Phytoremediation of diesel contaminated soil using urban wastewater and its effect on soil concentration and plant growth

Petroleum hydrocarbons in oil production may leak into the soil during extraction, purification, or transportation and contaminate surface and subsurface soils, as well as groundwater (Tahhan et al. 2011; LeFevre et al. 2012; Sajna et al. 2015; Ramadass et al. 2017). With recent technological developments, diesel has become a common organic pollutant in the environment (Moreira et al. 2011; Bęś et al. 2019). Diesel-contaminated soils and waters endanger human health, while also promoting the extinction of various plant species and reducing their productivity. Therefore, proper solutions are required to remove these contaminants (Glick 2010; Hentati et al. 2013; Kang et al. 2014; Sammarco et al. 2015).

In recent decades, various methods have been used to clean up soil contamination, including physical, chemical, and biological techniques. Phytoremediation is a low-cost and environmentally-friendly method that refers to the use of plants and associated soil microbes to reduce the volume, mobility or toxicity of contamination in soil and water (Khan et al. 2013; Afzal et al. 2014; Arslan et al. 2017; Hussain et al. 2018a).

The success of phytoremediation the cleanup of contaminated soils has been confirmed in several studies. Karimi (2007) showed a significant reduction in planted diesel-contaminated soil compared to unplanted soil. Prematuri et al. (2020) concluded that the roots of Asteraceae plants could degrade total petroleum hydrocarbons (TPHs) in contaminated soil, and Ikeura et al. (2019) used Italian ryegrass, zinnia, and alfalfa to reduce the concentration of diesel in soil. Recently, plant species such as tall fescue have been reported to be efficient candidates for TPH remediation (Liu et al. 2013; Wei et al. 2019). Wei et al. (2019) found that ryegrass, and tall fescue had the highest oil degradability in a group of six plants that were tested for the remediation of polluted soil. Tall fescue is a grass that is widely used for phytoremediation. Fast growth, deep grass root penetration, and a dense and extensive fibrous root system with a maximum surface area are its main properties compared to other plants (Hall et al. 2011; Wei et al. 2019). Grass pea is also considered an effective plant species in the remediation of contaminated soil (Hao et al. 2014). Grass pea is a legume belonging to the Fabaceae family, which grows in weak soils and under challenging conditions (e.g., water logging in extremely arid environments), and is resistant to insects and pests, and often used to remove metals (Vaz Patto et al. 2006; Sarkar et al. 2019).

Legumes could grow well in petroleum-contaminated soils with a high carbon-to-nitogen (C/N) ratio owing to their ability to harbor nitrogen-fixing bacteria and enrich the soil with nitrogen (Adam & Duncan 2003; Hall et al. 2011; Shan et al. 2014). Poaceae and Fabaceae families are influential in the phytoremediation of soils polluted with petroleum products (Borowik et al. 2019; Hawrot-Paw et al. 2019).

One of the ways to enhance bioremediation and phytoremediation is to add organic matter such as composts and sewage sludge to the soil. Organic substitutes improve the soil physicochemical properties due to their nitrogen, phosphorus, potassium, and micronutrients content (Kominko et al. 2018). The results of a study by Wyszkowski et al. (2020) showed that adding sewage sludge to the soil reduced the negative effect of diesel oil on the chemical composition of plants. The application of sewage sludge to the soil resulted in an increase in the average nitrogen, sodium and magnesium in the oat plant.

Alternatively, in developing and some advanced countries where there is competition among water consumers (for both industrial and agricultural uses), using treated wastewater for irrigation can be an acceptable solution. Therefore, in order to save fresh water and enhance the phytoremediation process, treated municipal wastewater was used to irrigate the plants in this study, which is the main novelty of our study.

Almost no research exists that directly evaluates the interaction of wastewater and petroleum products in phytoremediation. Use of wastewater can be a turning point in this research, mainly due to the scarcity of water resources, which is the current crisis in most Middle Eastern countries and Iran. The present study aimed to investigate the effects of treated urban wastewater on the phytoremediation of diesel-contaminated soil using tall fescue and grass pea in a pot experiment over two years.

