E ﬃ ciency of Five Selected Aquatic Plants in Phytoremediation of Aquaculture Wastewater

Featured Application: Authors are encouraged to provide a concise description of the speciﬁc application or a potential application of the work. This section is not mandatory. Abstract: The lack of clean water sources, due to the presence of pollutants in water, is a major issue in many countries, including Malaysia. To overcome this problem, various methods have been introduced, including phytoremediation treatment. Therefore, this phytoremediation study examined the ability of ﬁve aquatic plants— Centella asiatica , Ipomoea aquatica , Salvinia molesta , Eichhornia crassipes , and Pistia stratiotes —to remove three pollutants—total suspended solids (TSS), ammoniacal nitrogen (NH 3 -N), and phosphate—from aquaculture wastewater. Using wastewater samples, each containing 50 g of one of the plants, the pollutant levels were measured every two days for 14 days. The results showed a drastic decline in the concentration of pollutants, where C. asiatica was able to remove 98% of NH 3 -N, 90% of TSS, and 64% of phosphate, while I. aquatica showed the potential to eliminate up to 73% of TSS and NH 3 -N, and 50% of phosphate. E. crassipes drastically removed 98% of phosphate, 96% of TSS, and 74% of NH 3 -N, while P. stratiotes was able to eliminate 98% of TSS, 78% of NH 3 -N, and 89% of phosphate. S. molesta was e ﬃ cient in removing 89.3% of TSS and 88.6% of phosphate, but only removed 63.9% of NH 3 -N.


Introduction
Aquaculture is the breeding, rearing, and harvesting of aquatic life, such as fish, shellfish, prawns, algae, and other organisms living in an aquatic ecosystem. Aquaculture is one of the fastest-growing economies in the world food economy. Global aquaculture production has doubled in the past decade in terms of value and volume, and now supplies one-third of the seafood consumed worldwide [1]. Asia dominates this production, accounting for 88.91% by volume in 2014 [2]. There is no doubt that the contribution of aquaculture to the food supply will increase in the future due to the growing world population.
However, unsustainable aquaculture can give rise to new problems, such as water pollution, if the effluent and wastewater are not meticulously maintained. Water pollution caused by effluent from ponds is one of the main issues related to aquaculture [3,4]. Aquaculture has become large enough to have significant impacts on the environment, and some environmental concerns have been expressed by environmental activists and scientists [5]. In most freshwater aquaculture ponds, wastewater is generated before harvesting the stock, in which all of the particles and nutrients are

Aquatic Plants
C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes were used to determine the efficiency of each plant in absorbing and removing total suspended solids (TSS), ammoniacal nitrogen (NH 3 -N), and phosphate from aquaculture wastewater. These aquatic plants were filled in tanks containing distilled water for a week as an acclimatization period, before being exposed to contaminants from the aquaculture wastewater.

Analysis of Wastewater Quality
The initial water quality measurements were taken to determine the reduction rate after phytoremediation treatment. The dissolved oxygen (DO) level was determined using the YSI 5000 multiparameter instrument. Biochemical oxygen demand (BOD) was determined using the BOD 5 method [17]; the first DO reading was recorded on day one, and then the water samples were kept in a 20 • C incubator for five days. On the fifth day, the DO reading was taken again. The calculation for the determination of BOD 5 is as follows: TSS was determined using the gravimetric method, wherein a 0.45 µm filter paper was used to trap TSS. The filter paper was initially filtered through distilled water to make sure all salts and foreign substances were removed, and then it was dried in the oven at a temperature of 103-105 • C and weighed. The dried filter paper was then used to filter the wastewater sample, and then dried again at 80 • C to weigh the residual substances. The calculation for the determination of TSS is as follows: Total suspended solids (mg/L) = (A-B)/V × 1000 mL (2) where, A = Weight of filter paper after filtration (weight of filter paper + dried residue), mg B = Weight of filter paper before filtration (weight of filter paper), mg V = Volume of filtered water sample, mL For the phosphate parameter, the ascorbic acid method (reactive orthophosphate) was used, and a packet of PhosVer ® (ascorbic acid) reagent pillows was needed for each sample analysis. The NH 3 -N parameter was determined and recorded using the Nessler method and the HACH DR/2010 spectrophotometer [15]. All values were recorded in the form of ± standard deviation average and mean values. The parameters and methods involved are shown in Table 1.

