Physiological responses and algae inhibition of Pontederia cordata to simulated eutrophication and acid rain co-pollution

Abstract

Background

Eutrophication and acid rain are two threats that many water bodies must contend with. Eutrophication and climate change have accelerated widespread outbreaks of cyanobacterial blooms as both have become more severe. Pontederia cordata, a garden ornamental plant, can inhibit some algae growth and remove total nitrogen (TN) and total phosphorus (TP) from the water. In this study, we investigated how simulated acid rain and eutrophication co-pollution affected P. cordata's growth physiology and ability to inhibit algae growth.

Results

Under mild eutrophication (2 mg·L⁻¹ TN, 0.4 mg·L⁻¹ TP, and 15 mg·L⁻¹ COD_Mn) or weak acid rain (pH = 5.0), P. cordata alleviated the degree of cell membrane lipid peroxidation by stabilizing superoxide dismutase (SOD) and catalase (CAT) activities in the leaves, allowing for normal plant growth. Under mild eutrophication and acid rain conditions, cultured P. cordata water samples maintained strong algae inhibition by reducing the Chl a content and SOD activity of Microcystis aeruginosa cells. Compound stress where acid rain was the primary inhibitory factor along with moderate or severe eutrophication inhibited P. cordata growth, which probably reduced the input of algae-inhibiting allelochemicals, thus reducing its ability to inhibit algae.

Conclusions

In summary, P. cordata has application potential in mild eutrophic water and acid rain (pH ≥ 4). These findings provide guidance for further research on phytoremediation and algae control in scenarios of compound pollution.

Introduction

One of the top three ecological and environmental issues facing the planet today is acid rain. Known as wet deposition, it refers to rain, snow, fog, hail, or any other type of atmospheric precipitation with a pH of less than 5.6. Strong acid rain has a pH of less than 4.5, while weak acid rain ranges from 4.5 to 5.6. China is the third largest acid rain area in the world after Europe and North America, with 40% of its territory polluted by acid rain. China’s estimated annual economic losses caused by acid rain pollution are as high as 110 billion yuan. In 2016, the SO₂ emissions of India exceeded those of China. Up until 2030, SO₂ and NO_x emissions, which are India's primary contributors to acid deposition, are anticipated to rise (Andrade et al. 2020). Acid rain damages plants mainly through toxic effects on aboveground morphological structures, mineral element metabolism, antioxidant system, and photosynthesis. Andrade et al. (2020) found that simulated acid rain (SAR) (pH = 4.5) altered epidermal relief and resulted in the wilting of epidermal cells of Joannesia princeps. Verónica et al. (2020) examined the effects of SAR on Liquidambar styraciflua and Fraxinus uhde, the two most prevalent urban trees in Mexico City, and discovered that SAR (pH = 2.5) reduced the chlorophyll content in the leaves, altered cuticle scaling, and impacted the formation of epicuticular wax aggregates. In the leaves of two varieties of tomato, Debnath et al. (2018) showed that the contents of chlorophyll, carotenoid, total soluble protein, and soluble sugar decreased as the pH of SAR decreased, while the contents of hydrogen peroxide and lipid peroxide increased. Specifically, enzymatic and non-enzymatic antioxidants (total phenols, total flavonoids, and iron reduction) decreased at a pH of 2.5. The harmful effects of acid rain on plants are also reflected in its superposition with other pollutants. Academics from around the world are paying more attention to research on the combined stress of acid rain and other environmental factors (Du et al. 2022; Neves et al. 2009).

