Effects of nitrogen form on growth rate of the free-floating macrophyte Phyllanthus fluitans: Implications for nitrogen phytoremediation and invasive plant management

<!-- metadata commented in wiki content


<div id="_Hlk171546405" class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<big>'''The Effects of Nitrogen Preference of Nitrate (NO'''</big><span style="text-align: center; font-size: 75%;">'''<sub>3</sub><sup>-</sup>'''</span><big>''') Compared to Ammonium (NH₄⁺)'''</big></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<big>''' on the Growth of Phyllanthus fluitans.'''</big></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
'''Nathalie Zhang, Tian Jin, Ashley Quito Angamarca'''</div>
-->

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
'''Abstract'''</div>

This study investigated the influence of different ammonium to nitrate ratios on the growth of ''Phyllanthus fluitans ''to determine nitrogen preference. Excessive inorganic nitrogen is among the most prevalent pollutions in growing nations. Phytoremediation (using plants to clean pollution) is a leading method for preventing further damage from nitrogen pollution. This study can aid in determining phytoremediation suitability. In this study, the suitability of ''Phyllanthus fluitans'' for phytoremediation was determined by researching its nitrogen preference, adaptability, and growth. These physiological responses were tested through a 28-day trial (for Trial 1) and a 24-day trial (for Trial 2) with plants placed in separate nutrient solutions containing different ratios of ammonium to nitrate at 0.002M nitrate (0:1), 1:3, 1:1 (Trial 1 control), 3:1, 0.002M ammonium (1:0), and water (Trial 2 control). Data was collected by counting leaves, measuring fresh weight, dry weight, and mean root length. Results in both trials showed that plants had the highest fresh weight, dry weight, root length, and survival rate when in a 0:1 solution. This data, combined with a high mortality rate among all groups containing ammonium, strongly suggests ''Phyllanthus fluitans ''to have'' ''a preference towards nitrate. The results of this study provided insight into the role of ''Phyllanthus fluitans ''for phytoremediation and can help conservationists predict and prevent possible ''Phyllanthus fluitans ''invasions. In the future, further studies determining the phytoremediation suitability of alternative plants should be carried out and their results should be extrapolated for environmental conservation.

Keywords: Aquatic remediation, Floating plants, Nitrogen pollution, Nitrogen preference, Phytoremediation

==1 INTRODUCTION==


'''1.1''' Purpose and Importance'''

Nitrogen is an important component for all living organisms, especially plants. However, an overabundance of nitrogen in the forms, ammonium (NH₄⁺) and nitrate (NO<sub>3</sub><sup>-</sup>) can degrade water quality, make drinking water unsuitable for consumption, cause harmful algal blooms, dead zones, declining biodiversity, collapse nutrient cycles, develop water blooms, detriment agriculture, and contributes to fish deaths [1,2]. Excessive inorganic nitrogen is among the most prevalent water problems and can be the result of wastewater runoff from households, and farmlands [3]. Subsequently, increasingly stringent water pollution regulations (concerning multiple pollutants but with a strong emphasis on nitrogen) have been proposed and implemented worldwide [4]. To address the severe environmental determinants caused by nitrogen pollution, various measures need to be researched and developed to remediate eutrophic waters.

The ability of aquatic macrophytes (''Phyllanthus fluitans'' is considered a species of “aquatic macrophyte”) to remediate wastewater has been receiving increased attention due to macrophytes being able to take up and store large amounts of inorganic nitrogen [2]. The use of phytoremediation is among the most environmentally friendly and low-cost methods to remove pollutants from water and soil. Data from the results of this study aid in setting a standard for the potential effectiveness and application of ''Phyllanthus fluitans ''for phytoremediation [5]. Nitrogen preference is an important but significantly understudied factor in plant physiology, morphology, domination, and phytoremediation. In this study, the nitrogen preference of the aquatic floating plant, ''Phyllanthus fluitans'' was studied to elucidate its inclination for either nitrate or ammonium. This study also assessed the growth rate of ''Phyllanthus fluitans. ''This data is highly important because the growth rate of ''Phyllanthus fluitans ''can determine its suitability for phytoremediation (due to the strong correlation between growth rate and nitrogen uptake) [2]. Multiple studies done previously agree that studying the morphology, growth characteristics, physiology, and nitrogen preference of macrophytes can aid in engineering better phytoremediation systems.'' ''The results of this study can be applied to phytoremediation systems, improving their efficiency [3,6].

'''1.2''' Research question/hypothesis'''

The present study determined the effects of different ratios of nitrate (NO<sub>3</sub><sup>-</sup>) to ammonium (NH₄⁺) on the growth of ''Phyllanthus fluitans'' using the ratios of 1:3, 3:1, 1:0, 0:1, and 1:1 (control). A previous study was conducted researching the nitrogen preference of duckweed (a morphologically similar aquatic plant) [7]. This study compared the effects of varying nitrate to ammonium ratios (0:100, 75:25, 50:50, 25:75, and 0:100) in the free-floating macrophyte species, duckweed ''L. minor''. The results of the study demonstrated that the highest relative growth rate was at a ratio of 25:75 for ''L. minor''. Using data from these results, it was hypothesized that the highest plant growth would be observed in the group receiving a ratio of 1:3 (ammonium to nitrate). This hypothesis is also supported by previous research demonstrating toxicity symptoms when ammonium acts as the sole nitrogen source for ammonium-intolerant plants, but often a faster growth rate when used for ammonium-tolerant plants, or in conjunction with nitrate for ammonium-intolerant plants [3,6,8].

