Effects of microcystin-LR on purification of drinking water source and physiological response of Hydrocharis dubia (Bl.) backer

Effects of microcystin-LR on purification of drinking water source and physiological response of Hydrocharis dubia (Bl.) backer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of microcystin-LR on purification of drinking water source and physiological response of Hydrocharis dubia (Bl.) backer Junfei Liu, Yingbo Dong, Hai Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3066142/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The safety of drinking water source directly affects human health. Ensuring the safety of drinking water is critical. Microcystin-LR (MC-LR), a toxic and common pollutant, releases by algae into drinking water source and can impede the in-situ remediation effect of aquatic plant on drinking water source purification. However, the effect mechanism of the MC-LR on the purification of the drinking water by aquatic plant remains unclear. This study aims to explore the effects mechanism of MC-LR (0–10 µg L − 1 ) on drinking water source purification by Hydrocharis dubia (Bl.) backer . After an exposure experiment, the removal rate of NH 4 + -N, TP, and COD promoted significantly at low concentrations of MC-LR (< 1µg L − 1 ). And the removal rate of NH 4 + -N, TP, and COD increased from 78.9–90.7%, 70.0–93.2%, and 46.0–77.3%, respectively. With the increase of MC-LR concentration, the pollutant removal rate was obviously inhibited causing by concentration-dependent. Furthermore, the growth and development of the Hydrocharis dubia (Bl.) backer roots were significantly promoted at the concentration of 0.2 µg L − 1 . The length, tips, surface area, and average diameter of the root increased by 71.3%, 271.4%, 265.5%, and 113.0%, respectively. Chlorophyll contents under low-concentration MC-LR shows a 14.5%-15.7% promoting effect compared with the control group (CK). The activities of POD and CAT were also stimulated with the MC-LR increasing at low concentration. Notably, the MDA contents increased with increasing MC-LR concentration (p < 0.01). Therefore, the presence of MC-LR could affect the purification efficiency of Hydrocharis dubia (Bl.) backer in drinking water source. This study indicates the effect mechanism of MC-LR on drinking source water purification by Hydrocharis dubia (Bl.) backer . Aquatic plant Microcystin-LR Drinking water source Hydrocharis dubia (Bl.) backer Phytoremediation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction A widespread fertility of harmful cyanobacterial blooms in many marine and fresh-water ecosystems is serious causing by eutrophication and global climate change(Xu, Pang et al. 2021 ). Previous studies showed that the average days of cyanobacterial blooms in the United State increased from seven days per year under present climate to 18–39 days in 2090 causing by climate change(Chapra, Boehlert et al. 2017 ). The massive cyanobacterial existing in water environment can block the light and affect the growth of herbivorous zooplankton(Huisman, Sharples et al. 2004 ). In addition, some cyanobacteria cell lysis can release toxins (like Microcystin) into water that can cause digestive and neurological diseases(Zhang, Whalen et al. 2021 ). The Microcystin-LR (MC-LR), one of the most common Microcystin, is set to a guideline limit of 1.0 µg L − 1 in drinking water by the World Health Organization(Xue, Steinman et al. 2020 ). How to control the content of MC-LR in drinking water under guideline limit is an important work. To date, the studies show that most methods for algae removal would produce the harmful by-products, or the heavy metals accumulation such as Cu in ecosystems(Du, Li et al. 2022 ). Therefore, there is an urgent need for an effective and eco-friendly way to impede the proliferation of upcoming cyanobacterial blooms. Phytoremediation technology, an effective method of decreasing cyanobacteria, has gradually attracted more attention due to its advantages of low cost and the continuable ability of restoring drinking water source ecosystem. There are many aquatic plants that can purify water efficiently, of which the Hydrocharis dubia (Bl.) backer has strong tolerance to waste water, developed