Nile banks in Egypt

Increasing population growth, unexpected climate change and poor management of water resources are the main reasons for water scarcity, so efficient management of available water resources is needed, such as designing water harvesting structures, reusing wastewater, and improving the desalination plants especially in developing countries (Asthana & Shukla 2014). Recently, in non-urban areas of developing countries, the reuse of wastewater is presented as a perennial source of nutrients for agricultural production (Varkey et al. 2015).

Many types of industries, such as automobile and metal processing industries, are responsible for heavy metals contamination of our environment (Matouq et al. 2015). Heavy metals are toxic materials and harmful to the environment because they are non-biodegradable and toxic at trace concentrations (Ayyappan et al. 2005; Gautam et al. 2013). Metal toxicity causes many human diseases, such as cancer and nervous disorders (Nagjyoti et al. 2010; Lee et al. 2012). Removal of heavy metals from treated wastewater is required before its reuse in agriculture. Removal of heavy metals by traditional treatment methods such as precipitation, coagulation–flocculation, adsorption, and other advanced oxidation processes comprises a big cost (Lara et al. 2014). Therefore, it has become mandatory to induce a cost-effective green technology to remove these heavy metals and improve the effluent standards (Gonzalez et al. 2014).

Constructed wetlands (CWs) are designed and constructed to simulate the biological, chemical, and physical processes that happen in natural wetlands (Zhang et al. 2014). Wetlands are one of the most productive life support systems in the world and they are very important to mankind in socio-economic and ecological fields (Sanyal et al. 2015). Wetlands with macrophytes are natural systems that can be used for the treatment of domestic wastewater. Using macrophytes to treat wastewater is a cost-effective process due to a low cost of construction, operation, and maintenance. Phytoremediation is a known process that uses different types of plants to reduce and remove contaminants from soil and water (Akinbile & Yusoff 2012; Gupta et al. 2012; Kumar et al. 2012; Ugya et al. 2015; Lu et al. 2016; Prasad & Maiti 2016). Phytoremediation is also called green remediation, botano-remediation, agro-remediation, and vegetative remediation (Kumar & Chopra 2016).

Wetlands are capable of providing removal rates ranging from 60% to over 95% for many pollutants; they provide important functions such as habitat enhancement and they are less intrusive and provide a more environmentally sensitive approach to pollution abatement. One of the disadvantages of wetlands is that the reliability of their treatment systems can often be less consistent than that of traditional treatment systems, as nature can be unpredictable and external factors such as weather and pests often cause sites to display inconsistent contaminant removal rates (Davis 1995; Tousignant et al. 1999).

Ajibade & Adewumi (2017) evaluated the phytoremediation potential of three aquatic macrophytes (Commelina cyanea, Phragmites australis and Water Hyacinth (Eichhornia crassipes)) for treatment of municipal wastewater. Phragmites australis gave the highest removal efficiency for phosphate (85.8%).

This study aims to remove heavy metals from treated domestic wastewater using two types of plants that grow naturally on the River Nile banks in Egypt, namely Echinochloa pyramidalis and Ludwigia stolonifera, which were planted individually and in combination with different densities on the secondary treated wastewater from West Gerga wastewater treatment plant, Sohag, Egypt. These plants are undesirable, since they retard and increase the roughness of water flow. Egypt's government has already removed and disposed of these plants, which comprises a big cost without any benefits from these removed plants, to improve the performance of irrigation water distribution networks and enhance the maintenance working environment of irrigation structures.

Studies performed in previous works, the laboratory, and the field have provided encouraging insights into the suitability of aquatic plants to act as bio-indicators of water quality, especially in relation to the monitoring of trace metals.

Fawzy et al. (2012) investigated the concentrations of Cd, Cu, Pb, and Zn in sediments, water, and different plant organs of six aquatic vascular plant species: Ceratophyllum demersum; Echinochloa pyramidalis; Eichhornia crassipes; Myriophyllum spicatum; Phragmites australis and Typha domingensis growing naturally in the Nile system (Sohag Governorate), and reported that the concentrations of heavy metals in water, sediments, and plants possess the same trend: Zn > Cu > Pb > Cd, which reflects the bio-monitoring potentialities of the investigated plant species (Fawzy et al. 2012).

