Removal E ﬃ ciencies of Constructed Wetland Planted with Phragmites and Vetiver in Treating Synthetic Wastewater Contaminated with High Concentration of PAHs

: This study aimed to evaluate the capability of horizontal subsurface ﬂow constructed wetlands (HSFCWs) in treating contaminated wastewater with a high concentration of polycyclic aromatic hydrocarbons (PAHs) (Phenanthrene, Pyrene, and Benzo[a]Pyrene), using two plants, namely Phragmites and Vetiver . The investigated parameters were (1) PAHs uptake by the plants, (2) PAHs removal e ﬃ ciencies, (3) accumulated PAHs in the soil of CWs, (4) shoot / root concentration factor, (5) translocation factor, and (6) PAHs correlation to lipid contains in the plants. During the treatment period, the results showed that the highest concentration of Phenanthrene in the shoot and the root systems of Phragmites , was 229.3 and192 µ g / g; Pyrene was 69.1 and 59.2 µ g / g; and Benzo[a]Pyrene 25.1 and 20.2 µ g / g, respectively. Meanwhile, in the Vetiver shoot and root systems were Phenanthrene 87.5 and 64.1 µ g / g; Pyrene 63.2 and 42.1 µ g / g; and Benzo[a]Pyrene 21.3 and 27.3 µ g / g, respectively. The removal rates of Phenanthrene, Pyrene, and Benzo[a]Pyrene (PAHs compounds) by the CW planted with Phragmites were found to be 83%, 71%, and 81%, respectively, while the removal rates by CW planted with Vetiver were found to be 67%, 66%, and 73%, respectively. Moreover, the removal rates by unplanted CW were found to be 62%, 58%, and 55%, respectively. The results indicated that the HSFCW planted with Phragmites has an e ﬀ ective pathway to remove high concentrations of PAHs.


Introduction
The polycyclic aromatic hydrocarbons (PAHs) are major wastewater pollutants discharged mainly from petroleum refineries and petrochemical industries. Due to their benzene structures, PAHs compounds could cause several health hazards even at very low concentrations, while mutagenicity, carcinogenicity, and teratogenicity are on the top list of diseases caused by these pollutants [1,2]. Consequently, there is an increasing concern among the public regarding the environmental fate of PAHs. Thus, effective and safe remediation strategies are needed urgently in order to remove the PAHs from industrial wastewater. The new green technologies are alternative methods for wastewater treatment, which include the removal of toxic PAHs pollutants, such as

Preparation of Synthetic Wastewater
In order to reflect the actual situation, synthetic wastewater was prepared by dissolving three compounds of PAHs, supplied by Sigma-Aldrich GmbH (Munich, Germany), in acetone and then mixed with distilled water. The selected compounds are typically found in PAHs and have different characteristics, as shown in Table 1. The percentage of the three compounds of PAHs in the synthetic wastewater were 74.61% for Phenanthrene, 17.11% for Pyrene, and 8.28% for Benzo[a]Pyrene, and they were prepared according to the earlier research studies [14,15].

Plant Sampling and Analysis
After the commencement of the experiments, as presented in Figure 2, plant samples were removed from the CWs on days 7, 14, 28, 42, and 72, for biomass measurement. The removed plants were replaced with plants cultivated under same conditions (same species, age, and irrigation water). After removal, the plants were thoroughly washed with tap water to remove attached sediments, rinsed with deionized water, and then dried, using absorbent paper [16]. Parts above ground and underground were manually separated. The height of the individual plant was recorded during the treatment period. The corresponding fractions were dried in an oven at 80 °C for 48 h, and the dry weights were recorded.

Preparation of Synthetic Wastewater
In order to reflect the actual situation, synthetic wastewater was prepared by dissolving three compounds of PAHs, supplied by Sigma-Aldrich GmbH (Munich, Germany), in acetone and then mixed with distilled water. The selected compounds are typically found in PAHs and have different characteristics, as shown in Table 1. The percentage of the three compounds of PAHs in the synthetic wastewater were 74.61% for Phenanthrene, 17.11% for Pyrene, and 8.28% for Benzo[a]Pyrene, and they were prepared according to the earlier research studies [14,15].

Preparation of Synthetic Wastewater
In order to reflect the actual situation, synthetic wastewater was prepared by dissolving three compounds of PAHs, supplied by Sigma-Aldrich GmbH (Munich, Germany), in acetone and then mixed with distilled water. The selected compounds are typically found in PAHs and have different characteristics, as shown in Table 1. The percentage of the three compounds of PAHs in the synthetic wastewater were 74.61% for Phenanthrene, 17.11% for Pyrene, and 8.28% for Benzo[a]Pyrene, and they were prepared according to the earlier research studies [14,15].

Plant Sampling and Analysis
After the commencement of the experiments, as presented in Figure 2, plant samples were removed from the CWs on days 7, 14, 28, 42, and 72, for biomass measurement. The removed plants were replaced with plants cultivated under same conditions (same species, age, and irrigation water). After removal, the plants were thoroughly washed with tap water to remove attached sediments, rinsed with deionized water, and then dried, using absorbent paper [16]. Parts above ground and underground were manually separated. The height of the individual plant was recorded during the treatment period. The corresponding fractions were dried in an oven at 80 °C for 48 h, and the dry weights were recorded.

Preparation of Synthetic Wastewater
In order to reflect the actual situation, synthetic wastewater was prepared by dissolving three compounds of PAHs, supplied by Sigma-Aldrich GmbH (Munich, Germany), in acetone and then mixed with distilled water. The selected compounds are typically found in PAHs and have different characteristics, as shown in Table 1. The percentage of the three compounds of PAHs in the synthetic wastewater were 74.61% for Phenanthrene, 17.11% for Pyrene, and 8.28% for Benzo[a]Pyrene, and they were prepared according to the earlier research studies [14,15].