Set-up of the Phytoremediation Laboratory Experiment
Experiments were performed in rectangular tanks with a volume of 0.012 m 3 (40 cm length × 20 cm width × 15 cm depth). All the plants were washed, dried, and left in a tank filled with 15 L distilled water for a week. This step was necessary to make sure the plants were neutralized prior to the next step. Excess water was allowed to drain off and was dried with a dry cloth; then, 50 g of each plant was weighed before being placed in the aquaculture wastewater sample.
The wastewater sample was measured for 5 L for each tank where 50 g of plants per species were used to treat the 5 L wastewater. The first tank was filled with the measured wastewater along with C. asiatica, then another tank was filled with the wastewater sample without plants, as the control, and these steps were repeated with the other four plant species. Three replications for each species were prepared for an average reading and to assess the role of the plants in removing organic and inorganic pollutants. No new wastewater was added to the tanks throughout the experiment to allow a constant pattern of pollution reduction [18].
The levels of TSS, NH 3 -N, and phosphate in the wastewater samples were measured every two days, starting from day 0, to evaluate the changes in water quality and the reduction rate achieved by the phytoremediation process. Figure 1 shows a schematic diagram timeline of how the phytoremediation treatment was conducted in the laboratory under fluorescent tubes.
prepared for an average reading and to assess the role of the plants in removing organic and inorganic pollutants. No new wastewater was added to the tanks throughout the experiment to allow a constant pattern of pollution reduction [18].
The levels of TSS, NH3-N, and phosphate in the wastewater samples were measured every two days, starting from day 0, to evaluate the changes in water quality and the reduction rate achieved by the phytoremediation process. Figure 1 shows a schematic diagram timeline of how the phytoremediation treatment was conducted in the laboratory under fluorescent tubes.

Statistical Analysis
Mean values were recorded for the three replicates of each plant. Statistical analysis was performed using Statistical Package for the Social Sciences 23 (IBM SPSS 23) software, where a one-way ANOVA and an independent t-test were conducted. All data collected in this research were used to determine the significant differences between the plants in removing pollutants and excess nutrients. The comparisons of the means for each parameter after the 14-day phytoremediation treatment were calculated for p-values, and the significant level was determined at p < 0.05.

Initial Parameters of Aquaculture Wastewater before Phytoremediation
Based on the Environmental Quality (Sewage and Industrial Effluent) Regulations (2009), the initial physico-chemical parameters, including the temperature, pH, DO, TSS, and NH 3 -N, of the aquaculture complied with the required a and b standards. The same goes for the other parameters, including phosphate, turbidity, conductivity, total dissolved solids (TDS), and BOD 5 , which were taken during water sampling, were all at permissible levels. Table 2 shows the initial reading of the aquaculture wastewater parameters prior to phytoremediation process.

Phytoremediation of Aquaculture Wastewater
The removal rates of TSS, NH 3 -N, and phosphate were determined by using five selected aquatic plants as phytoremediation agents. These plants were chosen due to their great ability in removing pollutants from various wastewater. As stated by Manan et al., C. asiatica leaves, roots, and stems are good pollutant accumulation agents. Since it needs extra nutrients, such as zinc and lead, for biochemical processes, especially photosynthesis, the roots actively translocate nutrients to the leaves, making C. asiatica an efficient phytoremediation agent [19]. I. aquatica stems and leaves are also very efficient in absorbing nutrients and heavy metals, and use the pollutants as food in order to grow, which is why this species is known as a fast-growing plant [20]. Meanwhile, the roots of S. molesta are very fibrous, and the same goes for P. stratiotes, allowing it to easily and efficiently trap suspended solids in the water body. It is also a fast-growing plant; therefore, it needs a high amount of nutrients to stay healthy, alive, and grow. According to Ebel et al. [21], E. crassipes is one of the best macrophytes to be used as a phytoremediation agent because it removes pollutants from wastewater efficiently while having a high tolerance to the polluted environment [21].
According to Sa'at et al. [22], I. aquatica can reduce 92.6% of total suspended solids (TSS) and 82.7% of ammoniacal nitrogen (NH 3 -N) from palm oil mill wastewater. C. asiatica is able to trap 100% of TSS from water body and S. molesta's leaves and roots are highly efficient in accumulating and removing pollutants, especially heavy metals [22][23][24]. Akhtar et al. [25] stated that E. crassipes and P. stratiotes efficiently removed heavy metals without showing any kind of damage or bad reactions to the metals.