'''1.3''' Previous research'''

Due to the effects of nitrogen pollution and increasingly strict rules, researchers developed multiple methods in an attempt to remediate nitrogen. Common methods of nitrogen remediation include bacteria biofilm filters, stormwater retention ponds, adsorption, and wetlands [5,9,10]. Although many studies have confirmed the effectiveness of these methods, they come with limitations. For instance, bacteria biofilm filters and adsorption methods can be costly to set up and maintain [5].

Phytoremediation is used to describe the use of plants to remove contaminants and pollutants, it has been receiving increased attention from researchers and environmentalists due to its low cost, low maintenance, low risk, and high efficiency. Previous research indicates that phytoremediation often has synergistic effects with other remediation treatments, facilitating even greater effectiveness [9,10]. Phytoremediation systems are also effective for remediating pollutants other than nitrogen. This is relevant since researchers believe nitrogen pollution is linked to many other pollutants and/or water problems [11]. This ability gives phytoremediation an upper hand over alternative remediation methods limited to nitrogen reduction. Phytoremediation can effectively remove other pollutants including but not limited to, phosphorus, lithium, microplastics, cadmium, manganese, copper, zinc, and silver [11,12,13,14,15]. Phytoremediation also shows promise when used in aquaculture systems. Aquaculture is among the fastest-growing sectors in the world food economy and is expected to continue its expansion. Although aquaculture is an effective solution to supply seafood, unsustainable aquaculture can directly contribute to nitrogen pollution. Multiple studies show phytoremediation’s beneficial effects when integrated into aquaculture farms [5,10,16]. Phytoremediation shows promise in effectively remediating nitrogen but needs further research to improve its efficiency, maintenance, and widespread use. Nitrogen pollution is an increasingly relevant issue, and it is a necessity to create better systems to remediate nitrogen and decrease nitrogen pollution.

Nitrogen preference is an important but often neglected factor in plant ecosystems. It is generally defined as plants having certain forms of nitrogen (usually nitrate or ammonium) that they can absorb and assimilate best, in other words, a preference for specific types of nitrogen. Although there is a significant lack of research concerning nitrogen preference, previous studies show that it plays a key role in plant physiology, growth, nitrogen uptake, and dominance. The availability of the preferred type of nitrogen can result in domination or invasion by a plant species within an ecosystem [8]. Most aquatic plants prefer inorganic nitrogen in the form of ammonium [6]. This is due to the lower amount of energy needed for ammonium uptake and assimilation, often resulting in faster growth (relative to nitrate). Although this assumption does hold some truth, ammonium can cause severe toxicity symptoms when it is at the only nitrogen source available, especially at high concentrations. Hence, researchers hypothesize that plants adapted to a nitrate-dominant environment will struggle in mediums high in ammonium [8]. Nitrogen preference also has strong significance concerning nitrogen phytoremediation. Ammonium was also shown to be the main nitrogen-containing component in aquaculture wastewater, suggesting that plants with an ammonium preference will be more suitable for use in phytoremediation systems. Previous research shows ammonium to be the preferred source of nitrogen for ''Lemna minor,'' also commonly known as duckweed. The study of the nitrogen preference in duckweed later helped develop a newly established sustainable duckweed-based remediation system for aquaculture [16]. Researching the nitrogen preference in ''Phyllanthus fluitans'' could achieve similar significance due to the similar morphology of the 2 plants (both ''L. minor'' and ''Phyllanthus fluitans ''are considered “free-floating” and both are macrophytes). Ammonium was also shown to be the main nitrogen-containing component in aquaculture wastewater [16], suggesting that plants with an ammonium preference will be more suitable for use in phytoremediation systems.

'''1.4''' Free-floating macrophytes and Phyllanthus fluitans'''

''Phyllanthus fluitans'', commonly known as red root floater, is an aquatic floating plant originating from freshwater habitats of tropical and subtropical South America that is part of the Phyllanthaceae family. Floating plants have many beneficial uses such as wastewater treatment, biofuel production, ecotoxicological assessment, and aquaculture [17]. ''Phyllanthus fluitans ''also has the added benefit of being a valuable byproduct when used for remediation because of its ability to be used as animal feed. In European markets, suppliers can expect to receive 1 euro for every 67.3 grams of ''Phyllanthus fluitans ''sold. The value of ''Phyllanthus fluitans ''can help offset the high start-up cost of remediation systems and even be profitable [10]. Few reports, and even fewer studies have been conducted, on ''Phyllanthus fluitans.'' Researchers state that exploring and cultivating plants that lack proper research is of great significance for ecological restoration using phytoremediation [18]. Previous research suggests that floating macrophytes can adjust their root length to adapt to relative nitrogen concentrations in their environment; with lower concentrations floating plants grew longer, thicker roots, and with higher concentrations floating plants grew shorter, thinner roots [19]. This characteristic of their physiology strongly suggests that floating plants have wide adaptability and resilience. Plants that are resilient to unfavorable conditions and adapt to their environment are highly suitable for phytoremediation due to their lower chance to wither (further polluting already eutrophic environments) [5]. High growth rates (in macrophytes) are closely correlated with high nitrogen uptake and assimilation. Many researchers believe that a high growth rate is the most important factor when selecting plants for phytoremediation [2,11]. Floating macrophytes are intensively studied in several journals to have a higher growth rate than their emergent or fully submerged counterparts [3]. Subsequently, floating macrophytes with similar morphology and physiology to ''Phyllanthus fluitans'' are shown to not only be suitable for phytoremediation but also tend to outperform emergent or fully submerged plants when considering nitrogen remediation [2,3]. This study determined the suitability of ''Phyllanthus fluitans ''for phytoremediation by exploring its growth rate, adaptability, nitrogen preference, and resilience.