Plant Sampling and Analysis
After the commencement of the experiments, as presented in Figure 2, plant samples were removed from the CWs on days 7, 14, 28, 42, and 72, for biomass measurement. The removed plants were replaced with plants cultivated under same conditions (same species, age, and irrigation water). After removal, the plants were thoroughly washed with tap water to remove attached sediments, rinsed with deionized water, and then dried, using absorbent paper [16]. Parts above ground and underground were manually separated. The height of the individual plant was recorded during the treatment period. The corresponding fractions were dried in an oven at 80 °C for 48 h, and the dry weights were recorded.

Preparation of Synthetic Wastewater
In order to reflect the actual situation, synthetic wastewater was prepared by dissolving three compounds of PAHs, supplied by Sigma-Aldrich GmbH (Munich, Germany), in acetone and then mixed with distilled water. The selected compounds are typically found in PAHs and have different characteristics, as shown in Table 1. The percentage of the three compounds of PAHs in the synthetic wastewater were 74.61% for Phenanthrene, 17.11% for Pyrene, and 8.28% for Benzo[a]Pyrene, and they were prepared according to the earlier research studies [14,15].

Plant Sampling and Analysis
After the commencement of the experiments, as presented in Figure 2, plant samples were removed from the CWs on days 7, 14, 28, 42, and 72, for biomass measurement. The removed plants were replaced with plants cultivated under same conditions (same species, age, and irrigation water). After removal, the plants were thoroughly washed with tap water to remove attached sediments, rinsed with deionized water, and then dried, using absorbent paper [16]. Parts above ground and underground were manually separated. The height of the individual plant was recorded during the treatment period. The corresponding fractions were dried in an oven at 80 °C for 48 h, and the dry weights were recorded.

Relative Growth Rate
The relative growth rate (RGR) was based on total dry biomass, and it was calculated by using Equation (1) [17].
where RGR is the relative growth rate (g/plant/day); w1 and w2 are the primary and final dry weights, respectively, for a complete plant sample; and t1 and t2 are the treatment times in days.

PAHs Extraction
Liquid-liquid extraction method was used to extract PAHs from wastewater [18]. The process involved 100 mL of wastewater sample extracted with 30 mL of dichloromethane, and then poured into a separator funnel. The mixture was shaken vigorously for two minutes before two-phase separation. The extract was combined, filtered, and dried with concentrated anhydrous sodium sulfate by rotary evaporation at 40 °C. After removing the solvents, the residue obtained was dissolved in 2 mL of acetone and filtered through 0.45 mm polytetrafluoroethylene (PTFE) filters, and then injected for Gas Chromatography-Flame Ionization Detector (GC-FID) analysis.
The uptake of PAHs in plants was investigated by using the procedure as reported by previous research [19]. The samples of plants were dried at room temperature, before being grounded, homogenized, and sieved through a sieve No. 200 μm. Specifically, 1 g (dry weight) of the plant samples was extracted by ultra-sonication in 1:1 (v/v) resolution and mixed with dichloromethane and acetone. The solution was then decanted, collected, and replenished. The sample solution was sonicated for 1 h, and the solution was decanted, collected, and replenished again. This exact procedure was repeated three times.
The soil samples were dried at room temperature, until they reached a constant weight and sieved with a 200 μm sieve. The homogenized samples (2.0 g each) were extracted by ultra-sonication in 1:1 (v/v) resolution, of mixed dichloromethane and acetone. The solution was then decanted, collected and replenished. The sample solution was sonicated for 1 h, and the solution was decanted, collected, and replenished again. This procedure was also repeated for three times. The subsequent plant and soil samples concentration and cleansing procedures were similar as implemented for the wastewater samples.

Analysis of PAHs by GC-FID
The sample extracts (2 μL) were analyzed by using Gas Chromatography (model Hewlett-Packard (HP) 6890), equipped with a HP5973 mass selective detector and a DB-5 capillary column (30 mm × 0.25 mm × 0.25 m). The separation was achieved according to the following program: the initial oven temperature was 80 °C (held for one minute), and increased to 275 °C at 15 °C/min, held

Relative Growth Rate
The relative growth rate (RGR) was based on total dry biomass, and it was calculated by using Equation (1) [17].
where RGR is the relative growth rate (g/plant/day); w 1 and w 2 are the primary and final dry weights, respectively, for a complete plant sample; and t 1 and t 2 are the treatment times in days.

PAHs Extraction
Liquid-liquid extraction method was used to extract PAHs from wastewater [18]. The process involved 100 mL of wastewater sample extracted with 30 mL of dichloromethane, and then poured into a separator funnel. The mixture was shaken vigorously for two minutes before two-phase separation. The extract was combined, filtered, and dried with concentrated anhydrous sodium sulfate by rotary evaporation at 40 • C. After removing the solvents, the residue obtained was dissolved in 2 mL of acetone and filtered through 0.45 mm polytetrafluoroethylene (PTFE) filters, and then injected for Gas Chromatography-Flame Ionization Detector (GC-FID) analysis.
The uptake of PAHs in plants was investigated by using the procedure as reported by previous research [19]. The samples of plants were dried at room temperature, before being grounded, homogenized, and sieved through a sieve No. 200 µm. Specifically, 1 g (dry weight) of the plant samples was extracted by ultra-sonication in 1:1 (v/v) resolution and mixed with dichloromethane and acetone. The solution was then decanted, collected, and replenished. The sample solution was sonicated for 1 h, and the solution was decanted, collected, and replenished again. This exact procedure was repeated three times.
The soil samples were dried at room temperature, until they reached a constant weight and sieved with a 200 µm sieve. The homogenized samples (2.0 g each) were extracted by ultra-sonication in 1:1 (v/v) resolution, of mixed dichloromethane and acetone. The solution was then decanted, collected and replenished. The sample solution was sonicated for 1 h, and the solution was decanted, collected, and replenished again. This procedure was also repeated for three times. The subsequent plant and soil samples concentration and cleansing procedures were similar as implemented for the wastewater samples.