A significant amount of nitrogen and phosphorus nutrients have accumulated in rivers and lakes due to the increased discharge of domestic, industrial, and agricultural wastewater. That has led to an excess of nitrogen and phosphorus nutrients and a decline in the quality of the water. Eutrophication has become a focus of global water environmental governance and research. Additionally, eutrophication and climate change have catalyzed the expansion of cyanobacterial blooms in aquatic ecosystems worldwide (Huisman et al. 2018). In the 1980s, the scale of cyanobacterial blooms was 68% in 71 large lakes (each with an area of > 100 km²), which has continued to grow (Ho et al. 2019). The frequent occurrence of cyanobacteria blooms and the ongoing buildup of algal toxins threaten organismal and human health through the food chain, disrupt the functioning of aquatic ecosystems, and destroy the ecological balance of the water environment (D'Anglada 2021; Lee et al. 2017). Specifically, Microcystis aeruginosa is a common dominant species of cyanobacterial blooms in many eutrophic lakes (Princiotta et al. 2019). How to effectively control or eliminate the spread of algae is of great importance. Many physical and chemical algae suppression methods have been implemented, such as artificial mixing (Visser et al. 2016), ultrasound treatment (Kong et al. 2019), UV-activated persulfate (Wang et al. 2016a), and flocculants use (Wang et al. 2018). However, although these methods remove algae in the short term, they are expensive and cause secondary pollution. Mjelde and Faafeng (1997) found higher biomass densities of aquatic plants in 24 lakes in Norway, where lower phytoplankton densities led to better water quality. Indeed, aquatic macrophytes release allelochemicals via root secretion, leaching, volatilization, and the decomposition of plant residues in the water, which is an effective strategy for preventing algae development (Hilt and Gross 2008). Recently, macrophyte allelochemicals have attracted attention as substitutes for synthetic chemicals in cyanobacterial harmful algal bloom (CyanoHAB) control due to their environmental safety and higher biodegradability (Tazart et al. 2021). The inhibition of allelochemicals released by aquatic plants on the physiological metabolism, growth, and development of algae is mainly reflected in the following three aspects: (1) reduced chlorophyll content of algae cells and blocked photosynthetic electron transmission, which weakens the photosynthetic capacity and inhibits photosynthesis (especially photosystem II); (2) inhibited activities of antioxidant enzymes and adenosine triphosphate (ATP) in algal cells, which induce oxidative damage to algal cells and hinder their absorption of calcium, magnesium, and other mineral elements; and (3) increased destruction of the permeability of the cell membrane, which results in the exudation and loss of cell contents and even leads to programmed cell death (Wang et al. 2016b). Moreover, differences in the nutrient and pH levels exert important effects on plant allelopathy (Allen et al. 2017). According to the growth–differentiation balance hypothesis (GDBH), plants will decrease the input of allelochemicals as stress increases and their physiological functions are severely compromised, which will weaken allelopathy (Bauer et al. 2010).

Pontederia cordata belongs to the Pontederiaceae family and is a perennial emergent plant. Due to its developed roots, large biomass, appealing flowers, and long flowering period, this plant has high garden ornamental value. Additionally, this plant lowers the levels of total nitrogen (TN) and total phosphorus (TP) in eutrophic water, and its organic acid allelochemicals (succinic acid, cinnamic acid, and vanillic acid) have some effects that inhibit the growth of algae. It has a good application potential in the treatment of urban sewage (Qian et al. 2019; Tian et al. 2011). However, when faced with the dual stress of eutrophication and acid rain, the physiological response of P. cordata and corresponding effects on algae inhibition remain unknown. Thus, this study aimed to address the following research objectives: (1) to determine the effects of the compound stress of eutrophication and acid rain on the growth and photosynthetic capacity of P. cordata using the relative growth rate (RGR), root activity, and total chlorophyll (Chl T) content in the leaves; (2) to investigate the degree of membrane peroxidation and antioxidant responses under compound stress through estimating the relative conductivity (REC), malondialdehyde (MDA) content, and superoxide dismutase (SOD) and catalase (CAT) activities in the leaves; and (3) to elucidate the effects of various cultured water samples of P. cordata under different simulated eutrophication and acid rain treatments on the growth and physiological metabolism of M. aeruginosa by measuring the SOD activity, MDA content, and chlorophyll content of M. aeruginosa cells and the inhibition rates on algae. These results will provide a theoretical basis for future studies on phytoremediation and algae control under eutrophication and acid rain co-pollution.

Materials and methods

Location and plant materials

The present study was conducted in a greenhouse at the National Landscape Architecture Experimental Teaching Demonstration Center, Nanjing Forestry University, Nanjing, China (118° 49′ E, 32° 04′ N). P. cordata seedlings with a similar number of leaves and of the same height were selected for experimentation, and then planted in Hoagland nutrient solution. M. aeruginosa was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China), and then amplified and cultured in BG-11 medium. The culture conditions were 25 ± 2 °C, 3000 lx light, and a 12/12 h photoperiod. After the logarithmic growth period, the experiment began.

Eutrophication of water samples

The eutrophication of tested water samples prepared from C₆H₁₂O₆, CO(NH₂)₂, and KH₂PO₄ was set at four levels (E0, E1, E2, and E3) (Table 1). E0 was the control group. The TN and TP contents and the permanganate index (COD_Mn) of E0 were referred to the III water quality standard of GB3838-2002 (Ministry of Environmental Protection of the People's Republic of China 2002), which applies to centralized domestic drinking water. The TN, TP, and COD_Mn of E1 referred to the V-grade water quality standard, which applies to agricultural and general landscape water. In the E2, the TN and the TP were both 5 times of the V water quality standard; in the E3, the TN and the TP were both 10 times.