==2 METHODOLOGY ==

<span id='_zgy3qpwnaxgv'></span>The growth rate (and subsequent nitrogen preference, and vitality) of the plant species was observed for the duration of the experiment (28 days for Trial 1, 24 days for Trial 2). The data obtained from the investigation regarding changes in leaf growth, root growth; and wet weight was then compared to the control group (1:1 ammonium to nitrate for Trial 1, 0:0 water for Trial 2). The concentration of the nitrogen provided was based on the average nitrogen concentration in domestic wastewater due to this study’s focus on phytoremediation.

'''2.1''' Preparing stock solution'''

Stock solutions were added before being poured into individual containers for experimentation. Two food-grade plastic containers with a volume of at least 2 liters, were used for making and holding the stock solutions. To organize the experimental setup, two sets of tape (with the text “stock solution 1”, “stock solution 2” and “stock solution 3 (Trial 2)” written on them) were created and stuck onto each of the 2 containers with the corresponding group name. Stock solution 1 represents the ammonium solution, stock solution 2 represents the nitrate solution, and stock solution 3 represents the water solution. Both tanks were filled with 1.9 liters of distilled water and 2.22 mL of micronutrient mix (Tropica Plant Growth Premium Fertilizer; link: [https://tropica.com/en/plant-care/liquid-fertilisers/premium-nutrition/ https://tropica.com/en/plant-care/liquid-fertilizers/premium-nutrition/]), 2.5 mL of “Seachem Flourish Phosphorus” (a potassium phosphorus-based fertilizer; link: [https://www.seachem.com/flourish-phosphorus.php https://www.seachem.com/flourish-phosphorus.php]) (removed for Trial 2), 7.0 g of “Seachem Alkaline buffer” (a commercially available sodium bicarbonate-based supplement; Link: [https://www.seachem.com/alkaline-buffer.php https://www.seachem.com/alkaline-buffer.php]), and 20 g of “Seachem equilibrium” (changed to 2.5 g for Trial 2) (a commercially available supplement/fertilizer derived from potassium sulfate, calcium sulfate, magnesium sulfate, ferric sulfate, & manganese sulfate; link: [https://www.seachem.com/equilibrium.php https://www.seachem.com/equilibrium.php]). Both batches of solution were mixed with a stirring rod to dissolve solutes and make both solutions as homogeneous as possible. In the nitrate solution (stock solution 2) 3.3 grams of potassium nitrate were added. To the ammonium solution (stock solution 1) 2.02 grams of ammonium chloride was added (concentrations displayed in Table 1).

After both stock solutions were made (following the concentrations shown in Table 2) they were used to create separate groups using 100% virgin plastic containers (L 175 mm x H 80 mm x W 117 mm) to test the different ratios. To distinguish between each of the tested groups each had tapes at their sides with a corresponding text stating either “0.002M nitrate”, “0.002M ammonium”, “1:3 ammonium to nitrate”, “3:1 ammonium to nitrate”, “1:1 ammonium to nitrate” (as the control for Trial 1), and “water” (as the control for Trial 2). The number of solutions from the original stock solution and distilled water that were used for these separate ratios are stated in Table 2.

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
Table 1. Stock Solutions (* indicates changes made for Trial 2)</div>

{| style="width: 100%;border-collapse: collapse;" 
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Stock Solution
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Amount of Distilled Water (L)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Concentration of Nitrate (M)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Concentration of Ammonium (M)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Mass of Ammonium chloride (g)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Mass of Potassium Nitrate (g)
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|1
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|1.9
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0.02
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|2.02
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|2
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|1.9
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0.02
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|--------------
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|3.3
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|3 *
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|1.9
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|---------------
|}


<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
Table 2. Experimental Setup (* indicates changes made for Trial 2)</div>

{| style="width: 100%;border-collapse: collapse;" 
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Ratio of Ammonium to Nitrate
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Amount of solution from stock solution 1 (mL)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Amount of solution from stock solution 2 (mL)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Amount of solution from stock solution 3 (mL) *
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|Distilled water used to fill each tank (mL)
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|* Water (control for Trial 2)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|85
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|765
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0.002M nitrate (0:1)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|85
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|765
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|1:3
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|21.25
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|63.75
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|765
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|1:1 (control for Trial 1)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|42.5
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|42.5
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|765
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|3:1
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|63.75
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|21.25
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|765
|-
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0.002M ammonium (1:0)
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|85
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|0
|  style="border: 1pt solid black;text-align: center;vertical-align: top;"|765
|}


'''2.2''' '''Preparing specimens '''

All the plants that were used in the experiment were sourced from an online retailer and quarantined (with the same corresponding setup for the control) for 7 days to allow plants to heal from the damage of being shipped. Afterward, plants were chosen per container based on their separate examinations for health conditions regarding visible signs of nutrient deficiency, physical damage, invasion by epiphytes such as algae, possible signs of disease, etc. Trial 1 consisted of 6 samples per group, while Trial 2 consisted of 12 samples per group. All groups of plants were then gently tapped with a dry paper towel to ensure the removal of all surface liquids on the plants before measurement. Plants selected for all groups had a leaf amount of 2 to 6 leaves. The mass of the plants was taken using an analytical balance. The resulting mass should not differ more than ± 0.2 g between groups; this was done to ensure that the plants picked are made into equal groups with a similar mass.

