Comparative Account of Tolerance of Different Submerged Macrophytes to Ammonia Nitrogen in the Water Column: Implications for Remediation and Ecological Rehabilitation of Nutrient-Enriched Aquatic Environments

Abstract

This study aims to select the most suitable submerged plants for the remediation and ecological rehabilitation of nutrient-enriched aquatic environments. The experiment selected Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii as research objects. An artificial outdoor pot experiment was conducted with six different levels of ammonia nitrogen: 2, 4, 6, 8, 12, and 16 mg/L. The present study measured the physiological and growth parameters of submerged macrophytes under varying ammonia nitrogen concentrations. The response characteristics of plants to ammonia nitrogen stress were analyzed, and the tolerance thresholds of different submerged macrophyte species to ammonia nitrogen were determined. This enabled us to screen for ammonia nitrogen-tolerant pioneer species suitable for water ecological restoration in eutrophic water bodies. The experiment spanned 28 days. The results showed that the maximum suitable concentration and maximum tolerance concentration of ammonia nitrogen for Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii were 2, 4, and 4 mg/L and 4, 12, and 8 mg/L. Submerged plants can grow normally within their maximum ammonia nitrogen tolerance concentration. When the concentration exceeds the maximum tolerance level, the growth of submerged plants is severely stressed by ammonia nitrogen. Low ammonia nitrogen concentrations promote the growth of submerged macrophyte biomass and chlorophyll content as well as the accumulation of dry matter in plants, while high ammonia nitrogen concentrations damage the antioxidant enzyme system and inhibit the growth of submerged plants. The tolerance of the three submerged macrophytes to ammonia nitrogen is as follows: Myriophyllum verticillatum > Elodea nuttallii > Vallisneria natans. Therefore, Myriophyllum verticillatum should be chosen as the ammonia nitrogen-tolerant pioneer species in the ecological restoration of eutrophic water bodies. The research results can provide a theoretical basis for the application of aquatic macrophytes in the treatment of eutrophic water bodies and ecological restoration.

1. Introduction

In recent years, with the development of industry and agriculture and the acceleration of urbanization, large amounts of ammonia nitrogen nutrients have entered rivers and lakes, causing water eutrophication. This not only disrupts the structure and function of aquatic ecosystems but also severely hinders the sustainable development of the socio-economy, becoming a major environmental issue threatening the health of aquatic ecosystems and drinking water safety. According to the first-quarter 2025 national surface water environmental quality monitoring data from the Ministry of Ecology and Environment, ammonia nitrogen has become one of the primary exceedance indicators in major river basins, such as the Yangtze River, Yellow River, and Pearl River, and together with total phosphorus and chemical oxygen demand, it has exacerbated the risk of eutrophication and ecological degradation in key lakes. To systematically address the challenges of ammonia nitrogen pollution, the Ministry of Finance has allocated 56 billion yuan in special environmental governance funds, with 10.7 billion yuan allocated for water pollution prevention and control (second batch).

Currently, common restoration techniques for eutrophic water bodies, utilized both domestically and internationally, primarily include physical remediation, chemical remediation, and bioremediation [1]. Among these, bioremediation is favored for its high ecological efficiency and lower economic costs compared to other methods [2]. Planting submerged plants has emerged as a widely adopted bioremediation strategy for restoring degraded water bodies. As primary producers in aquatic ecosystems, submerged plants play a critical role in reducing nitrogen, phosphorus, and other nutrient concentrations within the water while releasing chemosensory compounds that inhibit algal growth. Additionally, they enhance the spatial ecological niches of underwater environments and provide essential habitats for aquatic organisms [3,4]. Research has demonstrated that nutrient salt removal efficiencies vary significantly across different types of aquatic plants. Specifically, emergent plants, floating plants, and submerged plants achieve removal efficiencies of approximately 5%, 25%, and 40%, respectively, with submerged plants proving to be the most effective in nutrient salt extraction [5]. Submerged species, including Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii, are frequently employed in water purification, submerged vegetation restoration, and eutrophic waterbody remediation due to their high adaptability to environmental conditions. Song Yuzhi et al. [6] explored the effects of Vallisneria natans on waterbody eutrophication through controlled indoor simulations while also examining its physiological responses to nitrogen and phosphorus nutrients during eutrophication. Their findings indicate that Vallisneria natans responds swiftly to variations in nitrogen and phosphorus concentrations in water bodies, showing optimal growth in eutrophic conditions. Additionally, Feng You et al. [7] conducted an indoor simulation with eight commonly used aquatic plants to evaluate their nitrogen and phosphorus absorption capacities in livestock and poultry wastewater as well as their purification efficiencies in waters with varying nitrogen and phosphorus levels. Their study found that fast-growing Myriophyllum verticillatum demonstrated superior purification capabilities compared to slower-growing species, such as Elodea nuttallii and Ceratophyllum demersum. The aforementioned studies illustrate that the restoration and reconstruction of aquatic vegetation dominated by submerged plants are a key approach to controlling and managing eutrophication in water bodies. Accordingly, this paper focuses on three submerged plant species with high tolerance to ammonia nitrogen, Vallisneria natans, Myriophyllum verticillatum [8], and Elodea nuttallii, which were selected as test materials.