Analysis of PAHs by GC-FID
The sample extracts (2 µL) were analyzed by using Gas Chromatography (model Hewlett-Packard (HP) 6890), equipped with a HP5973 mass selective detector and a DB-5 capillary column (30 mm × 0.25 mm × 0.25 m). The separation was achieved according to the following program: the initial oven temperature was 80 • C (held for one minute), and increased to 275 • C at 15 • C/min, held for one Sustainability 2020, 12, 3357 5 of 18 minute; then to 285 • C at 10 • C/min, held for one minute; and then increased to 295 • C at 5 • C/min, held for one minute. During the process, nitrogen was used as a carrier gas (1.5 mL/min) and makeup gas (35 mL/min). A 1.0 L aliquot of the extract was injected into the splitless mode. The injector was held at 250 • C and the detector at 300 • C [20].

Plants Growth with PAHs Contaminant
The plant's height is a general criterion indicating plant growth and also an important trait which reflects their growth and adaptive characteristics. The result of plant height in the constructed wetlands is illustrated in Figure 3. Plant height increased with the increased retention time, resulting in positive growth rates reflecting the ability of Phragmites and Vetiver to tolerate contaminated effluent with PAHs. For the period of the experiments, the heights of Phragmites and Vetiver were ranged from 129 to 143 cm and from 87 to 119 cm, respectively. Significant differences in heights between the two communities were found for both plants; F (287, 83) = 287.076; p = 0.005; Wilks' Λ = 0.126. for one minute; then to 285 °C at 10 °C/min, held for one minute; and then increased to 295 °C at 5 °C/min, held for one minute. During the process, nitrogen was used as a carrier gas (1.5 mL/min) and makeup gas (35 mL/min). A 1.0 L aliquot of the extract was injected into the splitless mode. The injector was held at 250 °C and the detector at 300 °C [20].