Due to their availability on the River Nile banks in Egypt, and their free cost, Echinochloa pyramidalis and Ludwigia stolonifera were used in the present study.

Batch experiments were configured at the same environmental conditions (i.e. weather, temperature, sunlight, wind speed, etc.), to avoid their influences on experimental results, from October 2017 to December 2017, using Echinochloa pyramidalis and Ludwigia stolonifera aquatic plants. Secondary treated effluent was collected from west Gerga wastewater treatment plant, located in Sohag city, Egypt. Batch tests were carried out with similar samples obtained during similar phases in the Chemical Department laboratories, Faculty of Science, Sohag University. Characteristics of the wastewater samples, obtained by using laboratory devices, are shown in Table 1.

In this study, constant concentrations of 5 mg/l of the tested metals (Cd, Ni, and Pb) were used as a mixture. Lead, cadmium and nickel solutions were prepared using Pb(NO₃)₂ (Sigma, St. Louis, MO A.R., 99.9%), Cd(NO₃)₂ (Aldrich Chemical Co., 98%), and Ni(NO₃)₂. The concentrations were calculated based on the individual element versus their compound form. The reagents were dissolved in distilled water to achieve the appropriate concentration level.

The experiments were executed at the Experimental Farm of the Faculty of Agriculture, Sohag University, Sohag, Egypt. In this study, an effluent from West Gerga wastewater treatment plant, with a constant artificial concentration of 5 mg/l of the tested metals (Cd, Ni, and Pb) was used as a mixture. The experimental setup consists of seven plastic tanks (50 cm length × 50 cm width × 96 cm height) with a working depth of 88 cm. Each tank has a wooden frame at the top to hold the plants up (Figure 1). Echinochloa pyramidalis and Ludwigia stolonifera plants were collected from the mainstream of the River Nile's east bank (N: 26° 33′ 29″; E: 31° 42′ 33″). The dimensions of these plants were not the same; the average dimensions, with a standard error of a single plant, were 43 ± 3.1 cm height and 8.5 ± 0.5 g fresh weights of Echinochloa pyramidalis and 56 ± 3.4 cm height and 38.6 ± 0.8 g fresh weight of Ludwigia stolonifera.

Acclimatization of the selected plants was done using distilled water for 7 days; the selected plants were thoroughly washed with freshwater before being placed in each experimental set. Then, 220 litres of secondary treated effluent was poured into each experimental set and subjected to 10 hours of sunlight daily throughout the length of the experiment. Distilled water was added at fixed intervals to compensate for the evaporation losses and keep the pH of the wastewater at a constant value.

The removal of Cd, Ni, and Pb was studied by planting the two plants each individually and both in combination, with different densities.

Four sets of experiments, as shown in Figure 2, were executed; the first set consisted of only E. pyramidalis plants with a maximum density, which could be fixed in the wooden frame, of 80 plants per square metre of wastewater surface. The second set consisted of only L. stolonifera plants, which were kept at a density of 80 plants per square metre. In the third set, three different experiments were carried out, each having a different density of combined plants (E. pyramidalis+L. stolonifera); the first density was 100% (i.e. 40 E. pyramidalis plants/m² and 40 L. stolonifera plants/m²), the second was 60% (i.e. 24 E. pyramidalis plants/m² and 24 L. stolonifera plants/m²), and the third was 20% (i.e. 8 E. pyramidalis plants/m² and 8 L. stolonifera plants/m²). In the fourth set, no plants were planted and it was used as an experimental control case for the determination of natural heavy metals removal.

For each experiment, three wastewater samples (triplicate) each of 15 ml volume were collected, at the same time of day from each compartment at different places within the root depth, at intervals of 1, 3, 5, 7 and 10 days (i.e. number of samples of each experiment, n = 15) for heavy metals analysis. Also complete plant samples, each weighing 5 grams, were collected at the beginning and at the end of each experiment.