The current research was conducted in pots during October-December 2018 and July-October 2019 at the greenhouse research center of Bu Ali Sina University, located in Hamedan, Iran (48° 32′ E, 34° 48′ N; 1,810 m above sea level). The greenhouse temperature was 21–33 °C, and the soil used for the experiment was collected from agriculture research farms.

For each treatment, the pots were filled with 12 kilograms of sieved soil (2 mm), which was contaminated with 1.5 and 3% of diesel oil. Diesel at different concentrations was mixed with acetone as a solvent in the ratio 3:1 (acetone: diesel) and uniformly sprayed with a pump sprayer into thin soil layers to obtain an even distribution. After pouring the mixture into the soil, it was stirred until it became homogeneous (Alavi et al. 2017; Al-Mansoory et al. 2017). The polyethylene pots (diameter: 25 cm, height: 27 cm) were filled with the diesel-contaminated soil and allowed to stabilize for three months before sowing seeds. The seeds of tall fescue and grass pea were provided by the Seed and Plant Improvement Institute of Karaj, Iran. Seeds were sown directly in the topsoil of each pot at a depth of 1.5–2.0 cm. After germination, the robust seedlings were chosen from each pot, and other seedlings were removed. The pots were rinsed with fresh-water and treated wastewater twice per week, and the bottom of the pots was closed to prevent drainage. Table 1 shows the physicochemical properties of the applied soil and water.

This experiment was a complete randomized design with three replications. The diesel oil treatments were carried out at three concentrations of DC (clean soil) (0%), D1.5 (1.5%), and D3 (3%; w_diesel/w_soil). The irrigation water treatments were freshwater (FW) and treated urban wastewater (TWW). Table 2 shows the experimental design of the study.

Plant and soil samples were taken 25, 40, 55, 70 and 85 days after planting. The soil diesel content was measured at two depths of 0–12 and 12–24 cm in each pot to obtain an average soil contamination. Samples were dried at room temperature and sieved (with a 2 mm sieve) before analysis. The shoots of the plants were harvested on each sampling day from the three replicates and separated from the soil meticulously.

Plant height (cm) was measured as the distance from the root to the highest leaf. Five of the tallest plants in each pot were selected, and the mean height was calculated. Plants were carefully washed using tap water. The washed samples were dried using absorbent paper. All the plants were dried in an oven at a temperature of 70 °C for 72 hours, and the shoot dry weight was measured and recorded using a digital balance (Choden et al. 2020).

The effects of treatments on diesel removal (%), residual diesel, and shoot dry weight were analyzed using analysis of variance (ANOVA) by the SPSS version 20 (IBM Statistics for Windows). Duncan's test was used to compare and rank the treatment means (P < 0.05).

In addition, t-test was employed for the comparative analysis of the data for two years. Correlations between the parameters were assessed using Pearson's correlation coefficient. In all the statistical analyses, the significance level was set at P < 0.05.

The diesel concentration at two depths in different treatments was monitored during the 85-day experimental period. In the tall fescue test in 2018 and within the first 40 days, the soil contamination of the bottom layer was significantly higher than the top layer with D3-TWW and D3-FW. From the third sampling, no significant difference was observed between the upper and lower soil layers in all treatments (Table 3).

Since hydrocarbons can move with water (Huguenot et al. 2015), some of the contamination in the upper layer is gradually leached to the deeper soil layer. The rhizosphere is a suitable environment for reducing contamination due to the interaction between the root exudates of plants and soil microorganisms (Bianchi & Ceccanti 2010; Ikeura et al. 2019). The results of this study clearly showed that plant development affected the residual diesel in the soil. At the end of the study period, it was observed that for D3-TWW-T (2019) and D3-FW-T (2019) the residual diesel in the bottom of the soil layer significantly decreased compared to the residual diesel in the top layer. In other treatments, no significant difference was observed between the contamination in the top and bottom layer (Table 3).