Plants Growth with PAHs Contaminant
The plant's height is a general criterion indicating plant growth and also an important trait which reflects their growth and adaptive characteristics. The result of plant height in the constructed wetlands is illustrated in Figure 3. Plant height increased with the increased retention time, resulting in positive growth rates reflecting the ability of Phragmites and Vetiver to tolerate contaminated effluent with PAHs. For the period of the experiments, the heights of Phragmites and Vetiver were ranged from 129 to 143 cm and from 87 to 119 cm, respectively. Significant differences in heights between the two communities were found for both plants; F (287, 83) = 287.076; p = 0.005; Wilks' Λ = 0.126. Based on the comparison between Phragmites and Vetiver plants grown in the CWs, it was found that the height of the Phragmites at the beginning was higher than Vetiver. The increase in the Vetiver height ratio was higher than Phragmites (44% and 19%). A comparison which was made for each plant with control plant of the same type had revealed that the growth trend is very close and the difference between the Phragmites and Vetiver is due to the different morphology of each plant. A similar trend was observed by other researchers as well [21,22]. An increase in plant height can lead to the presence of large amounts of biomass, which correspond to the significant removal of PAHs from liquid waste. Continuous increment in plant height of the CW indicates the ability of the plant species for shoot elongation and production of larger biomass.
Biomass response varied considerably among species. In this study, significant differences were observed between the two types of wetland plants in the biomass of shoots and root systems, and the magnitudes of the differences, were increased with increasing retention time, as shown in Figure 4. Meanwhile, the largest dry weights of the shoot and root systems of Phragmites were ranged from 28.7 to 45.1 and 18.1 to 27.5 g/plant, respectively. The dry biomasses of Vetiver shoot and root systems ranged from 15.3 to 18.1 and 9.6 to 12.3 g/plant, respectively. Meanwhile the comparison between shoot and root biomass to control plant observed low biomass in two parts of the plants during retention time. Perhaps this was caused by the toxic contaminants restrain on the proliferation of plant roots, its vitality, and microbes in the rhizosphere [23]. Moreover, a 4% decrease in root biomass Based on the comparison between Phragmites and Vetiver plants grown in the CWs, it was found that the height of the Phragmites at the beginning was higher than Vetiver. The increase in the Vetiver height ratio was higher than Phragmites (44% and 19%). A comparison which was made for each plant with control plant of the same type had revealed that the growth trend is very close and the difference between the Phragmites and Vetiver is due to the different morphology of each plant. A similar trend was observed by other researchers as well [21,22]. An increase in plant height can lead to the presence of large amounts of biomass, which correspond to the significant removal of PAHs from liquid waste. Continuous increment in plant height of the CW indicates the ability of the plant species for shoot elongation and production of larger biomass.
Biomass response varied considerably among species. In this study, significant differences were observed between the two types of wetland plants in the biomass of shoots and root systems, and the magnitudes of the differences, were increased with increasing retention time, as shown in Figure 4. Meanwhile, the largest dry weights of the shoot and root systems of Phragmites were ranged from 28.7 to 45.1 and 18.1 to 27.5 g/plant, respectively. The dry biomasses of Vetiver shoot and root systems ranged from 15.3 to 18.1 and 9.6 to 12.3 g/plant, respectively. Meanwhile the comparison between shoot and root biomass to control plant observed low biomass in two parts of the plants during retention time. Perhaps this was caused by the toxic contaminants restrain on the proliferation of plant roots, its vitality, and microbes in the rhizosphere [23]. Moreover, a 4% decrease in root biomass was seen in the root of F. arundinacea after mixing the PAH treatment [24]. Gao et al. [25] reported that PAH degradation was enhanced at the early stage and decreased as root exudates depleted. was seen in the root of F. arundinacea after mixing the PAH treatment [24]. Gao et al. [25] reported that PAH degradation was enhanced at the early stage and decreased as root exudates depleted.
(a) (b) The variation in morphology of Phragmites and Vetiver led to a distinct difference in the biomass of these plants, where the Phragmites was reported to be three times greater than Vetiver. Moreover, a clear difference could also be found between the biomass of both plants used in the treatment of PAHs with high concentrations and the biomass of control plant. The possible reasons for the low biomass growth ratio of the two plants compared to control plants are due to the effect of the high concentration of PAHs. Wang et al. [23] found that, when exposed to higher concentrations of diesel, reed plants would inhibit the synthesis of chlorophyll, thus reducing chlorophyll content in plants, as well as photosynthesis and inhibiting plant growth.
Based on the findings, it can be concluded that Phragmites and Vetiver plants were more tolerant to high concentration of PAHs 10,000 μg/L, as the biomass of the plants continued to increase during the retention time, albeit in small proportions of Phragmites and Vetiver plants, which may possess traits that enable them to cope with oxygen limitation in the root zone and also under unfavorable biochemical reduction processes. Additionally, interactions between the plant genotype and microorganism may cause significant differences in plant growth, for example, the effect on root morphology and microbial activity. These interactions may have positive or negative effects on the use of plant nutrients and metabolism [26]. Genes related to the degradation of hydrocarbons were generally expressed in the root zone contaminated with PAH compared to bulk soil, which could help to improve microorganisms associated with plant treatment systems [27].
The relative growth rate (RGR) of plants is a major component of fitness. In theory, the RGR of plants would be closely related to biomass allocation. The (RGR) of the shoot and root system of Phragmites and Vetiver in a constructed wetland for treating PAHs with high concentrations (10,000 μg/L) is shown in Figure 5. The results show that the RGR of both plants were affected by the high concentration of PAHs, where the RGR of both plants exhibited a lower outcome, which did not exceed 0.02 g. Phragmites shoot and root system reached the highest relative growth rate (0.019 and 0.016 g/d) at 14 days, and then gradually began to decrease toward the end of the treatment period. Andreolli et al. [28] observed a decrease in biomass, stem length, and dry root weight of PAH-treated plants when compared to the untreated ones. On the other hand, the RGR of Vetiver in the shoot and root systems was significantly lower than in the control plant, with the highest growth rate of the shoot system at seven days with 0.014 gd −1 and the highest growth rate in the root system at 14 days with 0.013 g/d. The variation in morphology of Phragmites and Vetiver led to a distinct difference in the biomass of these plants, where the Phragmites was reported to be three times greater than Vetiver. Moreover, a clear difference could also be found between the biomass of both plants used in the treatment of PAHs with high concentrations and the biomass of control plant. The possible reasons for the low biomass growth ratio of the two plants compared to control plants are due to the effect of the high concentration of PAHs. Wang et al. [23] found that, when exposed to higher concentrations of diesel, reed plants would inhibit the synthesis of chlorophyll, thus reducing chlorophyll content in plants, as well as photosynthesis and inhibiting plant growth.
Based on the findings, it can be concluded that Phragmites and Vetiver plants were more tolerant to high concentration of PAHs 10,000 µg/L, as the biomass of the plants continued to increase during the retention time, albeit in small proportions of Phragmites and Vetiver plants, which may possess traits that enable them to cope with oxygen limitation in the root zone and also under unfavorable biochemical reduction processes. Additionally, interactions between the plant genotype and microorganism may cause significant differences in plant growth, for example, the effect on root morphology and microbial activity. These interactions may have positive or negative effects on the use of plant nutrients and metabolism [26]. Genes related to the degradation of hydrocarbons were generally expressed in the root zone contaminated with PAH compared to bulk soil, which could help to improve microorganisms associated with plant treatment systems [27].
The relative growth rate (RGR) of plants is a major component of fitness. In theory, the RGR of plants would be closely related to biomass allocation. The (RGR) of the shoot and root system of Phragmites and Vetiver in a constructed wetland for treating PAHs with high concentrations (10,000 µg/L) is shown in Figure 5. The results show that the RGR of both plants were affected by the high concentration of PAHs, where the RGR of both plants exhibited a lower outcome, which did not exceed 0.02 g. Phragmites shoot and root system reached the highest relative growth rate (0.019 and 0.016 g/d) at 14 days, and then gradually began to decrease toward the end of the treatment period. Andreolli et al. [28] observed a decrease in biomass, stem length, and dry root weight of PAH-treated plants when compared to the untreated ones. On the other hand, the RGR of Vetiver in the shoot and root systems was significantly lower than in the control plant, with the highest growth rate of the shoot system at seven days with 0.014 gd −1 and the highest growth rate in the root system at 14 days with 0.013 g/d.

Concentration of PAHs in Phragmites and Vetiver Plant Species
The concentrations of the three compounds of PAHs in the selected plant species are presented in Figure 6. It was found that the concentrations in both plants had increased, and after a certain time, it started to decrease. The highest concentration of total Phenanthrene was obtained in the shoot system of Phragmites (Figure 6a), with an average value of 229.3 μg/g, and was in the root of the same plant with 192 μg/g. Meanwhile the highest concentration of Phenanthrene in the shoots and roots system of Vetiver (Figure 6b) was 87.5 and 64.1 μg/g, respectively.

Concentration of PAHs in Phragmites and Vetiver Plant Species
The concentrations of the three compounds of PAHs in the selected plant species are presented in Figure 6. It was found that the concentrations in both plants had increased, and after a certain time, it started to decrease. The highest concentration of total Phenanthrene was obtained in the shoot system of Phragmites (Figure 6a), with an average value of 229.3 µg/g, and was in the root of the same plant with 192 µg/g. Meanwhile the highest concentration of Phenanthrene in the shoots and roots system of Vetiver (Figure 6b) was 87.5 and 64.1 µg/g, respectively.