After washing, the plant samples were dried in a convection oven for 24 hours at 48 °C, and then ground and powdered using a mortar to ensure homogeneity for facilitating organic matter digestion. Dry plant materials were digested according to the wet digestion procedure involving concentrated nitric acid (Campbell & Plank 1998). Cd, Ni, and Pb concentrations in both wastewater and plant samples were measured using an Atomic Absorption spectrophotometer (PerkinElmer Analyst 400, USA). Measurements were repeated three times. Heavy metal concentrations were expressed as mg/l (ppm), for wastewater samples and mg/g of dry weight for plant samples.

Complete randomized design (CRD) was used with three replications for each treatment. Analysis of variance (ANOVA) was carried out, using Proc Mixed of SAS package version 9.2 (SAS 2008) and means were compared by Duncan test at 5% level of significance, to determine the effect of using aquatic plants, with different planted cases at various detention times, on the removal efficiency of heavy metals (Steel & Torrie 1981; SAS Institute 2008).

Finally, correlation analysis was performed to examine the relationship between the variables according to Spearman rank order correlation as heavy metal levels were the intendant variable and detention time was the dependent one.

Removal of Cd, Ni, and Pb due to natural processes such as degradation and precipitation was determined from control experimental sets without using plants, and results are shown in Table 2, which shows clearly the removal of a very little amount of heavy metals through the natural precipitation process. Keeping this result as a reference, two aquatic plant types; that is, Echinochloa pyramidalis and Ludwigia stolonifera, were used for the efficient removal of heavy metals. A significant enhancement in the removal of Cd, Ni, and Pb (Table 2) over the values of the control experimental set was observed when both plants were planted individually. The removal efficiency of Cd increases by about 34% and 44% for E. pyramidalis and L. stolonifera respectively; the removal efficiency of Ni increases by about 16% and 26% for E. pyramidalis and L. stolonifera respectively; and the removal efficiency of Pb increases by about 78% and 74% for E. pyramidalis and L. stolonifera respectively with respect to control experiments.

The possible processes concerned in heavy metals removal are metal consumption by these wetland plants, precipitation, and co-precipitation as insoluble salts and metal bound to the substrate (Brix 1994; Ranieri et al. 2011). Rhizosphere microbes play an important role in phytoremediation. Since the rhizosphere is the immediate vicinity of the root, the chemical and physical changes in that environment can easily affect heavy metal uptake by the plant. The rhizosphere of P. australis and T. latifolia provided the supporting media for the growth of microorganisms, which are the main places for heavy metals immobilization and uptake by plants (Jacob & Otte 2004).

When E. pyramidalis and L. stolonifera were grown in combination, there was a further increase in the removal of Cd and Pb compared to the plants grown individually. As is clear from the ANOVA analysis results shown in Table 2, the removal efficiency of Cd and Pb by a combination of the two plants increases by about 13% and 5% respectively with respect to that of the individual plants. Perhaps the reason for the improved removal of Cd and Pb is due to the change of the plants' behavior when grown in combination, which is known as synergistic effects of the plant species (Tripathi & Upadhyay 2003). The increase in the rhizosphere of plants increases the surface area of the rhizomes, which would further increase the rhizomes' cell wall ability to uptake metals through immobilization (Tsednee et al. 2012).

The solubilization and movement of phytosiderophores by plants result in a complex with free heavy metal ions that may be taken as a secondary cause for the increase in absorption performance (Hu et al. 2014). Removal of Cd and Ni was significantly higher in L. stolonifera culture than that of E. pyramidalis culture, whereas in E. pyramidalis culture removal of Pb was higher. The possible reason would be that the selected plant species may have different uptake efficiency for the heavy metals used. Similar findings were introduced by Mishra & Tripathi (2008), where significantly different removal efficiencies for Fe, Cu, Cd, Cr, and Zn using Pistia stratioties, Siprodela polyrrhiza and Eichhornia crassipes support the present observation (Mishra & Tripathi 2008). Generally, the variation of heavy metal concentrations in wastewater, through each experiment day, in relation to plant species (P) and detention time (T), as analyzed by the two way repeated measures ANOVA, was significant as is clear from the ANOVA – F values are shown in Table 3.