Nitrogen is a major contributor to the eutrophication of water bodies [9]. Studies have demonstrated that, as the concentration of nitrogen sources in aquatic environments increases, submerged plants exhibit heightened sensitivity to ammonia nitrogen, which imposes a significantly stronger stress effect compared to nitrate nitrogen [10]. Excessive absorption of ammonia nitrogen by submerged plants can lead to ammonia toxicity [11]. Moreover, the presence of ammonia nitrogen has been shown to inhibit the uptake of nitrate nitrogen [12]. Research by Jampeetong et al. [13] and Dai et al. [14] indicates that the removal rate of ammonia nitrogen by aquatic plants is significantly higher than that of nitrate nitrogen. The tolerance ranges and physiological responses of aquatic plants to nutrient concentrations in the water column and substrate vary under different habitat conditions. When the ammonia nitrogen concentration is below 15 mg/L, the efficiency of Vallisneria natans in removing ammonia nitrogen is significantly lower than that of Najas marina and Potamogeton perfoliatus, and the plants are able to grow normally. Conversely, when the concentration exceeds 15 mg/L, the chlorophyll and protein content decreases, thereby affecting the plants’ ability to carry out normal photosynthesis [15]. Zhou Jinbo et al. [16] found that the maximum tolerance concentrations of ammonia nitrogen for Vallisneria natans, Hydrilla verticillata, Ceratophyllum demersum, and Najas minor were 6, 6, 4, and 2 mg/L, respectively. Their relative growth rates followed a pattern of initially increasing and then decreasing with rising ammonia nitrogen concentrations. Once the maximum tolerance concentration was exceeded, submerged plants exhibited a decline in chlorophyll and soluble sugar content. At present, the purification effect of submerged plants on ammonia nitrogen in water bodies has been a focus of many scholars. However, limited attention has been given to the tolerance range of submerged plants under varying ammonia nitrogen concentrations and their physiological feedback responses. Typically, the concentration of ammonia nitrogen in eutrophic water bodies and aquaculture wastewater exceeds the thresholds defined by the Environmental Quality Standards for Surface Water’ for Class V water (2 mg/L) [17,18]. In practical applications, understanding the ammonia nitrogen tolerance of different submerged plant species allows for the determination of their adaptability under varying conditions. This knowledge facilitates the targeted planting of submerged species capable of withstanding specific ammonia nitrogen concentrations, thereby improving the management and remediation of eutrophic waters and aquaculture wastewater. This approach not only reduces the risk of plant mortality caused by indiscriminate planting but also offers high ecological benefits and cost-effective solutions.

In summary, relevant studies have mostly focused on the purification effect of submerged plants on ammonia nitrogen in water bodies, but there have been relatively few studies on the tolerance range of submerged plants under different ammonia nitrogen loads and on the physiological characteristics of submerged plants. Therefore, this study selected three submerged plants, Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii, each exhibiting distinct levels of pollution tolerance and competitive growth advantages, as subjects. A simulated outdoor aquatic ecosystem was constructed to evaluate the growth conditions and physiological responses of these submerged plants under varying ammonia nitrogen concentrations. By analyzing the response characteristics of the plants under ammonia nitrogen stress, this study aims to elucidate the mechanisms by which elevated ammonia nitrogen levels exert stress on submerged plants and to establish the tolerance thresholds of each species. The results are expected to identify ammonia nitrogen-tolerant pioneer species suited to the ecological restoration of eutrophic waters, thereby providing a theoretical foundation for the application of aquatic plants in the remediation and ecological rehabilitation of nutrient-enriched aquatic environments.

2. Materials and Methods

2.1. Overview of the Study Area

This experiment was conducted at the experimental base of Three Gorges University, located at Quanhe Reservoir in Dangyang City, Hubei Province, China (30.41° N, 111.38° E). Dangyang City lies within the transition zone between the mountains of western Hubei and the Jianghan Plain, resulting in significant topographical variation. The region features a subtropical monsoon climate characterized by four distinct seasons, high humidity, and synchronous rainfall and heat patterns. It also serves as a transition area between northern and southern climates [19]. The area experiences mild temperatures and abundant sunshine, with an average annual total of 1850 h of sunlight, a sunshine percentage of 41.8%, a frost-free period of approximately 268 days, and an average annual temperature of 16.4 °C.

2.2. Test Material

In this experiment, Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii were selected as test species. The submerged plants used in the experiment were purchased from the Jingzhou Aquatic Plant Cultivation Base. During short-distance transport, the plants were kept in water-filled foam boxes with ice packs to maintain a temperature between 15 °C and 25 °C. Soft materials were used to cushion the plants and prevent them from colliding with each other during transport. The plants were transplanted into an artificially created small pond and cultivated for one month to adapt. Vallisneria natans belongs to Hydrocharitaceae. It has no erect stem, and the basal leaves are strip-shaped, translucent, and serrated. It is distributed in the still waters of the country and is propagated by stolons. Myriophyllum verticillatum belongs to Haloragidaceae, with cylindrical stems and submerged leaves with 3-4 whorls and a pinnate deep fissure. It is a national second-class protected species, found in shallow water swamps, and relies on winter buds to overwinter. Elodea nuttallii belongs to Hydrocharitaceae. Its stem is brittle and easy to break. Its three-wheeled linear leaves are serrated. The male flowers are open and floating. It is native to America and has strong cold resistance. Before the experiment, the plants with good growth and consistent size were selected, and the roots were fixed by a planting ring and cultured in diluted improved Hoagland culture medium for 7 days. The chemical Reagents (analytically pure) used in this experiment were supplied by Sinopharm Chemicals agent Co., Ltd. in China. The formula is shown in

Table 1. Improved Hoagland nutrient solution.