Concentration of PAHs in Phragmites and Vetiver Plant Species
The concentrations of the three compounds of PAHs in the selected plant species are presented in Figure 6. It was found that the concentrations in both plants had increased, and after a certain time, it started to decrease. The highest concentration of total Phenanthrene was obtained in the shoot system of Phragmites (Figure 6a), with an average value of 229.3 µg/g, and was in the root of the same plant with 192 µg/g. Meanwhile the highest concentration of Phenanthrene in the shoots and roots system of Vetiver (Figure 6b) was 87.5 and 64.1 µg/g, respectively. (e) (f) The concentration of five hydrocarbons in the shoot system of Phragmites (Figure 6e) was 25.1 μg/g which was higher than the root system at 20.2 μg/g. In contrast, the concentration of B[a]P in the Vetiver (Figure 6f) shoot system was 21.3 μg/g and is lower than the root system of the same plant recorded at 27.3 μg/g. Sun and Zhou [29] also found that concentrations of B[a]P in the roots of two plants were higher than those in the aerial parts (stems, leaves, and shoots). Several studies demonstrated that the plants have a high ability to uptake Phenanthrene than Pyrene and B[a]P [29,30].
The characteristics of Phe compound, such as low molecular weight, high solubility in most lowpolar organic solvents, and high presence in synthetic wastewater in constructed wetlands, enable the uptake and degradation of these compounds in larger quantities. The Phragmites plants have high uptake ability compared to the Vetiver plants, due to the presence of strong rhizosphere. Binet et al. [31] found that phytoremediation of PAHs depends on the beneficial effects of plant rhizosphere on their dissipation. Under the influence of roots, the bioavailability of organic compounds can be increased in the soil of the rhizosphere through the activation of organic compounds derived from root secretions and the degradation of soil organic matter. Thus, these active organic chemicals can be absorbed by plant roots and transported to the top of the shoots [32]. However, it is difficult to compare the PAHs uptake of this study to other studies because most of the results obtained involved the PAHs uptake from media such as (soil/water),which has a low contamination rate, for example, the initial concentration of Pyrene was 200 mg/kg [33]; the sum of 16 PAHs in the influent was 786 ± 514 ng/L [9]; the average PAHs content of the raw sludge measured was 5.69 mg/kg [13], and the initial Phenanthrene concentration was 200 mg/kg [34].
The results of this study confirm that the ability of plants used in CW to absorb PAHs is highly depending on the PAHs concentration in the media in which the plants grow and also retention time [35,36]. For examples, the total concentration of 16 PAHs varied between 32 and 42 μg/kgd.m, and the accumulation of USEPA categorized 16 PAHs, which were examined in different parts of Glyceria maxima plants, and observed that PAHs in root, leaves, and stems were 85, 143, and 92 μg/kgd.m, respectively [36]. According to Sieciechowicz et al. [37], they found that the average content of 15 PAHs in the soil did not exceeded 700 μg/kgd.m, while the range in the leaves was 112-118 μg/kgd.m, which was found, on average, to be twice as high as that in stems, at 49-59 μg/kgd.m, and roots, at 65-69 μg/kgd.m. Plants may metabolize hydrocarbons in the common metabolic destinies, as well as The concentration of five hydrocarbons in the shoot system of Phragmites (Figure 6e) was 25.1 µg/g which was higher than the root system at 20.2 µg/g. In contrast, the concentration of B[a]P in the Vetiver (Figure 6f) shoot system was 21.3 µg/g and is lower than the root system of the same plant recorded at 27.3 µg/g. Sun and Zhou [29] also found that concentrations of B[a]P in the roots of two plants were higher than those in the aerial parts (stems, leaves, and shoots). Several studies demonstrated that the plants have a high ability to uptake Phenanthrene than Pyrene and B[a]P [29,30].
The characteristics of Phe compound, such as low molecular weight, high solubility in most low-polar organic solvents, and high presence in synthetic wastewater in constructed wetlands, enable the uptake and degradation of these compounds in larger quantities. The Phragmites plants have high uptake ability compared to the Vetiver plants, due to the presence of strong rhizosphere. Binet et al. [31] found that phytoremediation of PAHs depends on the beneficial effects of plant rhizosphere on their dissipation. Under the influence of roots, the bioavailability of organic compounds can be increased in the soil of the rhizosphere through the activation of organic compounds derived from root secretions and the degradation of soil organic matter. Thus, these active organic chemicals can be absorbed by plant roots and transported to the top of the shoots [32]. However, it is difficult to compare the PAHs uptake of this study to other studies because most of the results obtained involved the PAHs uptake from media such as (soil/water),which has a low contamination rate, for example, the initial concentration of Pyrene was 200 mg/kg [33]; the sum of 16 PAHs in the influent was 786 ± 514 ng/L [9]; the average PAHs content of the raw sludge measured was 5.69 mg/kg [13], and the initial Phenanthrene concentration was 200 mg/kg [34].
The results of this study confirm that the ability of plants used in CW to absorb PAHs is highly depending on the PAHs concentration in the media in which the plants grow and also retention time [35,36]. For examples, the total concentration of 16 PAHs varied between 32 and 42 µg/kg d.m , and the accumulation of USEPA categorized 16 PAHs, which were examined in different parts of Glyceria maxima plants, and observed that PAHs in root, leaves, and stems were 85, 143, and 92 µg/kg d.m , respectively [36]. According to Sieciechowicz et al. [37], they found that the average content of 15 PAHs in the soil did not exceeded 700 µg/kg d.m , while the range in the leaves was 112-118 µg/kg d.m , which was found, on average, to be twice as high as that in stems, at 49-59 µg/kg d.m , and roots, at 65-69 µg/kg d.m . Plants may metabolize hydrocarbons in the common metabolic destinies, as well as reactive Sustainability 2020, 12, 3357 9 of 18 products that may accumulate in tissues, e.g., roots. Predestination is possible if hydrocarbons are available in roots and migrate across the stem [36,38,39].