Comparison of different sampling days in each treatment within the two years of the tall fescue test showed that the concentration reduction of the topsoil in the D3-TWW-T treatment was slower than in the other treatments (Table 3).

Kayikcioglu (2012) concluded that treated domestic wastewater could decrease the activities of dehydrogenase and alkaline phosphatase, which are the essential enzymes involved in the degradation of TPH in soil. Soil microbial activity is estimated by dehydrogenase. Dehydrogenate transports hydrogen from an organic substrate to the mineral acceptor and acts as a biological oxidant of soil organic matter. Phosphatase is an index of the mineralization potential of organic phosphorus and biological activity in the soil, and plays a vital role in the biochemical mineralization of organic phosphorus (Curyło & Telesiński 2020).

The findings of the current research demonstrated that there was no significant difference in the residual diesel between the wastewater treatments at the beginning of the growth period (for both diesel treatments). In contrast, a significant reduction was observed in the soil diesel content with freshwater treatments at the beginning of the growth period. It can be concluded that the wastewater reduced the removal of diesel in the lower soil layer. The results of the grass pea test in 2018 (Table 4) indicated that there was no significant difference between the second to the fifth sampling time in the topsoil of the D3-TWW-G treatment. In other words, the interaction of wastewater and the high level of diesel contamination in the soil decreased the phytoremediation ability, thereby disrupting the reduction of soil diesel content from 40 days after planting (DAP) onward. Unlike the top soil, the level of diesel significantly decreased in the bottom layer of the D3-TWW-G treatment from the second sampling onward. In the grass pea test in 2019, the soil diesel content slightly decreased in the topsoil layer, while no significant difference was observed in this between the sampling times. In the bottom soil, the reduction rate was faster, and a significant difference was seen between different sampling times (Table 4). Differences in the diesel removal of soil layers may be due to limited root growth in pot experiments, which causes a difference in root density in soil profiles.

In all the tests, tall fescue and grass pea could reduce diesel in the contaminated soil over time. At the beginning of the growth period, the amount of residual diesel in the soil decreased rapidly, while the diesel removal rates declined with time, especially after 70 days. Our results are similar to several findings (Tang et al. 2010; Fahid et al. 2020), which could be attributed to the presence of light hydrocarbons in the initial days, as well as the nutrient availability of the plants at the beginning of the growth period.

The interactive effects of the irrigation water and diesel concentration were evaluated using two-way ANOVA, and a significant difference was observed between the two concentrations of diesel in all the treatments during the same year, and with the same plants (Table 5). Therefore, it could be said that the changes in diesel concentrations were influenced by phytoremediation, which is similar to the findings of Ossai et al. (2020). Shen et al. (2018) and Thomas et al. (2017) have reported that plant root exudates act as a substrate for soil bacteria and increase the degradation of organic contaminants. The low removal rate of diesel has been attributed to the decreased rhizobacteria population. By comparison, it has been shown that increasing the concentration of diesel can inhibit plant growth due to the osmotic effects on plant cells and the reduction of dissolved oxygen (Al-Baldawi et al. 2015); this finding has been confirmed in other studies (Tang et al. 2010; Ikeura et al. 2019). Hydrocarbon compounds may cause phytotoxicity due to the volatile components that move through plant cells. Moreover, the hydrophobic properties of petroleum compounds hinder the absorption of the water, nutrients, and oxygen required by plants (Fernández et al. 2011; Hussain et al. 2018b; Taheri et al. 2018). Xi et al. (2018) reported that residual soil diesel content increased, while the removal ratio decreased at higher initial diesel concentrations.

In the present study, no significant difference was observed among the TWW and FW treatments at the end of the phytoremediation of the two plant species. The lowest concentration of contaminants remaining in the soil was found with tall fescue in both the irrigation treatments. Untreated or inadequately treated wastewater could be an abundant source of nitrogen, phosphorus, and potassium for crop production, providing high nutrient availability to improve plant growth and reduce the effects of contamination. The growth of the plants that are irrigated with treated wastewater depends on the degree of the treatment and nutrient concentrations in the wastewater. In the current research, the results of the statistical comparison of the two years of the experiment confirmed that the residual diesel in the soil differed significantly between 2018 and 2019 in some of the treatments (Table 5).