Reagent	Concentration/mg·L−1	Reagent	Concentration/mg·L−1
CaCl2	22.20	H3BO3	0.830
MgSO4	24.65	MnSO4	22.300
NH4CI	6.80	ZnSO4	8.600
KH2PO4	2.65	CuSO4	0.025
EDTA	18.65	CoCI2	0.025
FeSO4·7H2O	13.90	Na2MoO4·2H2O	0.250

.

2.3. Experimental Design

The ammonia nitrogen tolerance test was conducted in pots with a bottom diameter of 0.26 m and a height of 0.31 m, each containing 22 L of dechlorinated tap water. A 6 cm layer of quartz sand was added to the bottom of each pot to secure the plants. Five plants, trimmed to a uniform length of 20 cm while retaining their root systems, were placed in each pot and then positioned outdoors for the experiment. Ammonia nitrogen concentrations were set at six levels, 2, 4, 6, 8, 12, and 16 mg/L, using ammonium chloride as the nitrogen source. These levels were labeled N1, N2, N3, N4, N5, and N6, respectively, while a control group (CK) contained no added ammonia nitrogen. Each treatment level included three replicates, with phosphorus concentration maintained at 0.02 mg/L, following lake eutrophication standards. The experiment spanned 28 days. The culture medium was replaced every 4 days to maintain the set ammonia nitrogen concentration, and distilled water was replenished every 2 days.

2.4. Assay of Ammonia Nitrogen Concentration and Chlorophyll Content

The method for determining ammonia nitrogen content uses the Nessler reagent colorimetric method [20]. A 50 mL water sample was pipetted into a colorimetric tube. Then, 1 mL of potassium sodium tartrate solution was added and mixed, followed by 1.5 mL of Nessler’s reagent with thorough mixing. After standing for 10 min, absorbance was measured at 420 nm in a 20 mm path length cuvette against a distilled water blank. Ammonia concentration was calculated using a standard curve.

Chlorophyll content was determined via acetone extraction. Fresh leaf fragments (0.1 g) were weighed into a pre-cooled mortar. After adding quartz sand and 2–3 mL of 80% acetone, the mixture was ground in an ice bath until homogenized. The homogenate was quantitatively transferred to a 25 mL brown volumetric flask, rinsed with 80% acetone, and diluted to volume. The extract was protected from light, incubated for 1 h, and then centrifuged (4000 rpm, 10 min). Absorbance of the supernatant was measured at 645 nm and 663 nm in a 1 cm cuvette against an 80% acetone blank. Chlorophyll concentrations were calculated from absorbance values.

2.5. Assay of Malondialdehyde Content and Enzyme Activity

Malondialdehyde (MDA) content was measured using the thiobarbituric acid (TBA) method [21]. Fresh leaf tissue (1 g, finely chopped) was homogenized with 2 mL of 10% trichloroacetic acid (TCA) and quartz sand in a mortar. After grinding to homogeneity, an additional 8 mL of 10% TCA was added, and grinding continued. The homogenate was centrifuged (4000 r·min⁻¹, 10 min), and the supernatant was collected. A 2 mL aliquot of supernatant was transferred to a clean tube, mixed with 2 mL of 0.6% TBA solution, and incubated in a boiling water bath for 15 min. The mixture was cooled rapidly to room temperature and centrifuged, and the supernatant absorbance was measured at 532 nm, 600 nm, and 450 nm. MDA concentration was calculated from the absorbance values.

Superoxide dismutase (SOD) activity was evaluated via the photochemical reduction method using nitrogen blue tetrazolium (NBT), and catalase (CAT) activity was determined using a UV-absorbance assay [22]. An aliquot of enzyme extract was added to a reaction mixture containing 50 mmol/L phosphate buffer (pH 7.8), 13 mmol/L methionine, 75 μmol/L NBT, 2 μmol/L riboflavin, and 0.1 mmol/L EDTA. After thorough mixing, reactions were incubated under 4000 lx illumination for 20 min. Reactions were terminated by light exclusion, and absorbance was measured at 560 nm. SOD activity was calculated from the absorbance values. Enzyme extract was added to the pre-chilled reaction mixture and mixed immediately, and the initial absorbance was recorded at 240 nm. The final absorbance was measured exactly 1 min post-mixing. CAT activity was calculated from the absorbance change.

2.6. Assay of Relative Growth Rate and Dry-Wet Ratio

Each measurement was performed in triplicate, and the average value was recorded. To determine the dry-wet ratio, plant samples were dried at 105 °C until a constant weight was achieved. The dry matter to fresh weight ratio was calculated by comparing the masses before and after drying [23]. The relative growth rate of the plants was calculated using the biomass values at the beginning and end of the experiment according to the following formula:

$$RGR={\displaystyle \frac{{{\displaystyle \ln W}}_2-{{\displaystyle \ln W}}_1}{t}}$$

(1)

where RGR represents the relative growth rate of bitterbrush, g/(g·d); W₁ denotes the initial biomass of bitterbrush, g; W₂ is the biomass of bitterbrush at the end, g; and t is the test period, d.