'''2.3''' '''Growth Conditions'''

To ensure that the only independent variable throughout the experiment was the tested ratios, outside factors were controlled through various methods. All the samples were placed in a three-tiered light rack alongside each other. Two long LED lights acted as the light source, controlled using an electrical timer to properly time the light from 9 am (EST) - 7 pm (EST) so that each group was provided with a controlled 10 hours of light per day. Additionally, factors that might influence the results such as plant weight, pH/nutrient levels, and temperature were controlled respectively by measuring all plants to create groups with similar mass, changing the water for a periodic amount of time, and maintaining all groups near each other to simulate similar temperatures conditions.

'''2.4''' '''Maintaining plants/experimenting'''

Trial 1’s sample size was 6 samples per group, while Trial 2’s sample size was 12 samples per group. After being measured, plants with 2 to 6 leaves were gently placed on the water's surface of each of the groups in their corresponding separate containers. Throughout the experiment, all groups were provided with 10 hours of light per day for 28 days while placed near one another to prevent inconsistencies in temperature for Trial 1. The same measures were taken for Trial 2, but in a 24-day duration. The plant medium/solution was replaced every Monday to maintain proper levels of nutrients with the same process repeated on maintained on the prepared pots and conditions. Plants were periodically removed and wrapped with a paper towel soaked in distilled water while this procedure was occurring. Throughout this procedure, all unexpected occurrences (deteriorating plant health, abnormal amount of dead plant matter, chlorosis, etc.) were recorded.

'''2.5''' '''Measuring Root Length & Leaf Numbers'''

The number of leaves of the different treatments was recorded at the beginning of the experiment to be later compared to the final results. This was followed by a weekly counting of the leaves manually every Monday, Wednesday, and Friday, with a separate smaller section on written notes on dying leaves, leaves lost (the number of dead leaves in each group was documented before taking measurements), leaves damaged, and buds growing. This acted as a precaution considering the delicateness of the plants and only samples of floating plants, with at least 1 healthy leaf, and a root system considered in the graph showing the numbers of total leaves per treatment. Additionally, the measurement of the longest root of each plant per treatment was taken using a ruler and recorded in centimeters using an Excel spreadsheet every Monday and Friday for Trial 1, but only every Monday for Trial 2 of the experiment's duration.

'''2.6''' '''Measuring Fresh and Dry Weight'''

Fresh weight was measured multiple times a week for a longitudinal representation of growth across the length of the study. The “fresh weight” of individual plants (separated by groups) was taken (every Monday and Friday for Trial 1, but every Monday for Trial 2), using an analytical balance. Aside from the recording of wet weight measurements from the damaged plants, this process involved carefully removing the separate groups of plants from their respective tanks and placing them on a wet paper towel which was soaked in plant growth medium to minimize shock. Plants were wrapped with a paper towel when measurements weren’t taking place. Preparing plants for measurement involved carefully removing the separate groups of plants from their growth medium and gently removing the liquids or foreign debris from the plants using a paper towel. Lastly, the dry weight was taken post-study using an electronic balance after the plants had been completely dried on a paper towel using an incubator at 37 to 40°C for 10 hours. The dry weight was taken separately between groups to provide a precise measurement in comparing the biomass between groups.

'''2.7''' '''Measuring Survival Number'''

The number of plants that were alive was counted every Monday, Wednesday, and Friday of the study period. They were distributed into a Microsoft Excel spreadsheet with their corresponding treatments and sample sizes. They were then calculated as a survival number percentage in which the same size number was divided by the initial sample size and the quotient was multiplied by 100. This was done by Microsoft Excel’s basic functions.

'''2.8''' '''Safety'''

Although most substances that were used in the experiment were mostly commercial and “not hazardous” under the criteria of the federal OSHA Hazard Communication Standard 29CFR 1910.1200, and Regulation (EC) No 1272/2008 (GHS), they can still be dangerous if exposed to for a long duration of time. These substances that can pose a threat include Seachem Flourish Phosphorus, Tropica Plant Growth Premium Fertilizer, Seachem Alkaline buffer, and Seachem equilibrium. However, ammonium chloride and potassium nitrate received a 2 and 1 NEPA health rating, respectively. To reduce the exposure risk, researchers washed exposed skin before and after experimentation, wore protective clothing and gloves when in contact with the material, and ensured that skin protection, eye protection, and protective clothing were put on precisely. During the experimental process, or when researchers came into contact with the material, researchers refrained from consuming any liquids or foods. It was also ensured that eye wash stations or safety showers were within the area in which the experiment was conducted. The products used were kept away from any heat in the case of gases being released and inhaled. Safety precautions and procedures were taken referencing the MSDS sheets. The plant used in this experiment poses no known health risks to humans (assuming it is not digested in excessive amounts).

==3 DATA ANALYSIS==

Data was collected and stored using Microsoft Excel. Images were taken using a smartphone and cropped to only exhibit the extent of plant material. The mean values were analyzed using Microsoft Excel. Data was shown using scatter plots and bar graphs in which the x-values were time in days and the y-values were mean fresh weight in grams, dry weight in grams, mean root length in centimeters, total leaf amount, and survival number as a percentage. Standard deviation, standard error of the mean, and error bars were added and determined using Microsoft Excel. Trial 1 figures used standard deviation as error bars while Trial 2 figures used standard error of the mean as error bars. To calculate significance, one-way ANOVA followed by Tukey HSD was done using [https://astatsa.com/OneWay_Anova_with_TukeyHSD/ https://astatsa.com/OneWay_Anova_with_TukeyHSD/]. Significance was determined by p-values, which were considered significant when the p-value was less than 0.05.