Table 1 Different elements' concentrations in tested water samples at various eutrophication levels

SAR preparation and application

The average annual pH of precipitation in the Yangtze River Delta of China is 4.87 ± 0.28 (Li et al. 2021). The S${\text{O}}_{4}^{2 - }$/N${\text{O}}_{3}^{ - }$ ranges between 2.4 and 2.9 (Chen 2018). To simulate acid rain, analytical pure H₂SO₄ and HNO₃ were applied to prepare acid rain mother liquor (pH = 1.0) with an S${\text{O}}_{4}^{2 - }$/N${\text{O}}_{3}^{ - }$ volume ratio of 3:1. Then, the pH of acid rain mother liquor was diluted to 4.0 and 5.0. Ultrapure water (pH = 7.0) was used for the control group.

Experimental design

Plants exposed to eutrophication and SAR spraying

The experiment was arranged in a factorial scheme that was completely randomized using two factors (eutrophication and pH), four eutrophication levels (E0, E1, E2, and E3), and three pH levels (7.0, 5.0, and 4.0) (Table 2). Three replicates from each of the 12 treatments were used, yielding a total of 36 experimental units. Plants with good growth and a biomass value of 80 g in Hoagland nutrient solution were selected, washed, and cultured in plastic buckets containing 1.5 L eutrophic water. Throughout the experiment, water samples were changed every 7 d. After two weeks, the plants were sprayed four times every 7 d with SAR or ultrapure water (10 mL per plant). The day of foliar spraying was regarded as day 1. During foliar spraying, the plastic buckets were completely covered with plastic film to prevent SAR spraying from affecting the pH of water samples. After 28 d, the cultured water samples were harvested for the M. aeruginosa inoculation test.

Table 2 Compound treatments of simulated acid rain (SAR) and eutrophication on Pontederia cordata

Algae inoculated in cultured water of plants exposed to eutrophication and sprayed with SAR

M. aeruginosa got exposed to different cultured water samples of plants mentioned above. The cultured water samples were filtered with medium-speed filter paper and a 0.45-μm microporous membrane to filter out protozoa and microorganisms. E0 + pH 7.0 was used as the control group. The initial OD₆₅₀ value of algae was 0.1. The day of inoculation was regarded as day 0, and the processing time lasted for 7 d. The algal density was measured on day 1, and algal cells were harvested for physiological index determination on days 1, 3, 5, and 7.

Measurement of indices

Related indicators of P. cordata

Determination of RGR: Plants were harvested on days 1 and 28, washed with ultrapure water, dried with filter paper, and weighed with an electronic balance. The RGR was calculated using the following equation: RGR = (W₁ − W₀)/W₀ × 100%, where W₁ is the initial fresh weight, and W₀ is the final fresh weight.

Root activity (Wang 2006): Fresh plant roots (0.3 g) were harvested on day 28 and then soaked in a mixture containing 0.4% 2,3,5-triphenyltetrazolium chloride (TTC) and phosphate buffer (1/15 M, pH = 7.0) for 1 h at 37 °C under continuous darkness. Afterward, 2 mL of 1 M sulfuric acid was added to terminate the reaction. Then, ethyl acetate and a small amount of quartz sand were added to the root samples. After grinding, the OD₄₈₅ of the red extract was measured to calculate the reduction strength of tetrazolium.

On days 7, 14, 21, and 28, the Chl T and MDA contents, SOD and CAT activities, and REC of P. cordata leaves were calculated following previously described methods (Wang 2006).

Chl T content: Leaves (0.1 g) were soaked in 10 mL of 95% ethanol at room temperature under continuous darkness for 24 h until the leaves were completely white. Afterward, the OD₆₄₉ and OD₆₆₅ of the supernatant were recorded using an ultraviolet–visible spectrophotometer (Lambda 25, Perkin-Elmer, Waltham, MA, USA).

SOD activity: Leaves (0.3 g) were soaked in 6 mL of phosphate buffer (0.05 M, pH = 7.8), then ground to a homogenate, and centrifuged at 4000 r·min⁻¹ for 10 min. The reaction system was exposed to 4000 lx light for 20 min, and the absorbance was measured at 560 nm.

CAT activity: The extraction method of the enzyme solution was the same regarding SOD. The reaction system was preheated to 25 °C. Then, 0.3 mL of 0.1 M H₂O₂ was added to the system, and the OD₂₄₀ was determined. Readings were recorded every minute for 3 min.