The preferential order of heavy metal removal in the present study was Pb > Cd > Ni in E. pyramidalis, L. stolonifera and their combination (Table 2). The possible reason for higher removal of Cd and Pb might be that emergent plants with a long root system have higher metal absorption capacity than that of free-floating plants. Sargın et al. (2015), using pollenechitosan microcapsules, observed lower removal of Cd (26.6%) and Ni (16.2%) compared with findings of the present work (Sargın et al. 2015). The present process performs greater Cd removal from wastewater (61.52%) than that observed by Ahmadi et al. (2014) while treating aqueous solutions with maghemite nanoparticles (20%) (Ahmadi et al. 2014). The greater removal of Cd may be due to the plants' greater uptake efficiency for metals when compared with the other advanced technologies. Abdul Syukor et al. (2016) carried out an experiment on the uptake of heavy metals by Typha angustifolia and Limnocharis flava plants and achieved 49.3% Cd and 54.7% Pb removal, while the present work achieved 61.52% and 93.22% higher removal for Cd and Pb when two plants (E. pyramidalis and L. stolonifera) were applied together (Abdul Syukor et al. 2016). While treating heavy metal contaminated wastewater with Phragmites australis and Typha latifolia, Kumari & Tripathi (2015) found a removal efficiency of 48.1% for Cd and 53.6% for Pb, which are 13.42% and 39.62% less than our findings in the present study, respectively (Kumari & Tripathi 2015).

Bello et al. (2018) investigated the phytoremediation ability of Phragmites australis to remove cadmium (Cd), lead (Pb), and nickel (Ni) from contaminated water, The results of the study showed that P. australis had a removal efficiency of 93% for cadmium, 95% for lead and 84% for nickel over a 6-week period, which was a higher removal efficiency than that by E. pyramidalis and L. stolonifera in the present study.

To study the effect of wastewater pH on the removal efficiency of heavy metals through precipitation and absorption, heavy metal concentrations and pH were measured during the experiments. Initially, pH of the secondary treated effluent wastewater was slightly alkaline (Table 1), and during the experiment varied from neutral to slightly alkaline (6.86 ± 0.01–7.82 ± 0.01). This change in pH may be due to the addition of oxygen (O₂) via the diffusion of the atmospheric O₂ and photosynthetic activity of plants. That variation of pH from neutral to slightly alkaline during the experiments implies that the heavy metals removal from the wastewater was achieved by metals solubilization in the rhizosphere and absorption through the plant roots.

Aquaculture with high pH and high concentration of heavy metal ions has more chances of precipitation as a dominant process of metal immobilization, where the presence of anions such as SO₄²⁻, CO₃²⁻, OH-, and HPO₄²⁻ facilitates the process (Hong et al. 2007; Ok et al. 2010).

Khan et al. (2009) reported a pH value equal to and greater than 7 was best for removing heavy metals from storm wastewater (Khan et al. 2009). Abid et al. (2015) reported that the optimum heavy metals removal efficiency using the phytoremediation process occurs in the alkaline pH range (Abid et al. 2015).

The relation of detention time and heavy metals removal was studied using Spearman rank order correlation. In all the planted experimental sets of wastewater, significant negative correlation coefficients of Cd, Ni, and Pb levels with retention period were determined, which ensured the positive relation of retention period and Cd, Ni, and Pb removal percentages at p < 0.001 (Table 4). Similar findings were observed by Mishra et al. (2009) for the removal of heavy metals from coal mine effluent (Mishra et al. 2009).