2.7. Statistical Analysis

Experimental data were processed and organized using Microsoft Excel 2016. Prior to analysis, all datasets underwent validation for normality and homogeneity of variance, with both assumptions satisfied (p > 0.05). One-way analysis of variance (ANOVA) was employed to examine the effects of different ammonium nitrogen concentrations on submerged macrophyte parameters: relative growth rate, dry/wet ratio, chlorophyll content, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and catalase (CAT) activity. Where significant differences were detected, Duncan’s multiple range test was applied for post hoc comparisons. Significant differences (p < 0.05) among ammonium concentration levels are denoted by lowercase letters in the figures. Pearson correlation analysis was conducted to assess relationships among the measured physiological indices. All statistical analyses were performed using SPSS Statistics 23.0, with data visualization implemented in Origin 2021.

3. Results

3.1. Effect of Ammonia Nitrogen Concentration on Relative Growth Rate of Submerged Plants

The impact of different ammonia nitrogen concentrations on the relative growth rate of submerged plants is shown in Figure 1. As shown in Figure 1, after 28 days of exposure to varying concentrations of ammonia nitrogen, the relative growth rates of Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii exhibited an initial increase followed by a decrease as the ammonia nitrogen concentration increased. When the concentration exceeded a certain threshold, the relative growth rates became negative. The peak relative growth rates differed among the submerged plants, with Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii reaching maximum values at 2 mg/L, 4 mg/L, and 4 mg/L, respectively, achieving growth rates of 0.17, 2.72, and 0.76 g/d. These values were significantly higher than those observed at other ammonia nitrogen concentrations (p < 0.05). When ammonia nitrogen concentrations were below these optimal levels, the plants showed positive growth rates, indicating they could grow normally within this range. However, above the threshold concentration, a decline in growth rates was observed. Specifically, Vallisneria natans and Elodea nuttallii displayed negative growth rates when ammonia nitrogen concentrations exceeded 8 mg/L, with minimum growth rates of −0.216 g/(g·d) and −0.159 g/(g·d) at 16 mg/L, respectively. In contrast, Myriophyllum verticillatum maintained a stable growth rate at 12 mg/L and 16 mg/L, suggesting a lower sensitivity to high ammonia nitrogen levels compared to Vallisneria natans and Elodea nuttallii. At these elevated concentrations, all three submerged plant species were unable to sustain normal growth due to the stress induced by the high ammonia nitrogen levels.

Analyzing the growth conditions of various aquatic plants revealed that Vallisneria natans shed all mature leaves at ammonia nitrogen concentrations of 8, 12, and 16 mg/L, suggesting that Vallisneria natans experiences substantial stress damage under high ammonia nitrogen levels. Myriophyllum verticillatum exhibited healthy growth across all treatment groups, indicating a greater adaptive capacity to different ammonia nitrogen concentrations. Elodea nuttallii showed no signs of mortality or decay in any treatment group, suggesting that it can survive long-term at various ammonia nitrogen levels.

3.2. Effect of Ammonia Nitrogen Concentration on Dry-Wet Ratio of Submerged Plants

The impact of different ammonia nitrogen concentrations on the dry-wet ratio of submerged plants is shown in Figure 2. The dry-wet ratio serves as an indicator of dry matter accumulation in plants, offering insight into plant growth conditions. As shown in Figure 2, after 28 days of treatment with varying concentrations of ammonia nitrogen, the dry-wet ratios of the aquatic plants in each treatment group differed significantly, displaying a trend of an initial decrease followed by an increase with rising ammonia nitrogen levels. Relative to the control group, the changes in the dry-wet ratio of Vallisneria natans were −2.4%, 7.2%, 9.2%, 11.2%, 15.2%, and 18.2%. At an ammonia nitrogen concentration of 2 mg/L, Vallisneria natans exhibited lower dry matter accumulation, indicating favorable growth conditions with efficient nutrient assimilation and uptake. Conversely, at 16 mg/L, Vallisneria natans showed the highest level of dry matter accumulation, suggesting that elevated ammonia nitrogen concentrations adversely impact dry matter accumulation, which may inhibit normal plant growth. Compared with the control group, the changes in the dry-wet ratios across the experimental groups of Elodea nuttallii were −9.6%, −2.1%, 1.3%, 7.3%, 9.5%, and 9.7% (p < 0.05). When the concentration was lower than 4 mg/L, the dry matter accumulation of Elodea nuttallii was less, Elodea nuttallii grew well in this range of ammonia nitrogen, and the assimilation and absorption of nutrients were more.

When the concentration of ammonia nitrogen was 16 mg/L, the dry matter accumulation of Elodea nuttallii was the highest, and the dry-wet ratio was 7.36%. The growth of Elodea nuttallii was inhibited at this concentration. Compared to the control group, the dry-wet ratios for each Myriophyllum verticillatum test group were −13.0%, −17.1%, −6.5%, 3.8%, 4.5%, and 8.9%. When ammonia nitrogen concentrations were below 6 mg/L, Myriophyllum verticillatum showed low dry matter accumulation, suggesting good growth conditions and efficient nutrient assimilation. However, maximum dry matter accumulation was observed at 16 mg/L, indicating growth inhibition at this concentration. For Myriophyllum verticillatum, the highest dry matter accumulation occurred at 16 mg/L, suggesting reduced nutrient assimilation and uptake at this ammonia nitrogen concentration and indicating that Myriophyllum verticillatum growth was significantly stressed by high ammonia nitrogen levels.