==4 DATA AND RESULTS==

[[Image:Draft_Zhang_868588273-picture-Group 1.svg|center|600px]]

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 1. Total Fresh Weight with Time - Trial 2. Day 0 to Day 24 is shown in a scatter plot (a), and Day 0, Day 14, and Day 24 are shown in a bar graph (b). Each colored line or column represents a different ratio of ammonium to nitrate. Sole NO₃ increased the total fresh weight the most, relatively. With treatments of 1:3 and 1:1 NH₄ to NO₃, from 0 days to 14 days, total fresh weight increased, but decreased from 14 days to 24 days. Sole ammonium had the lowest total fresh weight. Total fresh weight was used to have a better representation of the sample size. The letters represent statistical significance. Error bars represent ±SEM.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
 [[Image:Draft_Zhang_868588273-image5.png|324px]] </div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 2. Dry Weight Per Treatment Group - Trial 2. The end dry weight on the last day of the experiment for each ratio of nitrate to ammonium respectively. The end samples per treatment varied among water (10 samples), 0.002 M NO<sub>3</sub><sup>- </sup>(18 samples), 1:3 (8 samples), 1:1 (3 samples), 3:1 (3 samples), and 0.002 M NH₄⁺ (0 samples). The results indicate that the treatment group with sole nitrate treatment yielded the highest end dry weight.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">

[[Image:Draft_Zhang_868588273-picture-Group 4.svg|center|600px]]
</div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 3. Mean Root Length with Time - Trial 2. Day 0 to Day 4 is shown in a scatter plot (a), and Day 0, Day 14, and Day 24 are shown in a bar graph (b). The treatment of sole ammonium maintained the highest mean root length until Day 14 when it rapidly decreased due to the treatment's decreasing survival rate. On Day 24, sole nitrate, water, and 1:3 NH₄ to NO₃ treatments consecutively displayed the highest overall mean root length. The letters represent statistical significance. Error bars represent ±SEM.</span></div>


[[Image:Draft_Zhang_868588273-picture-Group 5.svg|center|600px]]

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 4. Total Leaf Amount with Time - Trial 2. Day 0 to Day 24 is shown in a scatter plot (a), and Day 0, Day 14, and Day 24 are shown in a bar graph (b). Results indicate that NO</span> <math display="inline">{_3}</math><span style="text-align: center; font-size: 75%;"> was the only group that had a significant increase. However, 1:1 and 3:1 NH₄ to NO₃ significantly reduced the total leaf amount. The sole ammonium group had the lowest total leaf amount. The letters represent statistical significance. Error bars represent ±SEM.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">

[[Image:Draft_Zhang_868588273-picture-Group 13.svg|center|600px]]
</div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 5. Image of Plants Taken on Different Days - Trial 2. Images of plants were taken on Day 0 (a), Day 7 (b), Day 14 (c), Day 21 (d), and Day 24 (e). Images of plants were taken every Monday throughout the study duration except the picture taken on the final day before the dry weight procedure. Algae growth was seen in ammonium to nitrate ratio treatments of 1:3, 1:1, and sole ammonium on Day 21. As the experiment progressed group photos from Day 0 to Day 24 demonstrated a decreasing sample size through surface area and worsening plant conditions in all groups except the sole nitrate and water treatments.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 6. Survival Number with Time - Trial 2. The sample size is a percentage in the scatter plot from Day 0 to Day 24. Each colored line represents a different treatment group of ammonium to nitrate. The sample sizes for all groups were maintained constant from Day 9 to Day 11 for all treatments aside from the sole nitrate treatment, with sample sizes showing considerable number differences from Day 16. The trend indicates that the treatment of sole nitrate generally maintained the highest sample size while the sole ammonium treatment showed the opposite trend, relatively.</span> 
[[Image:Draft_Zhang_868588273-picture-Group 12.svg|center|600px]]
</div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 7.''' '''Total Fresh Weight with Time - Trial 1. Day 0 to Day 28 in a scatter plot (a), and Day 0, Day 14, and Day 28 shown in a bar graph (b). Each line represents a different ratio of ammonium to nitrate. There were 6 samples per group, however, the sample size declined as time passed. Graphs displaying total fresh weight were added to give a better representation of growth rate and due to varying group sample sizes. Results indicate a major decrease in total fresh weight among all groups except for 0.002M nitrate. There was no significance in any groups compared to the control group due to the small sample size. Error bars represent ±STDEV.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
 [[Image:Draft_Zhang_868588273-image30.png|324px]] </div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 8. Dry Weight Per Treatment Group - Trial 1. Each colored column represents a different ratio of ammonium to nitrate in varying sample sizes of 0.002 M NO₃(6 samples), 1:3 (1 sample), 1:1 (1 sample), 3:1 (0 samples), and 0.002 M NH₄⁺ (3 samples). The results indicate that treatment groups with a sole source of nitrogen yielded the highest dry weights.</span></div>


[[Image:Draft_Zhang_868588273-picture-Group 14.svg|center|600px]]

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 9. Mean Root Length with Time - Trial 1. Fig. 3a shows the mean root length from Day 0 to Day 28 in a scatter plot. Fig. 3b shows the mean root length of Day 0, Day 14, and Day 28 in a bar graph. Each line and colored column represent a different ratio of ammonium to nitrate shown on the legend. There were 6 samples per group, however, the sample sizes declined as time passed. The trend shows that with all nitrogen applications, mean root length generally decreased. Although insignificant, the treatment group with sole nitrate yielded the highest average root length on Day 28. There was no significance in any groups compared to the control group due to the small sample size. Error bars represent ±STDEV. </span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">