The removal of Cd, Ni, and Pb from wastewater by E. pyramidalis and L. stolonifera at different retention times is shown in Figure 3, which illustrates the decrease in heavy metal concentration, for all the three metals, with detention time during 10 days of exposure. The possible reason for enhancement in heavy metals removal might be the continuous absorption of metals by plants. Similar findings by Kumari & Tripathi (2014) support the present observation (Kumari & Tripathi 2014).

Mass balance calculations of heavy metals at the end of the experiment for a contaminated sample are shown in Table 5. The values in Table 5 indicate that the loss of Cd, Ni, and Pb from the wastewater equals their net sorption in plants (mg), which was measured from the dry plant samples using an Atomic Absorption spectrophotometer, and loss due to natural precipitation (mg). Metal sorption for Cd and Ni was greater in L. stolonifera compared with E. pyramidalis, although this was not true for Pb. Natural metals precipitation (mg) occurs through the oxidation of materials in the presence of dissolved oxygen. It indicates the deposition of heavy metals in the form of oxidized salts on the bottom of the experimental set without any influence of experimental plants.

The accumulated heavy metals in E. pyramidalis and L. stolonifera plants were measured from the dry plant samples using an Atomic Absorption spectrophotometer at the initial time, after 1 day and 10 days, and the results are presented in Table 6. The table illustrates that the accumulation of Cd and Ni was higher in L. stolonifera than E. pyramidalis, while E. pyramidalis showed higher accumulation for Pb.

A comparison between the accumulated amounts of Cd, Ni and Pb within E. pyramidalis and L. stolonifera showed that Pb amounts were higher than that of cadmium, and Cd amounts were higher than that of Ni for the ten-day exposure. This may be a result of different response or tolerance of species to kinds of metals, and reflects the complexity of plant metal uptake under field conditions (Amany 2012).

In this study, the maximum removal efficiency of heavy metals by different intensities of the mixture of E. pyramidalis and L. stolonifera was at a density of 100%, while the minimum was at a density of 20%, and results are shown in Figure 4. The preferential order of the residual of heavy metals removal by the mixture of E. pyramidalis and L. stolonifera was Ni > Cd > Pb. The results showed a rapid reduction in metals residual since the first day exposure, with a different value for each metal type, since the results show the ability of both plants to absorb a larger amount of Pb in the first day than of Cd and Ni, while the metals removal continued over the remaining days of the experiment and decreased gradually by the end of the experiment for the three metals.

The possible reason for enhanced removal of Cd, Ni, and Pb might be the increase in the rhizosphere of these plants due to the increase of plant density, which results in more surface area of the rhizomes, which would further augment the rhizomes' cell wall capability to absorb metals through immobilization. Similar findings were observed by Saleh et al. (2017) for removal of cesium and cobalt radionuclides from simulated radioactive wastewater by Ludwigia stolonifera aquatic plants (Saleh et al. 2017).

In the present study, the heavy metals removal from secondary treated wastewater using two types of natural growing plants on the River Nile banks in Egypt, namely Echinochloa pyramidalis and Ludwigia stolonifera, was tested. Plantings these two plants in combination showed the highest removal of Cd and Pb, while L.stolonifera showed the highest removal of Ni. There was a positive relationship between detention time and removal of Cd, Ni, and Pb in the wastewater used for the experiment, which was confirmed by negative and significant correlation coefficients of Cd, Ni, and Pb concentrations with detention time at p < 0.001. Preferential order of heavy metals removal indicates higher affinities of Pb towards plants followed by Cd > Ni. Mass balance calculations showed that the loss of Cd, Ni, and Pb from wastewater was equal to their net sorption in plants and loss due to natural precipitation. The sorption ability of L.stolonifera is greater than that of E. pyramidalis for the tested heavy metals, except for Pb. The removal efficiency of heavy metals increases with the increase of plant density for both plant types.

The availability of aquatic plants and their free cost make their use in the wastewater treatment process to remove heavy metals an economically attractive alternative.

All relevant data are included in the paper or its Supplementary Information.