3.3. Effect of Ammonia Nitrogen Concentration on Chlorophyll of Submerged Plants

The impact of different ammonia nitrogen concentrations on the chlorophyll content of submerged plants is shown in Figure 3. As shown in Figure 3, after 28 days of ammonia nitrogen treatment, the chlorophyll content in Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii generally followed a trend of initially increasing and then decreasing with rising ammonia nitrogen concentrations. When the ammonia nitrogen concentration was below 2 mg/L for Vallisneria natans and 6 mg/L for Myriophyllum verticillatum, chlorophyll content displayed a consistent increase over time, reaching a peak on day 28 at these concentrations. At this peak, chlorophyll content in Vallisneria natans and Myriophyllum verticillatum was 0.40 mg/g and 0.14 mg/g higher, respectively, than in the control group on day 28, indicating favorable growth conditions within these ammonia nitrogen levels (p < 0.05). In the treatment groups with ammonia nitrogen concentrations of 4, 8–16, and 2–8 mg/L, chlorophyll content in the aquatic plants exhibited a pattern of increasing followed by decreasing. For Vallisneria natans and Myriophyllum verticillatum, chlorophyll content began to decline steadily at concentrations above 6 mg/L and 12 mg/L, respectively, reaching a minimum at 16 mg/L on day 28. At this point, chlorophyll content was 0.648 mg/g and 0.476 mg/g lower, respectively, than in the control group. When the ammonia nitrogen concentration was below 4 mg/L, 12 mg/L, and 8 mg/L for Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii, respectively, chlorophyll content in these submerged plants initially increased, indicating a short-term promotion effect. However, as exposure to high ammonia nitrogen concentrations continued, the plants were unable to withstand the prolonged toxicity. This resulted in a steady decline in chlorophyll content, with higher ammonia nitrogen concentrations accelerating the damage. Consequently, chlorophyll content in the plants continued to decrease under sustained high ammonia nitrogen stress.

3.4. Effect of Ammonia Nitrogen Concentration on Malondialdehyde (MDA) in Submerged Plants

The impact of different ammonia nitrogen concentrations on the MDA content of submerged plants is shown in Figure 4. As shown in Figure 4, increasing ammonia nitrogen levels led to an initial rise in MDA content in Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii, followed by a subsequent decline. At ammonia nitrogen concentrations of 2 mg/L for Vallisneria natans and 2–4 mg/L for Myriophyllum verticillatum and Elodea nuttallii, MDA content increased progressively over time, reaching maximum values of 1.703, 0.275, and 11.021 µmol/g, respectively. This indicates that low ammonia nitrogen concentrations exert a stress effect on submerged plants, with Elodea nuttallii exhibiting a stronger response to ammonia nitrogen stress than Vallisneria natans and Myriophyllum verticillatum (p < 0.05). When ammonia nitrogen concentrations exceeded 2 mg/L for Vallisneria natans and 4 mg/L for both Myriophyllum verticillatum and Elodea nuttallii, the MDA content in each submerged plant initially increased, reaching a peak between days 14 and 21, before subsequently decreasing. This shift suggests that, as ammonia nitrogen levels rose, the stress on the submerged plants intensified, with maximum MDA reduction observed at 16 mg/L. Specifically, at this concentration, MDA content decreased by approximately 67%, 47%, and 47% from peak values for Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii, respectively, with reductions of 0.652, 0.502, and 4.793 µmol/g compared to the control group on day 28. These findings indicate that all three submerged plant species experienced significant stress under high ammonia nitrogen conditions (p < 0.05).

3.5. Effect of Ammonia Concentration on Superoxide Dismutase (SOD) and Catalase (CAT) in Submerged Plants

The impact of different ammonia nitrogen concentrations on SOD activity in submerged plants is shown in Figure 5. As shown in Figure 5, the SOD activities of Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii initially increased rapidly with rising ammonia nitrogen concentrations, followed by a gradual decline. At ammonia nitrogen concentrations of 2 mg/L for Vallisneria natans and 2–8 mg/L for Myriophyllum verticillatum and Elodea nuttallii, SOD activities displayed an upward trend over time, peaking on day 28. Notably, Myriophyllum verticillatum exhibited a much stronger response within this ammonia nitrogen range compared to Vallisneria natans and Elodea nuttallii. The SOD activity in Myriophyllum verticillatum reached a maximum of 198.3 µmol/g at an ammonia nitrogen concentration of 8 mg/L on day 28, an increase of 161.077 µmol/g from day 7. This suggests that the plant’s defense system was activated early in the experiment, likely to mitigate cellular damage by enhancing SOD activity. When ammonia nitrogen concentrations exceeded 2 mg/L for Vallisneria natans and 8 mg/L for both Myriophyllum verticillatum and Elodea nuttallii, SOD activity initially increased and then declined over time, with peak activity shifting from day 21 to day 14. This suggests that the three submerged plants could adapt to a specific range of ammonia nitrogen levels; however, beyond this threshold, the plants’ defense systems were unable to effectively scavenge intracellular oxygen radicals, leading to an imbalance and progressively increasing cellular damage. Consequently, SOD activity declined. By the end of the 28-day experiment, the SOD activity of all three submerged plant treatment groups was significantly different from that of the control group (CK) (p < 0.05).