REC: The cell membrane permeability was measured using a conductometer. After the main vein and margin of leaves were removed, the leaves were punched into disks with a diameter of 6 mm. The leaf disks were soaked in 15 mL of distilled water with a conductivity (R₀) of 0 at room temperature for 24 h. Then, the conductivity (R₁) was measured with a conductivity meter (DDS-307 type, Inesa, Shanghai, China). Afterward, the disks were heated in a boiling water bath for 15 min; then the conductivity (R₂) was measured at a constant temperature (20–25 °C).

MDA content: A total of 2 mL of supernatant and 2 mL of 0.67% thiobarbituric acid (TBA) were mixed and boiled for 20 min. After quickly cooling, the mixture was centrifuged at 4000 r·min⁻¹ for 10 min. The absorbance was measured at 450, 532, and 600 nm.

Related indicators of M. aeruginosa

Determination of inhibition rates (IR) on algal infrared: BG-11 medium mixed with water samples in corresponding degrees was used as the control. The OD₆₅₀ was measured, as it reflects the algae density. The IR was calculated using the following equation: IR = (1 − N₁/N₀) × 100%, where N₁ and N₀ are the algae densities of the treatment and control groups, respectively.

The Chl a content in M. aeruginosa cells was measured using the hot ethanol method (Chen et al. 2006). First, microporous filter membranes enriched with algal cells were sheared, and then 8 mL of 90% hot ethanol solution was quickly added for extraction at 80 °C. After 2 min, the mixtures were immediately transferred to an ultrasonic cleaner and crushed in a 50-Hz ice bath for 40 min. After static extraction in the dark for 2 h, the mixtures were centrifuged at 4 °C and 5000 r·min⁻¹ for 12 min. The supernatant was added to 2 mL of 90% ethanol and centrifuged again. The absorbance was measured at 665 and 750 nm.

The SOD activity in algal cells was quantified using nitrogen blue tetrazole (NBT) colorimetry (Gao 2006). First, 10 mL of algal cell solution was centrifuged at 4 °C and 9000 r·min⁻¹ for 15 min. The supernatant was poured out, and 5 mL of buffer solution (0.05 M, pH = 7.8) was added. Then, the mixture was placed in an ultrasonic cleaner and crushed in a 50-Hz ice bath for 40 min. After centrifugation for 15 min, the reaction system was exposed to strong light for 20 min, and the absorbance was determined at 560 nm.

The MDA content in algal cells was measured using the TBA method (Wu and Ye 2016). First, 1 mL of supernatant and 2 mL of TBA solution were mixed and heated in a boiling water bath for 20 min. After cooling, the mixture was centrifuged at 4000 r·min⁻¹ for 10 min. Then, the OD₄₅₀, OD₅₃₂, and OD₆₀₀ of the supernatant were measured.

Data analysis

The data were analyzed using SPSS v26.0 software. Tukey’s test was used for multiple comparisons based on significant differences detected by the analysis of variance (ANOVA). Graphs were prepared using DataGraph v4.7.1 and Adobe Illustrator 2019.

Results

Growth and physiological responses of P. cordata to simulated eutrophication and acid rain

Relative growth rate (RGR)

Significant differences were detected in the RGR of P. cordata under different main effects of simulated eutrophication (Table 3). Compared to the control, no significant differences were detected in the RGR of P. cordata with E1 (P = 0.588), but significant decreases were detected in E2 and E3 (P < 0.05) (Table 4). This result indicated that mild eutrophication (E1) had no discernible inhibitory effects on plant biomass. Under various main effects of SAR, the RGR showed significant variances (Table 3). The RGR significantly decreased as the SAR concentration increased (P < 0.05), indicating that SAR spraying inhibited P. cordata growth.

Table 3 The variance analysis of the RGR, root activity, Chl T content, SOD activity, CAT activity, REC, and MDA content of P. cordata under the simulated acid rain spraying times, simulated acid rain (SAR) and simulated eutrophication

Table 4 The main effects of simulated acid rain spraying times, simulated eutrophication, or simulated acid rain (SAR) on Pontederia cordata

Root activity

Under the main effects of simulated eutrophication or SAR, significant variations in the root activity of P. cordata were found (Table 3). As the eutrophication level and SAR concentration increased, significant increases and decreases in the root activity of P. cordata were observed, respectively (P < 0.05) (Table 4). The root activity under different interactive effects of eutrophication and SAR presented the significant differences (F = 807.998, df = 11/35, P < 0.05; Fig. 1a). Compared to the control, significant increases in the root activity under compound stress only occurred in E1 + pH 7.0 (P < 0.05), which reached the highest value of 16.31 μg·g⁻¹·h⁻¹. In E0, E1, and E2, SAR application led to significant decreases in root activity when compared to the control. In E3, no significant differences in root activity were detected under the SAR treatment.