Assessment of PAH Removal Efficacy
The removal of three PAHs compounds from three CW systems is presented in Figure 7. The results clearly indicate that the removal rates of PAHs decreased with an increase in time in all CW systems. The results suggested that the removal degradation efficiencies were better and faster in CWs with plants.
The level of removal of phenanthrene in CWs planted with Phragmites ranged up to 91% at the beginning of treatment, and then it declined slowly, to 83%, by the end of treatment. The average removal efficiency of Phenanthrene in CW planted with Vetiver ranged from the first retention time to the end of treatment between 80% and 67%. Meanwhile, unplanted systems reached the removal efficiency for Phenanthrene between 76% and 62%, which was the lowest removal efficiency when compared to other systems. Ansari [40] found that, in the case of soil planted with B. juncea, Phenanthrene showed the highest degradations, at 88%, when compared to other PAH compounds, and most PAH degradation rates increased by 20-40%, compared to those in uncultivated soil.

Assessment of PAH Removal Efficacy
The removal of three PAHs compounds from three CW systems is presented in Figure 7. The results clearly indicate that the removal rates of PAHs decreased with an increase in time in all CW systems. The results suggested that the removal degradation efficiencies were better and faster in CWs with plants.
The level of removal of phenanthrene in CWs planted with Phragmites ranged up to 91% at the beginning of treatment, and then it declined slowly, to 83%, by the end of treatment. The average removal efficiency of Phenanthrene in CW planted with Vetiver ranged from the first retention time to the end of treatment between 80% and 67%. Meanwhile, unplanted systems reached the removal efficiency for Phenanthrene between 76% and 62%, which was the lowest removal efficiency when compared to other systems. Ansari [40] found that, in the case of soil planted with B. juncea, Phenanthrene showed the highest degradations, at 88%, when compared to other PAH compounds, and most PAH degradation rates increased by 20%-40%, compared to those in uncultivated soil. The Pyrene removal capacity of CWs planted with Phragmites, Vetiver, and unplanted system ranged from 87% to 71%, 72% to 66%, and 69% to 58%, respectively, with a significant difference (p < 0.05) between sampling days 7 and 72. The four-rings PAHs had low removal rates and proved to be more difficult to biodegrade than the three-ring PAHs. For this reason, the three-ring PAHs were preferred for the removal process, as indicated by Braeckevelt et al. [41]. The removal percentages of Benzo[a]Pyrene in CW with planted with Phragmites, Vetiver, and unplanted were 81%-86%, 73%- The Pyrene removal capacity of CWs planted with Phragmites, Vetiver, and unplanted system ranged from 87% to 71%, 72% to 66%, and 69% to 58%, respectively, with a significant difference (p < 0.05) between sampling days 7 and 72. The four-rings PAHs had low removal rates and proved to be more difficult to biodegrade than the three-ring PAHs. For this reason, the three-ring PAHs were preferred for the removal process, as indicated by Braeckevelt et al. [41]. The removal percentages of Benzo[a]Pyrene in CW with planted with Phragmites, Vetiver, and unplanted were 81-86%, 73-80%, and 55-64% respectively. The differences in reduction of PAHs between with and without plant were statistically significant (p < 0.05).
Based on the results, it was observed that lower PAHs removal was found in unplanted CWs as compared to planted CWs. This indicated that the presence of plants can facilitate the removal of PAHs from unplanted CW, which contained more roots, shoot biomass, and microorganisms that had boosted the treatment efficiency and believed to occur through a rhizosphere effect [42]. Furthermore, the flow in the SSF system is horizontal through the upheld media and includes aerobic, anoxic, and anaerobic zones. The aerobic zone is located around the roots that provide oxygen to the substrate zone [43].
Fountoulakis et al. [9] recorded that the average efficiency of PAHs removal was 79.2%, 68.2%, and 73.3% for SSF, FWS, and GF, respectively. Terzakis et al. [44] examined two FWS and two SSF pilot-size constructed wetlands treating highway runoff and found a comprehensive removal of 16 PAHs of 59%. Machate et al. [45] studied synthetic wastewater containing Phenanthrene, which was treated in the horizontal-ertical flow macrophytic-based treatment system and showed a 99.9% removal ratio.
Generally, treatments with different plant species will display different dissipation ratios. In this study, the performance of constructed wetland was clearly better with Phragmites. Fraser et al. [46] observed that Phragmites has a resilient rhizome system with high propagation ability, long growth period, strong adaptability, and strong resistance to pollution. This result is consistent with the findings obtained by Huang et al.'s study [47] in which they reported an improvement in the removal efficiency of Cu, Zn, Pb, and Cr compounds existing in the treated wastewater. Ballesteros et al. [48] concluded from their investigation that the CW system planted with Phragmites Kaka exhibited excellent and nearly complete removals, at an influent concentration level of 45 mg/L, suggesting that toxic and inhibitory effects depend on the system and that these effects were absent for this particular system at this contaminant level.