The impact of different ammonia nitrogen concentrations on the CAT activity of submerged plants is shown in Figure 6. As shown in Figure 6, CAT activities in Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii exhibited a trend of initially increasing and then decreasing with rising ammonia nitrogen concentrations. In the treatment groups where ammonia nitrogen concentrations were 2–6 mg/L for both Myriophyllum verticillatum and Elodea nuttallii, CAT activity increased over time. This pattern suggests that the ammonia nitrogen concentration induced stress in aquatic plants from the onset of the experiment, prompting an increase in CAT activity to scavenge oxygen radicals and thereby mitigate ammonia-induced damage. When ammonia nitrogen concentrations in the treatment groups were 2–8 mg/L for Vallisneria natans, 8–12 mg/L for Myriophyllum verticillatum, and 8–12 mg/L for Elodea nuttallii, CAT activity followed a pattern of an initial increase followed by a decrease. This response suggests that the three submerged plants can adapt to a specific range of ammonia nitrogen levels; however, beyond this threshold, the degree of damage deepens. At higher ammonia nitrogen concentrations of 12–16 mg/L for Vallisneria natans and 16 mg/L for both Myriophyllum verticillatum and Elodea nuttallii, CAT activity continued to decline over time, indicating that elevated ammonia nitrogen levels caused irreversible damage in the early stages, thereby limiting the plants’ ability to activate defense mechanisms against sustained stress. By the end of the 28-day experiment, the CAT activity of all three submerged plant treatment groups was significantly different from that of the control group (CK) (p < 0.05).

3.6. Analysis of Various Physiological (Chemical) Indicators of Submerged Plants Under Ammonia Stress

As shown in

Table 2. The correlation of physiological (biochemical) parameters of three submerged plants under ammonia nitrogen stress.

Submerged Plants	Parameter	Relative Growth Rate	Dry-Wet Ratio	Chlorophyll	MDA	SOD	CAT
Vallisneria natans	Relative growth rate	1
	dry-wet ratio	−0.901 **	1
	Chlorophyll	0.936 **	−0.991 **	1
	MDA	0.176	−0.453	0.342	1
	SOD	0.113	−0.431	0.343	0.797 *	1
	CAT	0.803 *	−0.930 **	0.917 **	0.505	0.589	1
Myriophyllum verticillatum	Relative growth rat	1
	dry-wet ratio	−0.964 **	1
	Chlorophyll	0.819 *	−0.761 *	1
	MDA	0.705	−0.760 *	0.843 *	1
	SOD	0.447	−0.321	0.773 *	0.581	1
	CAT	0.278	−0.123	0.455	0.267	0.879 **	1
Elodea nuttallii	Relative growth rat	1
	dry-wet ratio	−0.912 **	1
	Chlorophyll	0.896 **	−0.762 *	1
	MDA	0.719	−0.599	0.908 **	1
	SOD	−0.202	0.142	0.144	0.472	1
	CAT	0.842 *	−0.779 *	0.933 **	0.910 **	0.330	1

* Indicates significant correlation at the 0.05 level (two-tailed), ** indicates significant correlation at the 0.01 level (two-tailed).

, significant correlations were observed between six physiological (chemical) indicators of submerged plants under ammonia nitrogen stress. Relative growth rate and dry-wet ratio were both negatively correlated at a highly significant level (p < 0.01). Chlorophyll content in Myriophyllum verticillatum and Elodea nuttallii showed a positive, significant correlation with relative growth rate and a negative, significant correlation with the dry-wet ratio (p < 0.05), with Vallisneria natans exhibiting high significance for these correlations (p < 0.01). The correlation coefficients for Vallisneria natans were 0.843 and 0.908. CAT activity in Vallisneria natans displayed a significant positive correlation with relative growth rate (p < 0.05) and a highly significant correlation with both the dry-wet ratio and chlorophyll content (p < 0.01), with correlation coefficients of −0.930 and 0.917. For Elodea nuttallii, CAT activity showed a significant correlation with relative growth rate and the dry-wet ratio (p < 0.05), with correlation coefficients of 0.842 and −0.779, and a highly significant correlation with chlorophyll and MDA (p < 0.01), with correlation coefficients of 0.933 and 0.910, respectively.

4. Discussion

This study found that the relative growth rate, chlorophyll content, MDA content, and SOD and CAT activities of Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii increased first and then decreased with the increase in ammonia nitrogen concentration, while the change in dry-wet ratio was the opposite. The maximum suitable concentration and maximum tolerance concentration of ammonia nitrogen in Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii were 2 mg/L, 4 mg/L, and 4 mg/L and 4 mg/L, 12 mg/L, and 8 mg/L. When the concentration of ammonia nitrogen is lower than its maximum tolerated concentration, the plant can grow normally. Once the concentration of ammonia nitrogen exceeds the threshold, submerged plants will become severely stressed. The peak activity of chlorophyll a and antioxidant enzymes (SOD and CAT) will occur earlier than usual. The leaves of the plants will turn yellow and rot. In severe cases, the plants will die. The comprehensive comparison showed that the tolerance of Myriophyllum verticillatum to ammonia nitrogen was better than that of Vallisneria natans and Elodea nuttallii.