roots, fast growth and reproduction. Moreover, Hydrocharis dubia (Bl.) backer can play a pushing role in the stability of aquatic ecosystem and water quality purification(Liu, Pang et al. 2020 ). Previous studies have reported that Hydrocharis dubia (Bl.) backer has many adventitious roots that can insert into shallow water matrix and reproduce quickly in the center of open water. It can absorb nutrients such as N, P, and C, and compete with algae for light and space to inhibit the excessive reproduction of algae(Li, Wang et al. 2022 ). However, when cyanobacteria bloom in the drinking water source and release large amounts of MC-LR, Hydrocharis dubia (Bl.) backer directly contact with them and vulnerable to invasion(Li, Wang et al. 2022 ). In addition, there are few has focused on the effects of MC-LR on water purification of Hydrocharis dubia (Bl.) backer . MC-LR can affect physiological characteristics of aquatic plant, including root morphology, enzyme activities, malondialdehyde (MDA) content and photosynthesis parameters. It has been reported that at different exposure times or dosages, the effects of MC-LR on plants are varied(Li, Wang et al. 2022 ). MC-LR usually exists for months or even years in eutrophic water, chronic exposure may occur, which requires investigating the chronic effect of MC-LR on aquatic plants. And it can influence the nutrient removal efficiency of aquatic plant by inhibit the plant growth. In addition, MC-LR also has negative effects on chlorophyll content, net photosynthetic rate, and root system of plants(He, Wei et al. 2022 ). In many cases, the toxicity of MC-LR to aquatic plants is related to the induction of oxidative stress, which plays an important role in the mechanism of MC-LR biotoxicity. In recent years, MC-LR has passive effects on superoxide dismutase and peroxidase activities of aquatic plants, the oxidative damage caused by it to cells has attracted extensive attention to researchers. Additionally, MC-LR can induce cells to produce excessive reactive oxygen species (ROS). The oxidizing stress caused by MC-LR can change the contents of Glutathione (GSH) and other sulfhydryl-containing substances in cells, and induces changes in mitochondrial membrane permeability to make membrane potential depolarization. This process further result in cell damage, lipid peroxidation and even apoptosis, and increase the cytochrome release(Jiang, Zhang et al. 2022 ). Baranwal et al. indicated that the production and oxidative damage of ROS in organisms under pollutant stress may be a significant pathway of organisms poisoning(Baranwal, Kang et al. 2022 ). Therefore, it is necessary to pay attention to the influence of algae toxin on aquatic plants. Hydrocharis dubia (Bl.) backer has been widely applied in ecological restoration engineering, and their existence is often accompanied by the growth of algae. However, the effects of MC-LR released by algae on water purification and the physiological response of Hydrocharis dubia (Bl.) backer are still unclear. The present work aims to analyze the influence of MC-LR on the remediation of contaminated drinking water source by Hydrocharis dubia (Bl.) backer . And the physiological responses of Hydrocharis dubia (Bl.) backer , including plant root morphology and cell structure, chlorophyll and MDA content of leaves, and antioxidant enzyme system, will be revealed the influence mechanism of MC-LR on the drinking water source purification by Hydrocharis dubia (Bl.) backer . 2. Materials and methods 2.1 Plant and experimental design Hydrocharis dubia (Bl.) backer was purchased from Jinhua Farm Company (Zhejiang, China), and cultured in 1/5 Hoagland solution for 14 days. Hydrocharis dubia (Bl.) backer with similar growth were selected and pruned to the same root length and washed with water for subsequent experiments. The purification experiment of contaminated drinking source water was conducted in an artificial simulation. According to the water quality standards of China, the specific concentration of contaminated drinking water source is as follows ammonia (NH 4 + -N) 2.2 mg L − 1 , total phosphorous (TP) 0.4 mg L − 1 , and chemical oxygen demand (COD) 45 mg L − 1 . In accordance with that, 1.80 g NH 4 Cl, 0.42 g KH 2 PO 3 , 0.48 