Fig. 1

The interactive effects of simulated eutrophication and simulated acid rain (SAR) on the root activity (a), Chl T (b), SOD (c), CAT (d), REC (e), and MDA (f) of P. cordata harvested on day 28. Results are expressed as mean of triplicate samples ± standard error. Different lowercase letters indicate significant differences at P < 0.05

Chl T content

The Chl T content of P. cordata leaves under different main effects of simulated eutrophication showed the significant differences (Table 3). Compared to the control, no significant differences were detected in the Chl T content of P. cordata leaves in E1 and E2, but the Chl T content significantly decreased by 26.46% in E3 (P < 0.05). Significant variations in the Chl T content under different main effects of SAR were observed (Table 3). SAR (pH = 5.0 and 4.0) treatment led to a significant decrease in the Chl T content when compared to the control (P < 0.05). Significant differences were detected in the Chl T content under different main effects of SAR spraying time (Table 3). As the SAR spraying time increased, the Chl T content in the leaves increased at first and then decreased (Table 4). The Chl T content underwent significant changes as a result of various interactions effects of eutrophication and SAR for 28 d (F = 70.444, df = 11/35, P < 0.05; Fig. 1b). When sprayed with SAR for 28 d, the plants exposed to eutrophication exhibited reductions in the Chl T content when compared to the control. The Chl T content in E3 + pH 4.0 reached the lowest value (769.81 mg·g⁻¹), which was significantly lower than the control by 63.21% (P < 0.05).

SOD activity

Compared to the control, eutrophication and SAR exerted no obvious inhibitory effects on the SOD activity of P. cordata (Table 4). Significant differences were observed in the SOD activity under different main effects of SAR spraying time (Table 3). As SAR spraying time increased, the SOD activity in the leaves exhibited a significant upward trend (P < 0.05) (Table 4). Under various interactive effects of eutrophication and SAR for 28 d, significant variations in the SOD activity were identified (F = 309.917, df = 11/35, P < 0.05; Fig. 1c). SAR treatment in plants exposed to eutrophication for 28 d resulted in a significant decrease in the SOD activity when compared to the control (87.21 U·g⁻¹), which was similar to the Chl T content results. Additionally, as the eutrophication level increased, the SOD activity in the control exhibited a decreasing trend at first and then increased, while the pH 4.0 group increased at first and then decreased (P < 0.05). Moreover, when compared to the control, the SOD activities of plants in E1, E2, and E3 that were treated with SAR pH 4.0 were significantly higher than those treated with SAR pH 7.0 and 5.0 (P < 0.05).

CAT activity

Significant differences were identified in the CAT activity of P. cordata under different main effects of simulated eutrophication (Table 3). Compared to the control, no significant differences were detected in the CAT activity of P. cordata in E1 and E2, but the CAT activity significantly decreased by 29.73% in E3. Under various primary SAR effects, there were discernible changes in the CAT activity (Table 3). No significant differences were detected in the CAT activity between the SAR pH 5.0 and 7.0 treatments, while SAR pH 4.0 significantly decreased the CAT activity by 22.13% (P < 0.05). Significant variations in the CAT activity were found under several main effects of SAR spraying time (Table 3). As SAR spraying time increased, the CAT activity increased at first and then decreased, reaching the highest value at the third spraying time (Table 4). Significant differences were detected in the CAT activity under different interactive effects of eutrophication and SAR for 28 d (F = 55.807, df = 11/35, P < 0.05; Fig. 1d). The highest CAT activity value (2500.16 U·g⁻¹·min⁻¹) was detected in the control (P < 0.05). In E0, E2, and E3, the CAT activity in plants treated with SAR pH 4.0 significantly decreased by 22.51%, 29.98%, and 22.55%, respectively, when compared to that in plants treated with SAR pH 5.0 (P < 0.05). In E1, SAR treatment had no significant inhibitory effect on the CAT activity.

Relative conductivity (REC)

Under various main effects of simulated eutrophication, SAR, or SAR spraying time, significant changes were seen in the REC of P. cordata (Table 3). As the eutrophication level, SAR concentration, and SAR spraying time increased, the REC in P. cordata leaves significantly increased (P < 0.05) (Table 4). Significant differences were found in the REC under different interactive effects of eutrophication and SAR for 28 d (F = 942.548, df = 11/35, P < 0.05; Fig. 1e). In E0 and E1, the REC in the leaves treated with SAR pH 4.0 for 28 d was significantly higher than that in the leaves treated with SAR pH 7.0 and 5.0. In E2, the REC reached the highest value (67.80%) under SAR pH 5.0. In E3, the REC values of the three SAR treatments significantly increased, which were 3.28, 3.27, and 3.23 times higher than that of the control (P < 0.05).