The ability of tall fescue and grass pea in the remediation of the diesel-contaminated soil was also investigated. The obtained results indicated that tall fescue was more effective in diesel removal in both years of the experiment. Similar studies have also noted that the efficacy of grasses is due to their deep and extensive root systems with high surface areas and adequate distribution of the root exudates, which provide a proper substrate for bacterial colonization. This type of root system increases the availability of nutrients and facilitates contamination uptake (Frank & Dugas 2001; Parrish et al. 2004; Muratova et al. 2008; Cook & Hesterberg 2013; Gkorezis et al. 2016; Rodriguez-Campos et al. 2019).

In a study in this regard, Wei et al. (2019) stated that tall fescue could remediate 41 and 25% of the soil polluted with 0.5 and 4% crude oil, respectively. According to the tall fescue experiments in the present study, the diesel removal rate significantly reduced with increased diesel concentration. Notably, the type of irrigation water caused no significant difference in the diesel removal at any of the diesel concentrations. The grass pea plant was also able to reduce soil contamination in the present study. Legumes are capable of harboring nitrogen-fixing bacteria and producing nitrogen for the plant, which is beneficial for the fertilization of oil-contaminated soils that have a high C:N ratio (Hall et al. 2011; Balliana et al. 2017).

In the 2018 grass pea experiment in the current research, no significant difference was observed between the treatments at 40 DAP, while the D1.5 and D3 treatments at 55, 70, and 85 DAP differed significantly. The removal rates of diesel in the D1.5-FW-G (2018), D1.5-TWW-G (2018), D3-FW-G (2018), and D3-TWW-G (2018) treatments were estimated at 44, 44, 31, and 34%, respectively. Similar to the 2018 grass pea test, a significant difference was observed between the D1.5 and D3 treatments at 70 and 85 DAP for the 2019 grass pea test. In addition, the diesel removal rates of the D1.5-FW-G (2019), D1.5-TWW-G (2019), D3-FW-G (2019), and D3-TWW-G (2019) treatments were calculated to be 37, 41, 30, and 29%, respectively. Contrary to expectations, no significant difference was denoted between the diesel removal of the FW and TWW treatments since the TWW used in our experiment had low concentrations of organic matter, nitrogen, phosphorus and potassium.

Soil contaminated with 3% diesel affected plant growth and the leaves (irrigated with TWW or FW) had a lighter color compared to the control treatment. No visual symptoms were observed in the D1.5 treatments. Chlorosis and the yellowing of plant leaves were the visible result of chlorophyll deficiency in the present study. According to the literature (Al-Hawas et al. 2012; Baruah et al. 2014; Olaranont et al. 2018), increased oil contamination in soil is associated with reduced plant chlorophyll. The leaf greenness index of alfalfa, bristle grass (Xie et al. 2018), and European beech (Fagus sylvatica L.) leaves (Bęś et al. 2019) has also been reported to decrease with increased contamination.

Since low concentrations of diesel are reserved in soil particles by capillary forces, the movement of water and nutrients may not be prevented in soil, and the damaging effects on the plant may be minimized (Adieze et al. 2012). There were no significant differences between the TWW and FW treatments, but this difference in D1.5 and D3 treatments was significant.

Observations showed that the plants had reached the flowering stage, although some plant leaves were dry due to stress, and the number of flowers in contaminated soils was less than in the non-contaminated treatments. At the end of the grass pea experiment (85 DAP), it was observed that some of the DC treatments reached the podding stage, but this was not observed in the D1.5 and D3 treatments. The findings of Merkl et al. (2005) confirm the delayed flowering and fruit ripening of plants exposed to oil contamination.

Segura et al. (2001) used wastewater and groundwater for melon irrigation and reported no significant differences in the plant growth and production parameters in various treatments. Christou et al. (2014) compared the impacts of irrigation with treated wastewater and fresh well water on tomato crop productivity. They found that irrigation with wastewater did not significantly affect crop productivity in comparison to well water.