High ammonia nitrogen concentrations in water bodies can exert stress on aquatic plants, disrupting their normal physiological activities and contributing to the decline of aquatic vegetation during eutrophication in rivers and lakes [24,25]. Submerged plants, which grow primarily below the water surface, are particularly sensitive to ammonia nitrogen levels. A comprehensive analysis of physiological indicators throughout the test period revealed that the optimal ammonia nitrogen concentrations for Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii were 2 mg/L, 4 mg/L, and 4 mg/L, respectively. At or below these optimal concentrations, the relative growth rate, dry-wet ratio, chlorophyll content, malondialdehyde (MDA) levels, and enzyme activities (SOD and CAT) in submerged plants increased. When the ammonia nitrogen concentration does not exceed the maximum optimal concentration, the relative growth rate, dry-wet ratio, chlorophyll content, malondialdehyde (MDA) levels, and enzyme activity (SOD, CAT) of submerged plants show an upward trend. Within the maximum ammonia nitrogen tolerance range, submerged plants can maintain normal growth. Once the ammonia nitrogen concentration exceeds this threshold, both the peak chlorophyll a content and the peak activity of the antioxidant enzyme system occur prematurely, indicating that plant growth is severely stressed by ammonia nitrogen. A comprehensive comparison shows that Myriophyllum verticillatum has stronger tolerance to ammonia nitrogen than Vallisneria natans and Elodea nuttallii.

The maximum ammonia nitrogen tolerance concentrations for Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii were 4 mg/L, 12 mg/L, and 8 mg/L, respectively. When ammonia nitrogen concentrations were between the optimal and maximum tolerance levels, the relative growth rate of the submerged plants showed a decline. Vallisneria natans, in particular, exhibited a negative growth rate, with increased dry matter accumulation and reduced chlorophyll content compared to the control group. The chlorophyll peak, which initially occurred on day 28, shifted to day 14. This indicates that submerged plants have reached their tolerance limit and have rapidly activated their defense mechanisms. Plants respond to adverse environments by adjusting their metabolism, and the degree of stress experienced by submerged plants becomes more severe as ammonia nitrogen concentrations increase. At concentrations exceeding the maximum tolerance, Vallisneria natans and Elodea nuttallii displayed negative growth rates, while Myriophyllum verticillatum’s growth remained stable. In all three species, dry matter accumulation increased gradually, nutrient assimilation and uptake decreased, and chlorophyll content at the experiment’s end was significantly lower than in the control group. Under these conditions, the antioxidant enzyme defense system was unable to effectively scavenge intracellular oxygen radicals, preventing normal plant growth. The maximum tolerance capacity of submerged plants to ammonia nitrogen observed in this study was lower than that reported in similar studies [26,27], likely due to variations in tolerance ranges and physiological responses of aquatic plants to nutrient concentrations in the water column and substrate under differing habitat conditions.

Relative growth rate and dry-wet ratio are key indicators of plant growth conditions. Plants in good growth conditions, with higher nutrient assimilation and uptake, tend to accumulate less dry matter, resulting in a decrease in the dry-wet ratio. Conversely, as dry matter accumulation increases to support future growth, the dry-wet ratio rises [28,29]. In this study, the relative growth rates of the three submerged plant species initially increased and then declined with rising ammonia nitrogen concentrations, while the dry-wet ratios exhibited an opposite trend. This suggests that low ammonia nitrogen concentrations can enhance relative growth rates and dry matter accumulation in submerged plants, whereas high ammonia nitrogen concentrations have an inhibitory effect [30]. The relative growth rates and dry-wet ratios of Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii reached thresholds at ammonia nitrogen concentrations of 2 mg/L, 4 mg/L, 4 mg/L, and 2 mg/L, 6 mg/L, and 4 mg/L. Below these threshold concentrations, ammonia nitrogen promoted submerged plant growth, enhanced nutrient assimilation and uptake rates, and increased dry matter accumulation. However, at concentrations exceeding these thresholds, ammonia nitrogen had an inhibitory effect, imposing severe stress on the submerged plants and impairing normal growth. When ammonia nitrogen concentrations surpassed a critical limit, the relative growth rates of Vallisneria natans and Elodea nuttallii declined, eventually resulting in negative growth rates [31,32]. This suggests that Vallisneria natans is the most sensitive to ammonia nitrogen stress, followed by Elodea nuttallii, while Myriophyllum verticillatum demonstrated greater resilience, maintaining stable growth and showing a higher tolerance range for ammonia nitrogen than the other two plant species.