MDA content

The MDA content of P. cordata under different main effects of simulated eutrophication or SAR showed significant variations (Table 3). The change trends of the MDA contents of plants under compound stress were the same as REC and reached the highest value in E3 under SAR pH 4.0 treatment. Significant differences were identified in the CAT activity under different main effects of SAR spraying time (Table 3). As SAR spraying time increased, the change in the MDA content was similar to REC, showing a significant upward trend (P < 0.05) (Table 4). Under various interactive effects of eutrophication and SAR for 28 d, significant variations in the MDA content were found (F = 126.764, df = 11/35, P < 0.05; Fig. 1f). Compared to the control, the MDA content of plants treated with SAR pH 4.0 for 28 d and exposed to different levels of eutrophication significantly increased by 18.15–91.63% (P < 0.05). In E2 and E3, the MDA content of plants treated with SAR pH 5.0 and 4.0 was significantly higher than that of plants treated with SAR pH 7.0 (P < 0.05). E3 + pH 4.0 had the highest MDA content (28.62 nmol·g⁻¹).

Effects of simulated eutrophication and acid rain compound stress on P. cordata algae inhibition

Inhibition rate of M. aeruginosa

The inhibition rate on algae of various cultured water samples presented the significant differences (F = 30.936, df = 11/71, P < 0.05; Fig. 2). The inhibition rate on M. aeruginosa of the control reached the highest value (42.11%), which indicated that the inhibition effects on algal growth of cultured water samples from plants under compound stress were significantly reduced (P < 0.05). In E1, E2, and E3, significant differences were detected between the algal inhibition rates of cultured water samples from plants treated with SAR pH 5.0 and 4.0 (P < 0.05). Among these, E2 and E3 exhibited significant decreases in the algal inhibition rate when compared to pH 7.0 (P < 0.05). Additionally, under the same SAR concentration, the algae inhibition rates of cultured water samples significantly decreased as the eutrophication level increased (P < 0.05).

Fig. 2

Various cultured water samples from P. cordata under compound stress on the inhibition rate of M. aeruginosa. Results are expressed as mean of triplicate samples ± standard error. Different lowercase letters indicate significant differences at P < 0.05

Chl a content of algal cells

Significant differences were detected in the Chl a content of algae cells under different main effects of processing time or cultured water sample (F = 27.307, df = 3/287, P < 0.05; F = 20.461, d.f = 11/287, P < 0.05; Table 5). Over time, the Chl a content of algae cells significantly decreased at first and then increased, reaching the lowest and highest values on days 5 and 7, respectively (P < 0.05). In E0 and E3, the Chl a content significantly increased as the SAR concentration increased, reaching the highest value in E0 + pH 4.0. In E1 and E2, the Chl a content exhibited the opposite trend, reaching the lowest value in E2 + pH 4.0 (P < 0.05). On day 1, the Chl a content significantly increased by 13.60–17.48% in E0 + pH 4.0, E1 + pH 7.0, E1 + pH 5.0, and E2 + pH 7.0 when compared to the control, but significantly decreased by 12.61–22.31% in E2 + pH 5.0, E2 + pH 4.0, and E3 + pH 7.0 (Fig. 3a). On day 3, the Chl a content in E0 + pH 5.0, E0 + pH 4.0, and E3 + pH 4.0 significantly increased when compared to the control. On days 5 and 7, the Chl a content in E0 + pH 5.0, E0 + pH 4.0, and E3 + pH 4.0 exhibited the same change trends (P < 0.05).

Table 5 The main effects of processing time or cultured water sample on Microcystis aeruginosa

Fig. 3

Variations in the Chl a (a), SOD (b), and CAT (c) of M. aeruginosa cells in various cultured water samples from P. cordata under compound stress for 1, 3, 5, and 7 d, respectively. Results are expressed as mean of triplicate samples ± standard error. Different lowercase letters indicate significant differences at P < 0.05

SOD activity of algal cells

The SOD activity of algal cells under different main effects of processing time or cultured water sample showed significant differences (F = 13.043, df = 3/287, P < 0.05; F = 7.949, d.f = 11/287, P < 0.05; Table 5). The SOD activity of algae cells in cultured water samples of P. cordata reached the highest value on day 7, and its change trend was similar to the Chl a content. Compared to the control, the cultured water samples of P. cordata under compound stress exerted significant inhibitory effects on the SOD activity of algae cells (P < 0.05). Compared to E2, the SOD activity of algae cells in E0, E1, and E3 significantly decreased as the SAR concentration increased, reaching the lowest value in E3 + pH 4.0 (P < 0.05). On day 1, the SOD activities of algae cells in E0 + pH 5.0, E0 + pH 4.0, and E1 + pH 7.0 were not significantly different when compared to the control, while the SOD activities of other water samples significantly decreased (P < 0.05) (Fig. 3b). On days 3, 5, and 7, the highest SOD activity was detected in E0 + pH 7.0 (P < 0.05).