[[Image:Draft_Zhang_868588273-picture-Group 15.svg|center|600px]]
</div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 10. Total Leaf Amount with Time - Trial 1. Fig. 4a shows the total leaf amount from Day 0 to Day 28 in a scatter plot. Fig. 4b shows the total leaf amount on Day 0, Day 14, and Day 28 in a bar graph. Each line and colored column represent a different ratio of ammonium to nitrate shown on the legend. There were 6 samples per group, however, the sample sizes declined as time passed. Sole nitrate applications had the highest total leaf amount on Day 28. All other treatment groups decreased in total leaf amount as time passed. There was no significance in any groups compared to the control group due to the small sample size. Error bars represent ±STDEV.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">

[[Image:Draft_Zhang_868588273-picture- 1.svg|center|600px]]
</div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 11. Image of Plants Taken on Different Days - Trial 1. Images of plants were taken on Day 0 (a), Day 7 (b), Day 14 (c), Day 21 (d), and Day 28 (e). Images of plants were taken every Monday throughout the study duration of 28 days. Algae growth was seen in the treatments of 0.002M nitrate (Fig. 11a; Fig. 11c), 1:1 ammonium to nitrate (d), and 3:1 (e) groups. When treatment group photos were compared from Day 28 (e) to Day 0 (a), it was apparent that sample sizes decreased as plant conditions worsened.</span></div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
 [[Image:Draft_Zhang_868588273-image61.png|384px]] </div>

<div class="center" style="width: auto; margin-left: auto; margin-right: auto;">
<span style="text-align: center; font-size: 75%;">Figure 12. Survival Number with Time - Trial 1. The sample size is a percentage in the scatter plot from Day 0 to Day 28. Each colored line represents a different treatment group of ammonium to nitrate. The trend indicates that sample sizes for sole nitrate generally stayed the same throughout the experimental process. The trend also shows that the treatment group containing sole ammonium increased in sample size from Day 4, staying at a constant from Day 4 to Day 11, but decreased to half the initial sample size (from 6 to 3) near the end of the study. However, groups containing ratios of ammonium to nitrate depicted a decreasing survival percentage.</span></div>

==5 DISCUSSION==

''' '''<big>''' '''</big>The results of this study suggest that ''Phyllanthus fluitans ''has a strong preference for nitrate and an equally strong ammonium intolerance. This conclusion is supported by data from both Trials 1 and Trial 2; with both trials showing ''Phyllanthus fluitans ''to have the highest fresh and dry weight when nitrate was the sole nitrogen source (0.002M NO3<sup>-</sup>). Although Trial 1 gave intensive insight into ''Phyllanthus fluitans''’ physiology and phytoremediation suitability, it was unable to give definitive conclusions regarding the ''Phyllanthus fluitans''’ nitrogen preference due to a high mortality rate throughout most groups. After changing the methodology to decrease mortality, Trial 2 results both strengthened and conflicted with Trial 1’s findings, which allowed for conclusions regarding nitrogen preference to be made. Using data from Trial 1, the researchers of this study inferred that ''Phyllanthus fluitans''’ may have a higher tolerance for mediums containing sole nitrogen forms (either nitrate or ammonium). Although Trial 1’s data supported this preliminary hypothesis, it was made less credible when logistics regarding previous studies and evolutionary adaptation were questioned. The researchers of this study hypothesize that the inconsistent results regarding nitrogen preference were likely due to the addition of “Seachem equilibrium” in the growth medium (an additive that was significantly reduced in Trial 2). Although originally meant for providing calcium and magnesium, “Seachem equilibrium” contains a substantial amount of potassium. Previous research indicates that potassium in high enough concentrations can alleviate toxicity from ammonium and may even have synergistic effects [20]. Trial 2’s data regarding nitrogen preference was more robust due to a high mortality rate only occurring in selective groups and successfully disproved the former hypothesis. In Trial 2, the group receiving only nitrate (0.002M NO3⁻) displayed significantly higher fresh weight, dry weight, root length, and leaf number when compared to all other groups receiving either no nitrogen or nitrogen in ratios (ammonium to nitrate); indicating a strong nitrogen preference. Conversely, the group receiving solely ammonium displayed opposite patterns, with the entire group dying out at the end of the study of Trial 2. The conclusion of ''Phyllanthus fluitans''’ being intolerant to ammonium is further strengthened by Fig. 6 showing a disproportionately direct relationship between increased ammonium concentrations and lower survival rates. Additionally, the conclusion is made further evident by groups receiving ammonium having toxicity symptoms in the second half of Trial 2; with severity escalating with increasing ammonium concentrations (Fig. 5).