Reduction Rate of TSS
TSS are solid in a water body which includes a wide variety of materials, such as silt, decaying plants, animal matters, and other solid wastes [26]. Figure 2 shows the reduction rate of TSS using 50 g of C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes plotted against the days of treatment. It was found that P. stratiotes reduced TSS the most, with up to 98% reduction rate and a total of 43.3 mg/L of TSS by the 14th day of treatment.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 10 suspended solids in the water body. It is also a fast-growing plant; therefore, it needs a high amount of nutrients to stay healthy, alive, and grow. According to Ebel et al. [21], E. crassipes is one of the best macrophytes to be used as a phytoremediation agent because it removes pollutants from wastewater efficiently while having a high tolerance to the polluted environment [21]. According to Sa'at et al. [22], I. aquatica can reduce 92.6% of total suspended solids (TSS) and 82.7% of ammoniacal nitrogen (NH3-N) from palm oil mill wastewater. C. asiatica is able to trap 100% of TSS from water body and S. molesta's leaves and roots are highly efficient in accumulating and removing pollutants, especially heavy metals [22][23][24]. Akhtar et al. [25] stated that E. crassipes and P. stratiotes efficiently removed heavy metals without showing any kind of damage or bad reactions to the metals.

Reduction Rate of TSS
TSS are solid in a water body which includes a wide variety of materials, such as silt, decaying plants, animal matters, and other solid wastes [26]. Figure 2 shows the reduction rate of TSS using 50 g of C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes plotted against the days of treatment. It was found that P. stratiotes reduced TSS the most, with up to 98% reduction rate and a total of 43.3 mg/L of TSS by the 14th day of treatment.
Umar et al. [27] stated that the charges in the root systems of aquatic plants or macrophytes improve their ability to trap and attract particles, such as TSS, in a medium. Hence, the reduction of TSS by plants primarily depends on the plants' root systems, and plants with fibrous roots can accumulate more TSS than those with taproots. In the present study, 98% of the TSS was reduced by P. stratiotes, which has fibrous and long roots [27]. This result is also supported by the previous studies, which showed that P. stratiotes was able to increase water clarity by trapping suspended solids in the roots [28]. It is believed that this species is also able to remove heavy metals magnificently. Based on the index of bio-concentrating factors, P. stratiotes have been reported by Lu et al. to be hyperconcentrated in the roots area for heavy metals like Cr, Cu, Fe, Mn, Ni, Zn and Pb [29]. According to Akhtar et al. [25], 77.3% of Cr and 91.29% of Cu were removed during one month of treatment. Moreover, in E. crassipes, C. asiatica, I. aquatica, and S. molesta tanks, the TSS concentration levels were reduced to 96%, 90%, 73%, and 89.3%, respectively. I. aquatica possesses taproots; hence, it trapped the lowest amount of TSS among the five species. E. crassipes has fibrous roots, which explains why the concentration of TSS on the last day of treatment was significantly reduced. However, C. asiatica does not possess fibrous roots but effectively reduced TSS because it can be physically and biologically removed through filtration and sedimentation. TSS are trapped and Umar et al. [27] stated that the charges in the root systems of aquatic plants or macrophytes improve their ability to trap and attract particles, such as TSS, in a medium. Hence, the reduction of TSS by plants primarily depends on the plants' root systems, and plants with fibrous roots can accumulate more TSS than those with taproots. In the present study, 98% of the TSS was reduced by P. stratiotes, which has fibrous and long roots [27]. This result is also supported by the previous Appl. Sci. 2020, 10, 2712 6 of 11 studies, which showed that P. stratiotes was able to increase water clarity by trapping suspended solids in the roots [28]. It is believed that this species is also able to remove heavy metals magnificently. Based on the index of bio-concentrating factors, P. stratiotes have been reported by Lu et al. to be hyperconcentrated in the roots area for heavy metals like Cr, Cu, Fe, Mn, Ni, Zn and Pb [29]. According to Akhtar et al. [25], 77.3% of Cr and 91.29% of Cu were removed during one month of treatment.
Moreover, in E. crassipes, C. asiatica, I. aquatica, and S. molesta tanks, the TSS concentration levels were reduced to 96%, 90%, 73%, and 89.3%, respectively. I. aquatica possesses taproots; hence, it trapped the lowest amount of TSS among the five species. E. crassipes has fibrous roots, which explains why the concentration of TSS on the last day of treatment was significantly reduced. However, C. asiatica does not possess fibrous roots but effectively reduced TSS because it can be physically and biologically removed through filtration and sedimentation. TSS are trapped and accumulated when passing through the plant roots and eventually sink to the bottom by gravitational force or metabolized by microorganism [23]. The sedimentation process also helps to reduce TSS concentration by 60% in the control tank after 14 days, however, there was an increased TSS concentration for four days due to some disturbance to the tank on day 1 of experiment.
The graph plot of TSS reduction in Figure 2 shows that TSS consistently decreased with the help of phytoremediation agents and the reduction was very inconsistent without the aid of any plants. The plots of P. stratiotes and E. crassipes showed the most significant decreasing patterns, followed by those of C. asiatica, S. molesta and I. aquatica. However, starting from day 10, the concentration of TSS by most species tested in this experiment started to slowly decrease or stayed constant, possibly because it had already reached the maximum removal concentration.
A one-way ANOVA was conducted and showed a significant value (p < 0.05) for the reduction rate of TSS, from the first day of treatment until the end, by all five species. However, a statistical independent t-test showed no significant value (p < 0.05) between the five plants, since the difference between the highest and lowest TSS concentrations removed was only 9.6 mg/L. This proves that all five species practically have almost the same potential to reduce TSS.