MDA content of algal cells

Under the main effects of processing time, there were significant changes found in the MDA content of algal cells (F = 54.412, df = 3/287, P < 0.05; Table 5). Over time, the MDA content of algal cells exhibited the opposite trend as the SOD activity, reaching the highest and lowest values on days 3 and 7, respectively (P < 0.05). Under compound stress, no significant differences were detected in the MDA content of algal cells when compared to the control (F = 0.218, df = 11/287, P = 0.996; Table 5). On day 1, when compared to the control, the MDA contents in E0 + pH 5.0 and E2 + pH 7.0 significantly increased by 12.5% and 2.5%, respectively (P < 0.05), while no significant differences were detected in other water samples (Fig. 3c). On days 3 and 5, the highest MDA content of algal cells appeared in E0 + pH 7.0 and E3 + pH 4.0. On day 7, when compared to the control, the MDA content in the six water samples from E1 and E2 significantly increased but significantly decreased in E0 + pH 4.0 and E3 + pH 4.0 (P < 0.05).

Discussion

Chlorophyll, a photosynthetic pigment, serves as the building block for capturing light energy in plant leaves, and the amount of it reflects a plant's capacity for photosynthesis. In this experiment, under mild (E1) and moderate (E2) eutrophication, no significant change was detected in the Chl T content of P. cordata leaves when compared to the control (E0) (Table 4). This result indicated that P. cordata absorbs N and P elements normally and consistently within a range of nutrient concentrations, ensuring the biosynthesis of chlorophyll in the leaves. Excessive concentrations of N and P prevented chlorophyll synthesis or accelerated its decomposition (Table 4), resulting in limited photosynthesis and biomass growth. Additionally, the Chl T content in the leaves significantly decreased under SAR pH 5.0 and 4.0 (Table 4), which was consistent with a previous study on two tomato cultivars (Debnath et al. 2018). This might be as a result of H⁺ accumulating in plant tissues and taking the place of Mg²⁺ in the chlorophyll molecules, thereby reducing the chlorophyll content by converting chlorophyll into pheophytin (Eguagie et al. 2016; Pham et al. 2021). The degree of plant damage can be directly characterized by changes in biomass when the plant is under stress. Similar to the research done by Pham et al. (2021) on soybean (Glycine max), plant biomass decreased as the SAR pH value rose. Therefore, we speculated that SAR decreased H⁺-ATPase activity in the plasma membrane of P. cordata, impeded the transmembrane chemical gradient of H⁺, and consequently prevented the electrical gradient. That ultimately destroyed the energy source of plant growth, and thereby inhibited the accumulation of biomass (Falhof et al. 2016).

