3.1 Weather Conditions
Precipitation was higher in the months of summer than in the winter months. Weather measurements, therefore, exhibited typical tropical conditions of climate in the Pakistan except the very low temperatures in January and December 2015 and as well as unusually high precipitation in March and July 2015 (Fig 6).
3.2 Percentage removal of metals in HFCW
The influent varied during the course of the research, starting at 500 L/day for the first five months, and was then changed to approximately to 216 L/day (9 L/h) for the remainder of the study. Mean annual effluent flow rates for the control/unplanted system was approximately equal to the inflow. The planted systems had lower and more variable mean outflow rates than their unplanted counterparts due to the higher evapotranspiration rates during the peak growing season. It is expected that more mature systems planted with Vetiveria zizanioides and Chloris Gyana would exhibit greater water losses by evapotranspiration. Variation in electrical conductivity was observed due to presence of plants in both HF1 and HF2 than control (Table 2). The two treatment systems removed the Cu, Zn and Pb to slightly fluctuating grades as compared to the control (Fig 7). In horizontal flow system HF1, the mean effluent Zn concentrations were 6.24 mg/L. These concentrations were less than what would typically be expected for a horizontal flow wetland treating domestic wastewater in different studies (Badejo et al. 2015; Leung et al. 2016). However, the effluent concentration of Zn in HF2 was significantly low (p<0.05) was due to difference in plantation. Eucalyptus with sand and zeolite in HF2 showed better removal efficiency than the grass species of HF1. Different studies showed that the woody plants including Eucalyptus have capacity to accumulate more metals (Kang et al. 2018; Shukla et al. 2011). The average effluent concentration in HF1 of Cu and Pb were 0.055 mg/L and 0.508 mg/L respectively and was likely a result of the tolerant plants; Vetiveria zizanioides and Chloris Gyana species. This was in line with the experience from other studies of horizontal flow wetlands with sand as the main filter media (Abou-Elela et al. 2013; Bohórquez et al. 2016; Zhou et al. 2018). However, the HF2 exhibited significantly low (p<0.05) annual mean effluent concentrations of the Cu and Pb. The possible reason could be that both of HF systems were operated with HRT of 7 days, demonstrating the possible effect of hydraulic loading rate on the removal of metals from the leachate. It also showed that the HRT of 7 days in both systems have had enough of dissolved oxygen for efficient removal of metals. Moreover, aerobic conditions promoted the removal of the metals by plants. Other studies have also shown that the aerobic conditions were effective in removal of different organic and inorganic pollutants (Kahl et al. 2017; Behrends and Lane, 2009).
In contrary temperature had risen from May as shown in the Fig 6 which could have affected the performance of HF1 by marginalizing the proliferation of Vetiver zizanioides and Chloris gyana which might considered weaker as compared to woody plants. In HF2, the annual mean percent mass removal rate of Zn was 83%, Cu 94% and Pb 87% were efficient as compared to the control respectively (Fig 1 & Fig 7). However, performance of Eucalyptus was steady and was not affected by the temperature (Shukla et al. 2011).
3.3 Role of plants and substrates in treatment efficiency of HFCW
The role of plants was an addition to improve the removal of metals; dissolved oxygen and favorable environmental conditions including adequate contact time to reveal the full potential of the wetland technology. Therefore, selection of plants, substrates and influent concentration define its efficacy. The mentioned mechanisms and design factors are also reported in recent reviews as the key driver of treatment efficacy for different pollutant and contrarily reported first time for metal removal. In general, plants in surface flow treatment wetlands were reported to stabilize the surface of beds, facilitate physical filtration, mitigate (Huma and ilyas, 2017). This was especially true for the treatment of domestic wastewater, where nutrient loadings and subsequent oxygen demands were higher orders of magnitude than what was observed in natural environments and watercourses. Vymazal, 2011 and Wu et al. 2015 considered plant nutrient uptake as of major importance whereas Bakhshoodeh et al. (2016) compared planted and unplanted HF pilot systems (control in present study) and also, found that planted beds consistently achieved better removal of metal than unplanted beds. It is also worth highlighting that the plant species Vetiver grass in the study of Bakhshoodeh et al. (2016) showed exceptional performance of the metal removal from leachate compost and results were in line with the present work. Plants contributed significant proportion of the overall removal in the systems due to favorable climatic conditions. The more considerable effect of plants on treatment efficacy was the evapotranspirative water loss; specifically observed in HF2. The significant loss of water to plant evapotranspiration was important to consider; up to 50% of the inflow could be lost to evapotranspiration during the summer months (Nivala et al. 2018). This has repercussions for any effluent standards that are concentration-based. However, in the present study the system have good percentage removal rates, due to synergistic combination of plants and substrate.
Metal accumulation was also analyzed in plants and concentration of Zn and Pb were found in greater amount in roots compared to shoots in Vetiveria zizanioides, Chloris gyana in HF1 and Eucalyptus globulus in HF2 (Fig 4). The lower concentrations in shoots in the present study was in agreement with the speculation that plants confined the transfer of metals into their shoots to evade the impending toxic effects of high metal concentrations on their photosynthetic organs. Copper was the only metal to be present in high concentration in shoots because Cu was considered as an essential macronutrient in plant growth and metabolic processes (Megateli et al. 2009) in low concentration but it is toxic to plant’s growth in higher concentration. However, the average accumulation of Cu was high in V. zizanioides>C. gayna>E. globulus, Zn was accumulated in C. gayna>V. zizanioides >E. globulus and Pb in C.gayna>V. zizanioides>E. globulus. The results also revealed higher accumulation of Zn in the substrates of both wetlands; HF1 and HF2 respectively (Fig 5). Therefore, substrates were considered as primary sink of the wetlands where roots of plants also flourished. Therefore, the Zn accumulation was greater primarily in substrates than roots and comparatively negligible amount had been translocated to shoots. These facts indicated that the appropriate selection of the plant species and substrates could be vital to improve the efficiency of metal removal (Chazarenc et al. 2010).
Substrates provided an optimum growth medium for plants adaptation, enhancing the removal efficiency of both HFCW. In both HFCW (HF1 & HF2), Zn was highly absorbed by the substrates (Fig 2) and less accumulated by the plants (Fig 3), also supported by PCA of HF1. There is no statistically significant difference between both HFCW for Cu and Pb absorption by substrates whereas, elemental analysis of substrates revealed high concentration of Zn (Fig 5). Sand amended with zeolite is considered as better medium for plant growth and also, absorbent for heavy metals (Nosrati et al, 2021)
3.4 Principal Component Analysis
Principal component analysis (PCA) showed significant relation between different variables. In Fig 7, the axes F1 and F2 accounted for 100% variability between plants, substrates and percentage removal in HF1. The analytical results revealed the correlation of Chloris gyna with the percentage removal of Pb whereas, substrate revealed the correlation with Zn removal. Conversely, Zn was distinctively separate from Chloris gyna and Vetiveria zizanioides. These observations demonstrated that the grass plant species and substrates both played important role in removal of metals from leachate.
In Fig 7, the axes F1 and F2 showed 100% variability between Eucalyptus, substrates and percentage removal. The percentage removal of Zn was detected to be near to substrates while Cu and Eucalyptus were clustered together because Eucalyptus has more affinity for copper accumulation. Moreover, removal of Pb has no significant correlation with plants and substrates.