Although most plants prefer being in environments with substantial amounts of hardness-increasing ions such as calcium and magnesium (micronutrients that play large roles in plant physiology) [21], some plants may be intolerant to these ions in high concentrations. Using Trial 1 data, and anecdotal findings, the researchers of this study hypothesized that ''Phyllanthus fluitans ''may exhibit a sensitivity to mediums with moderate and high levels of general water hardness. In hopes to confirm the plausibility of this hypothesis, and to decrease mortality the addition of “Seachem equilibrium” (a general water hardness booster) was reduced from 10 grams to 2.5 grams, in Trial 2. As shown by the contrast between Fig. 6 and Fig. 12, the survival rate was increased for most groups in Trial 2. Although this does not prove ''Phyllanthus fluitans’'' intolerance to water hardness'' ''(due to the other changes made in Trial 2; namely, increased sample size and reduced data collection frequency.), there is a high possibility that water hardness was the main driver behind the high mortality rate in Trial 1. This is believed to be the case due to multiple anecdotal accounts in online forums, and video streaming sites showing ''Phyllanthus fluitans’'' intolerance to high general hardness. Additionally, researchers of this study found similar responses to water hardness when cultivating plants for the present study. Another important change of Trial 2 was the increased sample size (from 6 to 12 plants). Although there were drastic differences between groups in Trial 1, statistical significance was not found due to the low sample size and high mortality rate. Increased sample size, in tandem with Trial 2’s lower mortality (compared to Trial 1), facilitated data significance, which strengthened the findings of the present study.

''Phyllanthus fluitans’ ''suitability for phytoremediation can be assessed using results from both trials. Researchers state that phytoremediation-suitable plants need to adapt and survive in a variety of conditions, even those unfavorable to them. Conversely, plants that cannot survive in a variety of conditions are known to be poor at phytoremediation, often further polluting the already eutrophic wastewater [5]. Previous research shows the adaptability of floating macrophytes by their ability to change root length to accommodate differing conditions [19]. Although this same pattern was observed when studying ''Phyllanthus fluitans’, ''adapting root length was insufficient to prevent mortality amid unfavorable conditions (examples in the present study being water hardness and ammonium intolerance) (Fig.10; Fig. 12). Most researchers believe that plants with a preference for ammonium will be most suitable for phytoremediation due to the abundance of ammonium in wastewater [16]. As mentioned previously, ''Phyllanthus fluitans’ ''likely has a strong preference for nitrate and an intolerance for ammonium. This subsequently makes ''Phyllanthus fluitans’ ''a poor candidate for phytoremediation due to its preference not being in line with the contents of wastewater. Additionally, since increases in fresh weight were not seen with any group containing ammonium, significant amounts of nitrogen were likely not uptaken nor assimilated in those groups. This inference was made evident by previous studies showing a direct correlation between biomass growth and nitrogen removal [2]. ''Phyllanthus fluitans’'' water hardness sensitivity, ammonium intolerance, and strong nitrate preference indicate poor adaptability to environmental water chemistry and subsequent poor phytoremediation suitability.

Contrasting the beneficial phytoremediation use of select floating macrophyte species, they are also known to be invasive in environments with ample light and excess nutrients [17]. ''Phyllanthus fluitans ''has spread to natural bodies of water mainly in Florida, where researchers fear that its fast, aggressive growth could destroy native aquatic life and become as problematic as other invasive floating plants [22]. The results of the present study suggest that ''Phyllanthus fluitans’ ''are unlikely to become invasive in water bodies with moderate or high levels of water hardness and/or low pH (where nitrification is reduced or eliminated). However, ''Phyllanthus fluitans’'' still has the potential to become highly problematic in places where its optimal conditions (low water hardness, nitrate as primary nitrogen source, elevated levels of macronutrients, and ample light) are met due to its possibility for an immensely high growth rate; as shown with the nitrate group more than doubling its biomass at the end of Trial 2’s 24-day period (Fig.1). Environmentalists or other members in charge of managing invasive species can apply the findings of this study to predict and prevent possible ''Phyllanthus fluitans ''invasions.

==6 CONCLUSION==

Results from this study suggest that ''Phyllanthus fluitans'' possess a strong preference for nitrate, ammonium intolerance, and water hardness sensitivity. These findings subsequently gave incredible insight into ''Phyllanthus fluitans''’ phytoremediation suitability and invasive potential. Due to its poor physiological responses to environmental stress in nonoptimal water conditions and nitrate preference, ''Phyllanthus fluitans''’ is likely a poor candidate for phytoremediation. However, in optimal conditions, ''Phyllanthus fluitans''’ has the potential to become highly invasive, as made evident by its high growth rate. Outside of this study, future research should focus on determining the phytoremediation suitability of alternative plants and apply species-specific data to improve phytoremediation systems and manage invasive species.

==ACKNOWLEDGEMENTS ==

We thank Dr. L. Wang, Dr. J. Cohen, Ms. Zhu, Dr. S.X. Lin, Mr. Z. Liang, Ms. R. DePierto, Mrs. N. Jaipershad, Dr. D. Marmor. Funding comes from the FLHS Science Research Program.

==REFERENCES ==

[1] NOAA, “What is eutrophication?,” ''National Oceanic and Atmospheric Administration'', 2017. Retrieved from [https://oceanservice.noaa.gov/facts/eutrophication.html https://oceanservice.noaa.gov/facts/eutrophication.html].

[2] F. Su, Z. Li, Y. Li, S. Li, H. Chen, P. Zhuang, and F. Wang, “Removal of Total Nitrogen and Phosphorus Using Single or Combinations of Aquatic Plants,” ''International journal of environmental research and public health'', vol. 16, no. 23, pp. 4663, 2019.

[3] A. Jampeetong, H. Brix, and S. Kantawanichkul, “Effects of inorganic nitrogen forms on growth, morphology, nitrogen uptake capacity and nutrient allocation of four tropical aquatic macrophytes (Salvinia cucullata, Ipomoea aquatica, Cyperus involucratus and Vetiveria zizanioides),” ''Aquatic Botany'', vol. 97, no. 1, pp. 10-16, 2012.