Photosynthesis is among the most sensitive physiological processes affected when aquatic plants are stressed by ammonia nitrogen, with chlorophyll content in plant leaves serving as a key indicator of photosynthetic efficiency and plant health [33]. In this study, low concentrations of ammonia nitrogen led to a short-term increase in chlorophyll content, potentially due to enhanced chlorophyll synthesis facilitated by the available ammonia nitrogen or as a response by submerged plants to mitigate nutrient stress. However, this hypothesis requires further research. Conversely, high ammonia nitrogen concentrations imposed stress on submerged plants, inhibiting chlorophyll synthesis, reducing photosynthetic efficiency, and ultimately impacting plant growth and health [34]. The stress mechanism in submerged plants exposed to high ammonia nitrogen concentrations may be attributed to increased algal biomass in the water or the high ammonia nitrogen levels themselves. Elevated ammonia nitrogen can lead to significant carbohydrate consumption in plants due to ammonia assimilation, reducing carbohydrate reserves. This depletion occurs because the total conversion rate of ammonia nitrogen into amino acids or amides may be lower than the uptake rate or the rate of cellular ammonia production through amino acid pathways [35]. Additionally, ammonia nitrogen accumulation can hinder the uptake and transport of Mg²⁺, which in turn impairs photosynthesis and adversely affects plant growth and reproduction [36]. However, the predominant stress mechanism in various habitats remains underexplored and warrants further investigation.

Research has shown that antioxidant enzyme activity within plant defense systems provides a sensitive indicator of the extent of damage aquatic plants experience under stress [37,38]. Malondialdehyde (MDA) serves as an indicator of membrane lipid peroxidation, with changes in its content reflecting the extent of membrane system injury under adverse conditions [39]. Malondialdehyde (MDA), a key product of membrane lipid peroxidation, accumulates in cells, leading to cross-linking and polymerization of proteins, nucleic acids, and other macromolecules, which is cytotoxic. Therefore, MDA is commonly used as an indicator of membrane lipid peroxidation. In contrast, superoxide dismutase (SOD) and catalase (CAT) represent the first line of defense in the plant antioxidant system, capable of removing excess superoxide anions from cells. These enzymes work in synergy with other antioxidants to mitigate damage from external stressors, making SOD and CAT activity levels reliable indicators of damage severity in aquatic plants under adverse conditions [40,41]. At the start of the experiment, membrane lipid peroxidation (MDA) levels in Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii increased with rising ammonia nitrogen concentrations across all treatment groups. The increase in MDA was notably higher in Elodea nuttallii under low ammonia nitrogen concentrations compared to Vallisneria natans and Myriophyllum verticillatum, indicating that different ammonia nitrogen levels imposed stress on these submerged plants. This stress disrupted the cellular free radical metabolism balance, leading to an excessive production of oxygen radicals. This overproduction, in turn, triggered or intensified membrane lipid peroxidation, resulting in continuous MDA generation. The accumulation of MDA damages cellular membranes; interferes with photosynthesis, respiration, and other cellular metabolic processes; and, in severe cases, can lead to cell death [42]. Under normal conditions, SOD and CAT activities in plant leaves are maintained at stable levels, balancing with reactive oxygen species. However, when submerged plants are subjected to ammonia nitrogen stress, they activate the antioxidant enzyme defense system to mitigate damage. By increasing SOD and CAT activities, the plants work to eliminate membrane peroxidation products, scavenge oxygen radicals, and maintain plasma membrane stability. Once ammonia nitrogen concentrations exceed the plants’ tolerance thresholds, however, MDA content, SOD activity, and CAT activity begin to decline over time. This indicates that high ammonia nitrogen levels cause irreversible damage, preventing the plants from fully activating their antioxidant defense systems to counter prolonged ammonia stress and hindering their ability to remove excess lipid peroxidation products.

This study investigated the tolerance of various submerged plants to ammonia nitrogen using artificial outdoor potting experiments and identified suitable ammonia-tolerant pioneer species for water ecological restoration in eutrophic bodies of water. However, due to the more complex natural conditions of such bodies of water, the ammonia-tolerant pioneer species identified in this study still need to be verified and expanded upon in actual engineering applications.

5. Conclusions

(1)

The optimal and maximum tolerance concentrations of ammonia nitrogen for Vallisneria natans, Myriophyllum verticillatum, and Elodea nuttallii were 2 mg/L, 4 mg/L, and 4 mg/L (optimal) and 4 mg/L, 12 mg/L, and 8 mg/L (tolerance). Submerged plants were able to grow normally within their maximum ammonia nitrogen tolerance concentrations. However, once these concentrations were exceeded, the peak enzyme activity in the antioxidant defense systems of the submerged plants occurred earlier, indicating that their growth was under severe stress from the elevated ammonia nitrogen levels.

(2)

The relative growth rate, chlorophyll content, MDA levels, and SOD and CAT activities in the three submerged plants initially increased and then declined with rising ammonia nitrogen concentrations, while the dry-wet ratio showed the opposite trend. Low ammonia nitrogen levels promoted biomass growth, chlorophyll synthesis, and dry matter accumulation, whereas high concentrations caused irreversible damage, limiting antioxidant defenses and preventing the removal of excess lipid peroxidation products.

(3)

Ammonia nitrogen tolerance among the three submerged plants was ranked as follows: Myriophyllum verticillatum > Elodea nuttallii > Vallisneria natans. Based on the ability of high ammonia nitrogen tolerance, we conclude that Myriophyllum verticillatum is a good candidate aquatic plant for treating high ammonia nitrogen wastewater in constructed wetland systems.