g KNO 3 , and 12.00 g C 6 H 12 O 6 ·12H 2 O were dissolved and mixed into a 1 L system, then diluted 300 times (the purity of the above reagents was analytical pure). The MC-LR were purchased from Beijing Puhuashi Technology Development Co., Ltd. In each group, the same weight of plants was added and exposed into simulated contaminated drinking water source under different MC-LR concentrations (0, 0.1, 0.2, 1, 2, 5, 10 µg L − 1 ); The removal efficiency of pollutants in test water was fast, so the experiment of MC-LR on water purification was carried out for 3 days, and aeration for 2 hours every 12 hours; Three replicates were prepared for all treatments. After the experiment, representative concentrations were selected and the following physiological reaction experiments were carried out. Physiologic response experiments were exposed to different concentrations of MC-LR and maintained in artificial contaminated water under the described laboratory conditions for a total exposure period of 14 days. Each treatment had three replicates, and each replicate contained two plants of similar size. A control treatment was set up using a culture medium and plants without MC-LR. The plants were harvested after 3 days and 14 days of exposure. The 3 days was set to study the sensitivity and adaptive response in plants to MC-LR stress. To assess plant physiological stress response, malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) activity, and chlorophyll content as markers of defense and phytotoxicity were analyzed and evaluated. As roots take time to establish deep root growth, the evaluation of their physical and chemical properties was carried on after 14 days. During the experiment, half of the water in the treatment system with the contaminated drinking water source is replaced every day. They were first rinsed with double distilled water to remove the adsorbed substance from their surface and then packed individually for the estimation of various parameters. 2.2 Pollutants measurements Nessler’s reagent colorimetric method and Ammonium Molybdate Spectrophotometry were adopted in the determination of NH 4 + -N content and TP content respectively. COD content was determined by Fast Digestion Spectrophotometry. 2.3 Measurement of plant root growth In order to evaluate plant response to different MC-LR exposure, root morphology and change in cell structure were measured after 14 days of exposure. The length, tips, surface area and average diameter of the root of Hydrocharis dubia (Bl.) backer were measured by root scanner (LC4800P) and analyzed by WinRHIZO Pro 2005b, manufactured by REGENT from Canada. Scanning electron microscopy (SEM/JSM-6510A, manufactured by Japanese Electronic Company) was used to observe the cell change within the cell structure of the roots before and after exposure. 2.4 Photosynthetic pigment measurements The chlorophyll Meter (TYS-A) was used to analyze the relative chlorophyll contents (SPAD). The average value of the leaves measured everyday was assessed as the chlorophyll contents of the current treatment group. 2.5 Measurement of the antioxidant system To assess plant physiological stress response, malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) activity, and chlorophyll content as markers of defense and phytotoxicity were analyzed and evaluated. The SOD enzyme activity was determined by the nitroblue tetrazolium photochemical reduction method (Badger and Andrews, 2002). The POD activity was analyzed by guaiacol method of Espie G S and Canvin D T with minor modifications (Espie and Canvin, 1997 ). The CAT activity was measured by UV absorption method. As reference to Cavender-Bares K with minor modifications (Cavender-Bares et al., 1999). The MDA content of leaves was determined by thiobarbituric acid (TBA) colorimetry, which refers to Aravind and Prasad (Aravind and Prasad, 2003 ). The antioxidant capacity of leaves in different treatment groups was tested. 0.2 g of Hydrocharis dubia (Bl.) backer was homogenized with 2 mL of 50 mM PBS (pH 7.8). The mixture was centrifuged at 12000 g and 4°C for 15 min, and the supernatants were used for enzyme assays (Zhang et al., 2007 ). 