Reduction Rate of NH 3 -N
The concentration of NH 3 -N in wastewater can be unpredictable due to the decomposition of organic waste matter and nitrogen fixation processes [30]. Figure 3 shows the reduction rate of NH 3 -N using C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes plotted against the days of the treatment. C. asiatica reduced the most NH 3 -N concentration by the end of the experiment compared to the other four species, reaching 98% reduction. C. asiatica is known as a medicinal plant for its high nutritional value and because of its ability to take in organic substances and nutrition from the environment, which explains why it serves as an excellent phytoremediation agent [31].
This study shows E. crassipes and P. stratiotes serve more effectively as phytoremediation agents in catfish aquaculture wastewater than in tilapia fish aquaculture wastewater. The reduction rates of NH 3 -N by E. crassipes and P. stratiotes in catfish aquaculture wastewater reached until 74% and 78% respectively, compared to a reduction rate of only 69% and 65% respectively, in tilapia fish aquaculture wastewater [32].
The total average removed concentrations of NH 3 -N in the wastewater sample after 14 days of treatment with C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes were 4.07 mg/L, 2.87 mg/L, 2.65 mg/L, 3.3 mg/L, and 3.1 mg/L, respectively. Although the percentage of removal and the total average concentration showed some distinct differences, statistical independent t-tests showed the opposite, and the value was not significant (p < 0.05). However, these species are believed to have high oxygen transfer abilities, providing suitable environments for bacterial nitrification while enhancing the removal of NH 3 -N [33]. In addition, all species reached a constant concentration of NH 3 -N after the 12th day of treatment because they had reached their removal capacity. The concentration in the control tank was higher than those obtained in the tanks with plants at the end of the experiment. N using C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes plotted against the days of the treatment. C. asiatica reduced the most NH3-N concentration by the end of the experiment compared to the other four species, reaching 98% reduction. C. asiatica is known as a medicinal plant for its high nutritional value and because of its ability to take in organic substances and nutrition from the environment, which explains why it serves as an excellent phytoremediation agent [31]. This study shows E. crassipes and P. stratiotes serve more effectively as phytoremediation agents in catfish aquaculture wastewater than in tilapia fish aquaculture wastewater. The reduction rates of NH3-N by E. crassipes and P. stratiotes in catfish aquaculture wastewater reached until 74% and 78% respectively, compared to a reduction rate of only 69% and 65% respectively, in tilapia fish aquaculture wastewater [32].
The total average removed concentrations of NH3-N in the wastewater sample after 14 days of treatment with C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes were 4.07 mg/L, 2.87 mg/L, 2.65 mg/L, 3.3 mg/L, and 3.1 mg/L, respectively. Although the percentage of removal and the total The increasing concentrations of NH 3 -N at day 10 by S. molesta were caused by the death of the plants and their debris. Plant decomposition is a major cause of increased ammonia content in wastewater because the nitrogen compounds in the organic matter re-enter the water and are broken down by microorganisms, known as decomposers. The decomposition process produces ammonia, which undergoes nitrification. The decomposition of ammonia, however, yields hydrogen and nitrogen, and is an important process in nature [30]. The removal of NH 3 -N was also caused by the biological mechanisms in nutrient intake and ammonia absorption [34]. The other possible reason for the NH 3 -N removal was the high growth of denitrifying bacteria [35,36].