Accumulation of PAHs in the Soil of Constructed Wetland
Stabilization and adsorption onto substrates are considered to be the most potential removal processes for PAHs. The accumulation of Phenanthrene, Pyrene, and Benzo[a]Pyrene in the CWs soil is shown in Figure 8. Phenanthrene recorded the highest accumulated concentration in the soil, compared to other compounds, owing to the highest ratio in the influent concentrations. Same trend was observed by Zhang et al. [49]. As observed, the accumulation of Phe in two planted CWs was significantly less than unplanted CWs, giving Phragmites 2.9-12.4 µg/g, Vetiver 3.7-16.9 µg/g, and unplanted system 5.5-19.5 µg/g. Plant roots provide readily degradable carbon, which is energy that generally increases microbial activity in the soil, which may lead to further degradation of organic pollutants through direct metabolism or a combined metabolism [50,51]. A similar trend was observed for accumulation of B[a]P in the unplanted CWs soil, which ranged from 0.47 to 1.02 μg/g which was significantly greater than the B[a]P in the CWs soil planted with Phragmites (0.26-0.65 μg/g) and accumulation of B[a]P in CWs soil planted with Vetiver (0.35-0.82 μg/g). The remediation for B[a]P required long periods of degradation due to its less water solubility and the difficulty in controlling the conditions, thus preventing degradation [52].
It is worth noting that, in light of the corresponding experiences, there was a significant difference between the concentrations of the three PAHs compounds in the soils of both unplanted CWs and planted CWs with Phragmites and Vetiver. The different accumulation between the three compounds of PAHs in CWs soil was probably related to different initial concentrations of these The accumulation of Pyrene in soils, especially in unplanted CW from 1.4 to 4.7 µg/g significantly increased with increment time of treatment. Meanwhile the low accumulation was observed in CW soil planted with Phragmites during the retention time, which ranged from 0.3 to 2.1 µg/g, as compared to the Pyrene concentration in the CW soil planted with Vetiver at 0.8-3.1 µg/g. The Pyrene was more persistent in unplanted CW soils than planted CWs, due to the presence of plants that improve the soil structure and thus ventilation and hydrological properties.
A similar trend was observed for accumulation of B[a]P in the unplanted CWs soil, which ranged from 0.47 to 1.02 µg/g which was significantly greater than the B[a]P in the CWs soil planted with Phragmites (0.26-0.65 µg/g) and accumulation of B[a]P in CWs soil planted with Vetiver (0.35-0.82 µg/g). The remediation for B[a]P required long periods of degradation due to its less water solubility and the difficulty in controlling the conditions, thus preventing degradation [52].
It is worth noting that, in light of the corresponding experiences, there was a significant difference between the concentrations of the three PAHs compounds in the soils of both unplanted CWs and planted CWs with Phragmites and Vetiver. The different accumulation between the three compounds of PAHs in CWs soil was probably related to different initial concentrations of these compounds, which have been identified depending on their presence in industrial wastewater, and due to the different characteristics of each compound. The low molecular weight of Phe could increase its movement under saturated conditions in the CWs, considering that water solubility is an important factor influencing PAH mobility in CWs [53]. Meanwhile, Pyr and B[a]P with four and five rings were prevalent in the particle stage and can generally be deposited in increasing proportions [54]. According to Gao and Zhu [55] and Huesemann et al. [11], the accumulated PAHs in soils that contain high octanol-water partition coefficients (K ow ) can be related to the soil organic matter, which makes it not readily available for uptake by the root. The increased dissipation of PAHs in planted CWs versus unplanted CWs soils can be attributed to plant uptake and plant biodegradation. Cottin et al. [56] found that the organic matter accumulated on the top of aged VFCW had a great adsorption capacity and could degrade PAHs with slow biodegradation kinetics.

Bioconcentration Factor and Translocation Ability of PAHs
Plants' uptake of Phe, Pyr, and B[a]P were evaluated by calculating SCF and RCF [57], as presented in Tables 2 and 3. In this study, the SCF and RCF ranged from 0.02 to 0.03 and 0.01 to 0.03 for Phe in the Phragmites, while in Vetiver, they ranged from 0.01 to 0.01 and 0.00 to 0.01, respectively, suggesting that accumulation of Phe in Phragmites was greater than those observed in Vetiver and SCF was higher than RCF. The high SCF values may be due to relatively lower molecular weight and higher water solubility of Phe.
Pyrene varied significantly among different plant species. The range of SCF and RCF for Pyrene in Phragmites were found to be 0.02-0.04 and 0.02-0.04, respectively, while in Vetiver, their ranges were found to be 0.02-0.04 and 0.01-0.02, respectively. Based on the results shown in Tables 2 and 3, the SCF and RCF of the Phragmites had recorded high values of Pyrene when compared to Vetiver. The higher values in Phragmites may be due to plant ability. Cui et al. [13] found that P. Australis accumulated PAHs effectively compared with native plants, and the contents of middle-MW and high-MW PAHs in leaves were much higher than those in roots and stems.
The SCF and RCF of the B[a]P were the highest in the Vetiver plants, ranging from 0.04 to 0.05 and 0.06 to 0.07, while the lowest values were in the Phragmites plants, ranging from 0.02 to 0.03 and 0.01 to 0.02, respectively. The Vetiver was characterized by a higher ability to accumulate the B[a]P than Phragmites; therefore, the RCF of the Vetiver was higher compared to SCF. The same trend was observed in the study by Sivaram et al. [58], which found that the SCF and RCF of Benzo[a]Pyrene in Vetiver were 0.002 and 0.08, respectively. Sun et al. [29] likewise recorded the concentrations of B[a]P in the roots of two plants, where they were found to be greater than those in aerial parts.  As derived from the results, the values of SCF for Phenanthrene and Pyrene compounds were higher when compared with Benzo[a] Pyrene compound. Moreover, the levels of the Phenanthrene and Pyrene compounds in the shoot and root of Phragmites were greater than those in Vetiver plants. In contrast, the concentration of Benzo[a]Pyrene in the Vetiver was higher than that in the Phragmites. Differences in the biomass of the shoot and root system among plant cultivars are potential reasons for the variation in SCF and RCF, which have a direct contribution to PAHs degradation, as reported Huang et al. [59].
The translocation factor (TF) is used to analyze the ability of plants to transport PAHs from the root to shoot system of plants. The TF values for three PAHs in all treatments were higher than 1, except B[a]P in Vetiver, as i was less than 1. Higher values of TF indicate better translocation capacity of PAHs from the roots to shoot tissues. Sun et al. [20] also noted that the TF was used to evaluate the effectiveness of a plant in transferring a chemical from soil to roots and from roots to plant parts. The values of Phenanthrene in both species (Phragmites and Vetiver) were compared with the other two compounds, and it was found that it had gradually increased with the retention time in Phragmites, while it cautiously decreased with the retention time in Vetiver, which was found to be 1.19-1.50 and 1.43-1.70, respectively. The TF value of Pyrene in both plants declined with increasing retention time and found to be in both plants, 1.16-1.25 and 1.35-1.59, respectively. It was observed that the RCF of Phenanthrene was much higher than that of Pyrene; RCF for Phenanthrene uptake by ryegrass was greatly smaller than RCF for Pyrene uptake. The higher K ow value of Pyrene than that of Phenanthrene could be on account of the observed results [30].
TF values for Benzo[a]Pyrene varied between plants, where values slowly decreased over time in Phragmites, from 1.73 to 1.23, while in Vetiver, TF was <1.0 and increased significantly, from 0.65 to 0.71. A similar trend was observed by Sun et al. [20], as they found that the value of TF in most treatments was less than 1.0. Low TFs indicated that T. patula and M. jalapa took up B[a]P from the soil and transferred it to the aboveground parts, with lower efficiency with increasing time. The difference in variation trends between the three PAHs compounds may be attributed to the physical and chemical properties of these compounds [60].