2.6 Statistical analysis All data are the means of three independent replicates and are expressed as means ± standard deviation. A one-way analysis of variance (ANOVA) using SPSS statistical software version 22.0 was used to analyze the data. 3. Results and discussion 3.1 Effects of MC-LR on the pollutant purification capacity of Hydrocharis dubia (Bl.) backer In this study, the effects of different MC-LR concentration on the drinking source water purification by Hydrocharis dubia (Bl.) backer were revealed by measuring the removal efficiency of ammonia nitrogen, phosphorous and COD. As shown in Fig. 1 , the low MC-LR concentrations (0.1-1 µg L − 1 ) increased the removal of pollutants by Hydrocharis dubia (Bl.) backer (p < 0.01) in comparison with the controls, especially for COD. Within this concentration range, the removal rates of NH 4 + -N, TP, and COD under optimal condition increased from 78.9%, 70.0% and 46.0–90.7%, 93.2% and 77.3%, respectively. The effluent concentrations of NH 4 + -N, TP, and COD were 0.2–0.4 mg L − 1 , 0.02–0.05 mg L − 1 , and 12.1–28.8 mg L − 1 . When the MC-LR concentration was between 1 µg L − 1 and 10 µg L − 1 , the removal rates of NH 4 + -N, TP, and COD decreased respectively to 70.1%, 84.9%, and 28.7% with the increase of MC-LR concentration. The effluent concentrations of NH 4 + -N, TP, and COD were 0.6 mg L − 1 , 0.06 mg L − 1 , and 26 mg L − 1 , respectively. It might because the toxicity of MC-LR at concentrations above 1 µg L − 1 exceeds the tolerance threshold of Hydrocharis dubia (Bl.) backer , resulting in the tissue growth of Hydrocharis dubia (Bl.) backer was inhibited. In this case, the roots had difficulty absorbing and utilizing the nutrients in the water, and the TP, TN and COD removal efficiencies decreased. The similar results were found by other studies in the decreasing of fresh weight for Iris pseudacorus L (Wang and Wang 2018 ), negative effect on the photosynthesis (Pflugmacher, Jung et al. 2006 ) and restrain the development of root tissue of the aquatic plant under superfluous MC-LR. The results indicate that low contents (0.1-1 µg L − 1 ) are in favor of the purification effect of eutrophic water by Hydrocharis dubia (Bl.) backer and a passive influence for Hydrocharis dubia (Bl.) backer remediation with the concentration of MC-LR between 1–10 µg L − 1 . Among the concentration scope of 0.1-1 µg L − 1 , the groups of 0.2 µg L − 1 and 0.5 µg L − 1 concentration were representative. From Fig. 1 , the MC-LR concentration of 1–10 µg L − 1 inhibited the purification of eutrophic water, of which 1 µg L − 1 and 5 µg L − 1 concentration was representative. Thus, four representative concentrations (0.2, 0.5, 1 and 5 µg L − 1 ) were selected to study the physiological response of Hydrocharis dubia (Bl.) backer . 3.2 Effects of MC-LR on the root growth The root growth of aquatic plants is related to the nutrient absorption ability of the root system. The function of roots is main to absorb the water and dissolved inorganic salts and transfers them to the interior of the aquatic plant for growth(Li, Gu et al. 2020 ). Moreover, the roots also have the functions of supporting, breeding, and storing of synthesizing organic substances. Hence, the growth of roots directly affects the growth of plants. Table 1 shows the effect of MC-LR on the root growth of Hydrocharis dubia (Bl.) backer seedlings. As illustrated in Table 1 , chronic exposure to different concentrations of MC-LR induced an obvious difference on the root growth of Hydrocharis dubia (Bl.) backer (p 0.5 µg L − 1 >1 µg L − 1 (p < 0.01). Obviously, the root growth rate of Hydrocharis dubia (Bl.) backer was the fastest under 0.2 µg L − 1 MC-LR treatment, the average root length, root tip, root surface area and average diameter were 5.62 cm plant − 1 , 5 tips plant − 1 , 2.667 cm 2 plant − 1 , and 1.397 mm plant − 1 , respectively. Compared with the CK, the average diameter and surface area of the root, root length and root tips of Hydrocharis dubia (Bl.) backer increased by 113.0%, 265.5%, 71.3% and 271.4%, respectively. The poisonousness of the hydroponic solution exceeds the tolerance of Hydrocharis dubia (Bl.) backer under the MC-LR concentration of 5 µg L − 1 , which the growth of the root was severely inhibited (p < 0.01). Compared with the CK, the root length, root tips, root