Reduction Rate of Phosphate
In wastewater, phosphates are transformed into orthophosphate and are available for plant uptake as a source of nutrients; however, this may cause a nutrient imbalance and an imbalance in the material cycling processes of the natural phosphorus cycle in the ecosystem [37]. Controlling excessive phosphorus content in wastewater is a key factor in deterring the eutrophication of surface water. Its excessive presence may cause losses of livestock and introduce potentially lethal algal toxins into drinking water, which are very bad for aquaculture industries and humans alike [38].
E. crassipes is the best plant among the five species to treat phosphate in aquaculture wastewater, as shown in Figure 4. Since organic phosphate acts as a nutrient for plants, E. crassipes was highly efficient in absorbing phosphate for its nutritional needs, with a reduction rate almost reaching 100% and a total of 0.34 mg/L phosphate reduced after 14 days of phytoremediation treatment. This species has long fibrous roots capable of providing extensive surface area for the plant to carry out physical, chemical and microbial processes for nutrient absorption and nitrification processes [18]. In addition, [39] reported E. crassipes reduced 50.04% of phosphate from an aquarium with a population of Sarotherodon melanotheron (blackchin tilapia) within 12 days of treatment. efficient in absorbing phosphate for its nutritional needs, with a reduction rate almost reaching 100% and a total of 0.34 mg/L phosphate reduced after 14 days of phytoremediation treatment. This species has long fibrous roots capable of providing extensive surface area for the plant to carry out physical, chemical and microbial processes for nutrient absorption and nitrification processes [18]. In addition, [39] reported E. crassipes reduced 50.04% of phosphate from an aquarium with a population of Sarotherodon melanotheron (blackchin tilapia) within 12 days of treatment.  Based on the graph of the phosphate concentrations, I. aquatica was very inconsistent and removed the least amount of the pollutant. However, the concentration of phosphate decreased at days 4, 6, and 12. S. molesta had the most consistent phosphate removal rate, but the readings reached a maximum removal concentration on the 10th day and stayed constant until day 14. The concentration of phosphate at the end of the experiment in the control tank was the highest compared to tanks with plants, because phosphate is essential for plants' life-sustaining reactions, which include energy transfer, protein activations, and regulation of metabolic processes [40,41]. That could explain the difference between the control tank with no plants for absorption, and the tanks with five macrophytes species.
An independent t-test analysis showed no significant value (p < 0.05) between the phosphate reductions by all five species after 14 days, except for E. crassipes and I. aquatica, since the highest average removal value was 0.34 mg/L by E. crassipes, with I. aquatica being the lowest-0.17 mg/L. The efficient reduction of phosphate from the wastewater comes from plant uptake and the presence of diverse substrates [42]. This pollutant is the major constituent in adenosine diphosphate (ADP) and adenosine triphosphate (ATP), which acts as a building block for nucleic acids, sugar phosphates, nucleotides, and many more, especially during the active growth of plants. Phosphates are crucial for energy storage and transfer in photosynthesis and respiration, essential for normal growth, maturity, and root system development, but too much phosphate in a plant's system may be lethal [40][41][42][43]. In addition, some macrophytes need phosphate more than other macrophytes, depending on the plants' type, requirements and its surrounding environment.
The results found in the present study were in line with other previous studies [9,[44][45][46][47][48]. A similar pattern of results was obtained to indicate the ability of phytoremediation in removing different pollutants. Phytoremediation for treating wastewater remains popular until now due to it being cost-effective, environmentally friendly, and sustainable with low maintenance.

Conclusions
In this study, the effectiveness of five aquatic plants-C. asiatica, I. aquatica, S. molesta, P. stratiotes, and E. crassipes-in removing nutrients from aquaculture wastewater was investigated. Through phytoremediation, the reduction rates of three pollutants-TSS, NH 3 -N, and phosphate-were studied. These aquatic plants performed well towards the reduction and removal of excessive nutrients from the wastewater, without being physically affected by them. It was found that P. stratiotes is the best eliminator of TSS from aquaculture wastewater, with C. asiatica being the best to reduce NH 3 -N, and E. crassipes the most efficient for phosphate removal. Overall, the results indicate that all five species have their own effectiveness and are somewhat efficient as phytoremediation agents, since the reduction rates after 14 days of treatment were greater than 50%, for suspended solids content, ammoniacal nitrogen, and phosphate.