In the 2019 tall fescue test in the present study and similar to the 2018 test, the dry matter in the D1.5 treatment was significantly heavier than the D3 treatment. Different from the 2018 test, the heaviest dry weight in the 2019 test was not achieved from the control treatment, and the highest yield was obtained in the D1.5 treatment.

Some studies have indicated that plant growth is better in soils with low contamination than in uncontaminated soils (Gaskin et al. 2008; Adieze et al. 2012). Sharifi et al. (2007) stated that low oil concentration in soil (1% w/w) could stimulate the growth of some plant species and increase the shoot height and weight of plants compared to treatment without oil. One of the reasons for proper plant growth in soils with low oil concentrations could be the presence of sulfur and its conversion into sulfate (S0₄) by thiobacillus bacteria so that it could be used by plants (Adieze et al. 2012).

In both tests of grass pea in the current research, the dry weight of the D1.5 treatment was significantly higher than the D3 treatment. In the uncontaminated soils, the dry weight of TWW was significantly higher than FW (Figure 4). Other studies have also confirmed increased plant growth following the use of wastewater (Bedbabis et al. 2010; Belhaj et al. 2016; Pham et al. 2018; Bakari et al. 2019; Tran et al. 2019).

Pearson's correlation coefficient was used to determine the correlations between plant height, plant aerial biomass, and diesel removal parameters during the 85 days of the experiment (Table 6). In the tall fescue test (2018), the diesel removal rate was significantly and positively correlated with plant height (r = 0.72–0.88; p < 0.01) and plant aerial biomass (r = 0.85–0.97; p < 0.01). Similar results were also observed in the 2019 tall fescue test, indicating that the removal rate of diesel from soil was positively correlated with plant height (r = 0.72–0.88; p < 0.01) and plant aerial biomass (r = 0.88–0.96; p < 0.01).

Strong and positive correlations were observed between diesel removal, plant aerial biomass, and plant height in the first test of the grass pea assessment (r = 0.87–0.96 for biomass, r = 0.77–0.85 for height; p < 0.01), and diesel removal had positive correlations with the plant aerial biomass and plant height in the second test (r = 0.84–0.95 for biomass, r = 0.81–0.92 for height; p < 0.01). Furthermore, the Pearson's correlation coefficient results indicated that the removal of diesel from soil was correlated with plant biomass and plant height, which is in congruence with the previous studies in this regard (Maqbool et al. 2012; Ikeura et al. 2019).

According to the results, grass pea and tall fescue have a tolerance potential to diesel concentrations of 1.5 and 3% (w/w). The comparison of the top and bottom soil layers indicated that more diesel was removed from the rhizospheric soil than the non-rhizospheric soil. Moreover, the comparison of the FW and TWW treatments showed that wastewater slowed down the soil diesel content remediation rate. The maximum diesel removal rate was 54% in tall fescue and 44% in grass pea, obtained in the D1.5 treatment. Assessment of shoot dry weight and plant height in various treatments also demonstrated that the increased concentration of diesel had a negative significant effect on plant growth. The tall fescue dry weight in the D1.5 treatment was significantly higher than in the other treatments, which may be due to the use of sulfur in the diesel of the plant or the stimulation of plant growth in low-dose diesel contaminated soil (Adieze et al. 2012).

The monitoring of visual symptoms indicated the yellow color of the plant leaves, slowed growth, delayed flowering, and podding in the D3 treatment compared to in the D1.5 and DC treatments. However, no significant difference was seen in the appearance of the FW and TWW treatments. Results demonstrated that the application of wastewater with low concentrations of organic matter, nitrogen, and phosphorus did not cause a significant increase in plant growth and remediation efficiency. Our findings indicated that the plant species used in this study were effective in soil phytoremediation. However, more studies are needed to decide if these plants are usable for phytoremediation of diesel contaminated soil at the field scale. Since the difference between freshwater and wastewater was not significant, we recommend using wastewater for phytoremediation purposes to prevent agricultural water shortages.

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

The authors declare there is no conflict of interest.