[4] L. J. Fox, P. C. Struik, B. L. Appleton, and J. H. Rule, “Nitrogen phytoremediation by water hyacinth (Eichhornia crassipes (Mart.) Solms),” ''Water, Air, and Soil Pollution, ''vol. 194, pp. 199-207, 2008.

[5] N. U. M. Nizam, M. M. Hanafiah, I. M. Noor, and H. I. A. Karim, “Efficiency of Five Selected Aquatic Plants in Phytoremediation of Aquaculture Wastewater,” ''Applied Sciences'', vol. 10, no. 8, pp. 2712, 2020.

[6] A. Jampeetong, T. Sripakdee, T. Khamphaya, and S. Chairuangsri, “The Effects of Nitrogen as NO3-and NH4+ on the Growth and Symbiont (Anabaena azollae) of Azolla pinnata R. Brown,” ''Chiang Mai University Journal of Natural Sciences'', vol. 15, no. 1, pp. 11-20, 2016.

[7] F. Petersen, J. Demann, D. Restemeyer, A. Ulbrich, H. W. Olfs, H. Westendarp, and L. J. Appenroth, “Influence of the nitrate-n to ammonium-n ratio on relative growth rate and crude protein content in the duckweeds Lemna minor and Wolffiella hyaline,” ''Plants'', vol. 10, no. 8, pp. 1741, 2021.

[8] S. Boudsocq, A. Niboyet, J. C. Lata, X. Raynaud, N. Loeuille, J. Mathieu, M. Blouin, L. Abbadie, and S. Barot, “Plant Preference for Ammonium versus Nitrate: A Neglected Determinant of Ecosystem Functioning?,” ''The American Naturalist'', vol. 180, no. 1, pp. 60-69, 2012.

[9] M. Borin, and M. Salvato, “Effects of five macrophytes on nitrogen remediation and mass balance in wetland mesocosms,” ''Ecological Engineering'', vol. 46, pp. 34-42, 2012.

[10] T. Irhayyim, G. Beliczky, and M. Bercsényi, “Nutrient bioremediation efficiency of bacterial biofilms and plant based biofilters in a recirculating common carp (Cyprinus carpio L.) culture system,” ''Iranian Journal of Fisheries Sciences'', vol. 20, no. 3, pp. 828-845, 2021.

[11] J. T. Spangler, D. J. Sample, L. J. Fox, J. S. Owen Jr., and S. A. White, “Floating treatment wetland aided nutrient removal from agricultural runoff using two wetland species,” ''Ecological Engineering'', vol. 127, pp. 468-479, 2019.

[12] A. I. Török, A. Moldovan, E. Kovacs, O. Cadar, A. Becze, E. A. Levei, and E. Neag, “Lithium accumulation in Salvinia natans free-floating aquatic plant,” ''Materials'', vol. 15, no. 20, pp. 7243, 2022.

[13] O. Olkhovych, N. Svietlova, Y. Konotop, O. Karaushu, and S. Hrechishkina, “Removal of metal nanoparticles colloidal solutions by water plants,” ''Nanoscale Research Letters'', vol. 11, pp. 1-7, 2016.

[14] U. Rozman, A. J. Kokalj, A. Dolar, D. Drobne, and G. Kalčíková, “Long-term interactions between microplastics and floating macrophyte Lemna minor: The potential for phytoremediation of microplastics in the aquatic environment,” ''Science of the Total Environment'', vol. 831, pp. 154866, 2022.

[15] Y. S. Chang, Y. J. Chang, C. T. Lin, M. C. Lee., C. W. Wu, and Y. H. Lai, “Nitrogen fertilization promotes the phytoremediation of cadmium in Pentas lanceolata,” ''International Biodeterioration & Biodegradation'', vol. 85, pp. 709-714, 2013.

[16] S. Paolacci, V. Stejskal, D. Toner, and M. A. Jansen, “Wastewater valorisation in an integrated multitrophic aquaculture system; assessing nutrient removal and biomass production by duckweed species,” ''Environmental Pollution'', vol. 302, pp. 119059, 2022.

[17] M. J. McCann, “Response diversity of free-floating plants to nutrient stoichiometry and temperature: growth and resting body formation,” ''PeerJ'', vol. 4, pp. e1781, 2016.

[18] C. Gu, F. Li, J. Xiao, S. Chu, S. Song, and M. H. Wong, “A novel submerged Rotala rotundifolia, its growth characteristics and remediation potential for eutrophic waters,” ''Scientific Reports'', vol. 9, no. 1, pp. 14855, 2019.

[19] Y. Jin, Q. Zhang, L. M. Zhang, N. F. Lei, J. S. Chen, W. Xue, and F. H. Yu, “Distinct responses of frond and root to increasing nutrient availability in a floating clonal plant,” ''Plos one'', vol. 16, no. 10, pp. e0258253, 2021.

[20] G. Fang, J. Yang, T. Sun, X. Wang, and Y. Li, “Evidence that synergism between potassium and nitrate enhances the alleviation of ammonium toxicity in rice seedling roots,” ''Plos one'', vol. 16, no. 9, pp. e0248796, 2021.

[21] P. J. White, and M. R. Broadley, “Calcium in plants”, ''Annals of botany'', vol. 92, no. 4, pp. 487-511, 2003.

[22] G. J. Wilder, and M. P. Sowinski, “Phyllanthus fluitans Benth.(Euphorbiaceae). A newly reported invasive species in Florida,” ''Wildland Weeds Fall'', vol. 13, no. 4, pp. 14-15, 2010.