Reactive oxygen species (ROS), such as the superoxide anion (${\text{O}}_{2}^{ - }$), hydrogen peroxide (H₂O₂), hydroxyl radical (HO), and singlet oxygen (¹O₂), can build up excessively in response to stress. This interfered with the normal physiological metabolism of plants by increasing membrane permeability, causing lipid peroxidation of the cell membrane, and allowing some nutrient ions to exosmose through the membrane. The MDA is the final product of plant cell membrane lipid peroxidation and the REC is a crucial index for assessing the stability and integrity of plant cell membranes. In this study, SAR pH 4.0 and E3 + pH 4.0 both resulted in significantly increased REC and MDA content in P. cordata leaves (Fig. 1; Table 4). These results may be explained by the following: (1) strong acid rain directly raised the extracellular H⁺ concentration, which led to higher H⁺ concentrations inside of cells. This imbalanced the metabolism of ROS by increasing the production of ROS relative to its clearance. (2) Elevated cytosol protons under strong acidic conditions caused Ca²⁺ to be lost from the cell membrane through replacement, which compromised the stability of the membrane system. (3) Strong acid rain stress enhanced the acid phosphatase activity of P. cordata leaves, promoted the degreasing reaction of the phospholipid bilayer in the membrane, and destroyed the membrane system's integrity (Ren et al. 2018). The SOD acts as the first line of defense against oxidation and catalyzes the disproportionation of ${\text{O}}_{2}^{ - }$ to generate H₂O₂ and O₂, while CAT catalyzes the rapid decomposition of H₂O₂ into H₂O and O₂ (Bezerril Fontenele et al. 2017). The synergistic effects of SOD and CAT lessen the buildup of oxygen free radicals and prevent the conversion of ${\text{O}}_{2}^{ - }$ and H₂O₂ into highly destructive HO molecule via the Haber–Weiss and Fenton reactions. As a result, free radical damage to biological macromolecules like nucleic acids, proteins (enzymes), and biofilms is avoided or reduced. In this study, the MDA content and SOD and CAT activities of P. cordata leaves did not differ from the control when mild eutrophication was present (Table 4). That indicated the plants maintained the antioxidant capacity by stabilizing the SOD and CAT activities, which is an ecological response strategy used by P. cordata to adapt to mild eutrophication. Severe (E3) eutrophication, strong acid rain, and compound stress all led to inhibited SOD and CAT activities, intensified oxidative damage, and inhibited metabolic activities, as evidenced by increased REC and MDA contents, as well as diminished allelopathic algal inhibition effects in the leaves (Fig. 2).

Allelopathy is an environmental adaptation mechanism produced by plants to enhance their competitiveness and survival during evolution. Allelochemicals released by macrophytes directly inhibit the growth of adjacent phytoplankton in terms of cell density, providing conclusive evidence of allelopathy (Fernandez et al. 2016; Nakai et al. 1999). According to Tian et al. (2011), the cultured water of P. cordata exerted a strong inhibitory effect on M. aeruginosa under normal growth conditions. In this study, under mild eutrophication, SAR had no discernible inhibitory effects on the allelopathic algal inhibition ability of P. cordata, which remained a high level. Under moderate and severe eutrophication, SAR was the main reason for the reduced allelopathic algal inhibition capacity (Fig. 3). According to the GDBH (Bauer et al. 2010), P. cordata allocates more of its limited life resources to self-repair processes in order to withstand oxidative stress under the compound stress of moderate or severe eutrophication and acid rain. That revealed this plant modifies its resource inputs of allelochemicals in response to changes in the growth environment. Previous studies reported that active allelopathic components, such as flavonoids, tannins, ρ-coumaric acid, and vanillic acid, released by aquatic plants significantly decreased the Chl a content in M. aeruginosa cells (Tazart et al. 2019; Zhang et al. 2010). This was in line with the results of this study, which indicated that the P. cordata allelochemicals in cultured water samples interfered with the photosynthesis of algae cells by inhibiting the Chl a content. That was one mechanism through which P. cordata inhibited M. aeruginosa growth. Additionally, algal cells' SOD activity was low and their MDA content was high on days 1 and 3 (Table 5), demonstrating that the oxidative stress induced by the allelochemicals released by P. cordata under compound stress may lead to: (1) leakage of Cu, Zn, Fe, and other metal ions from algal cells, which inhibited the antioxidant enzyme activity of algal cells (Zuo et al. 2016); or (2) leakage of cell components, such as proteins, nucleic acids, and inorganic ions, the enhancement of proton influx, increased cell membrane permeability, and damage to the cell membrane (Campos et al. 2009). On day 7, the Chl a content and SOD activity of algal cells significantly increased (Table 5). Zuo et al. (2016) discovered that allelochemicals easily degrade, in light of the fact that allelopathy effectiveness necessitates the continuous and stable release of allelochemicals. Therefore, we speculated that P. cordata allelochemicals at low concentrations caused by degradation exerted hormesis effects on the Chl a content and SOD activity of M. aeruginosa at this time (Calabreseci 1999).

Conclusions

Under mild eutrophication (E1, 2 mg·L⁻¹ TN, 0.4 mg·L⁻¹ TP, and 15 mg·L⁻¹ COD_Mn), P. cordata grew normally. Under compound stress of mild eutrophication (E1) and SAR (pH = 4 and pH = 5), cultured water samples of P. cordata maintained strong algal inhibitory effects in terms of reduced Chl a content and SOD activity. The compound eutrophication and SAR stress decreased the Chl T content and SOD and CAT activities in P. cordata, giving rise to oxidative damage. Under moderate (E2) and severe (E3) eutrophication, the SAR accounted for the reduced allelopathic algal inhibition capacity of the plants. Generally, P. cordata possesses essential landscape effects and ecological benefits in mildly eutrophic waters of areas afflicted with acid rain.