Impact of Lipid Content on PAHs Accumulation
The results of the correlation between three PAHs compounds and lipid content in Phragmites and Vetiver plants are shown in Figure 9. Schober et al. explain the correlation coefficients are translated into descriptors. A correlation between two variables is considered negligible if the absolute value of the correlation is between 0 and 0.10; weak correlation ranges between 0.10 and 0.39; moderate correlation is between 0.40 and 0.69; strong correlation is between 0.70 and 0.89; and very strong correlation is between 0.90 and 1.0 [61]. The different lipid content of plant tissue may influence the PAHs distribution between plant tissues after uptake by the plant. The correlation coefficient between Phenanthrene concentration and lipid content was strong when Phragmites was used with r 2 = 0.7595. Meanwhile, the same compound had a moderate correlation coefficient with lipid content in Vetiver with r 2 = 0.5839. For hydrophobic substances, such as PAHs, the root uptake is evident and is in a strong correlation with the root lipid content [62].
The correlation between the concentration of Pyrene and the lipid content in Phragmites was strong with r 2 = 0.7429, and when the Vetiver was used, it had a moderate correlation with r 2 = 0.5503. The amount of uptake depended primarily on the lipid content of plant roots, in which protein, fats, nucleic acids, cellulose tissues, and other components all contain lipophilic components, which appear to be the primary domains where PAHs will accumulate once they penetrate plant root cells [55].
On the other hand, the correlation between Benzo[a]Pyrene concentration and lipid content for Phragmites plants was found to be strong with r 2 = 0.7108, while the same correlation was moderate for Vetiver with r 2 = 0.6248. Significant correlations between the PAH content and content of lipids were found only during the phase of full plant maturity [63].
Plant composition, in particular, lipid content affects the absorption of organic contaminants [30,64]. Based from a study by Gao et al. [65], the accumulation of PAHs in plant tissues, with range associated with plant fat content, had significant associations between Phenanthrene and Pyrene concentrations and lipid contents in the root. The limited PAHs uptake by roots of the plant was affected by the lipid contents. Lin et al. [66] showed how the plant lipid content was useful in predicting the accumulation of PAHs. The variation and accumulation of PAHs in plants may also be observed by the difference in plant lipid content when Phragmites and Vetiver plant species are used in CW. The accumulation of PAHs depends mainly on the plant lipid content, which was found to vary among plant species used in the CWs [55].

Conclusions
This study investigated the effectiveness of constructed wetland (CW) in removing harmful pollutants such as PAHs compounds from industrial wastewater. A total of 20 wetland models were used in the investigation. The models were planted with Phragmites and Vetiver plant species in order to access their role in removing PAHs compounds from the industrial wastewater. Synthetic wastewater was prepared for this purpose. Hence, the main conclusions of this study can be summarized as follows. The comparison between unplanted wetland and planted wetland revealed the important role of Phragmites and Vetiver plants in removing PAHs compounds from the industrial

Conclusions
This study investigated the effectiveness of constructed wetland (CW) in removing harmful pollutants such as PAHs compounds from industrial wastewater. A total of 20 wetland models were used in the investigation. The models were planted with Phragmites and Vetiver plant species in order to access their role in removing PAHs compounds from the industrial wastewater. Synthetic wastewater was prepared for this purpose. Hence, the main conclusions of this study can be summarized as follows. The comparison between unplanted wetland and planted wetland revealed the important role of Phragmites and Vetiver plants in removing PAHs compounds from the industrial wastewater. The maximum difference in removal rates between planted constructed wetland and unplanted constructed wetland was found to be 21% Phenanthrene, 13% Pyrene, and 30% Benzo[a]Pyrene, confirming the role of vegetation in removing pollutants from wastewater in constructed wetlands. A linear relationship was observed between the concentrations of PAHs compounds and lipid content in the Phragmites and Vetiver plants used in CWs. The general relationship can be given as y = a + bx, where "y" is the concentration of PAHs compound and "x" is the lipid content; "a" is the intercept on the axis of PAHs compounds concentrations, and "b" is the slope of the line. The system of the unplanted constructed wetland has the acceptable ability in removing PAHs compounds from wastewater where the removal rate is mainly dependent on the type of PAHs compound. The average percentage removals of PAHs compounds (Phenanthrene, Pyrene, and Benzo[a]Pyrene) by unplanted CWs were found to be 62%, 58%, and 55%, respectively.