surface area and root average diameter decreased by 15.2%, 7.1%, 37.0% and 25.9%, respectively. The results indicate that a certain degree of self-protection of the root was established under the low-concentration MC-LR. The Hydrocharis dubia (Bl.) backer seedlings can absorb more nutrients through root extension and maintain a normal growth and development under low toxin concentration environment than that of suitable environment. Therefore, the low-concentration MC-LR plays a positive role in the growth of Hydrocharis dubia (Bl.) backer roots and a promoted absorption of pollutants in eutrophic water. Scanning electron microscopy (SEM) images of Hydrocharis dubia (Bl.) backer roots are shown the same results in Fig. 2 . The root epidermal cells of the CK were flat and the cells were no visible damage. The degree of epidermal damage of the roots increased by the MC-LR concentration. When Hydrocharis dubia (Bl.) backer seedlings were exposed to 5 µg L − 1 MC-LR, the root epidermal cells began to collapse and the cell wall was distorted. It could be seen that high concentration of MC-LR has obvious inhibitory effect on root growth. In summary, when the concentration of MC-LR exceeds the tolerance of Hydrocharis dubia (Bl.) backer seedlings, root tissue structure would be destroyed. And the low concentration (0.1-1 µg L − 1 ) can stimulate the root tissue and the morphology structure of the root remains unchange. Table 1 Effect of MC-LR on root growth of Hydrocharis dubia (Bl.) backer . MC-LR concentration (µg L − 1 ) Root length (cm plant − 1 ) Root tips (tips plant − 1 ) Root surface area (cm 2 plant − 1 ) Average diameter (mm) 0 3.28 ± 0.122 14 ± 0.814 0.675 ± 0.020 0.656 ± 0.018 0.2 5.62 ± 0.065 52 ± 1.632 2.467 ± 1.646 1.397 ± 0.009 0.5 4.15 ± 0.057 56 ± 2.449 0.765 ± 0.012 0.586 ± 0.007 1.0 3.49 ± 0.073 37 ± 2.229 0.393 ± 0.035 0.358 ± 0.008 5.0 2.78 ± 0.064 13 ± 0.816 0.425 ± 0.022 0.486 ± 0.009 3.3 Effects of MC-LR on photosynthetic pigments Photosynthetic pigment is one of the criteria for evaluating plant growth. According to the chlorophyll content of the CK in Fig. 3 (a), the photosynthesis of Hydrocharis dubia (Bl.) backer without MC-LR was significantly affected (p < 0.01). It could be seen from Fig. 3 (a) that a significant increase in the chlorophyll content was observed in leaves of Hydrocharis dubia (Bl.) backer at 1 d after 0.2 and 0.5 µg·L − 1 MC-LR treatment (p > 0.01). With the exposure time under MC-LR increasing, chlorophyll contents of Hydrocharis dubia (Bl.) backer in each treatment group continued to decrease, but still increased in varying degrees compared with the CK. The results showed the reduction rates of chlorophyll contents under the influence of low-concentration MC-LR (< 1µg L − 1 ) and high-concentration MC-LR (≥ 1 µg L − 1 ) were 42.0%-43.2% and 50.1%-56.2% respectively, while the reduction rate of chlorophyll content in the CK was 57.7% (Fig. 3 (b)). Overall, chlorophyll contents under the influence of low-concentration MC-LR were higher than those of other treatments (p < 0.01) and showed a 14.5%-15.7% promoting effect compared with the CK, which promoted the ability of plants to photosynthesize. This result was consistent with Section 3.2 . Plants exposed to environmental stress can produce ROS such as H 2 O 2 and O 2 − . Excessive ROS production may destroy cell membranes and lead to membrane lipid peroxidation and oxidative stress(Song, Yin et al. 2007 , Alzandi and Naguib 2020 ). MC-LR induces oxidative stress and inhibits plant growth through the accumulation of reactive oxygen species (ROS) in plants(Cao, Rediske et al. 2018 ). Therefore, when Hydrocharis dubia (Bl.) backer was exposed to high concentrations of MC-LR, the decrease in chlorophyll content might be due to the accumulation of reactive oxygen species (ROS), leading to cell damage and accelerating the decomposition of chlorophyll(Li, Riaz et al. 2022 ). 3.4 Effects of MC-LR on MDA content Membrane lipid peroxidation of plant tissues is shown in Fig. 4 . MDA content is often used to indicate the degree of oxidative damage in plants(Kumar and Prasad 2018 ). After one day exposure, the MDA content in leaves of Hydrocharis dubia (Bl.) backer showed no significant changes (Fig. 4 ), which slightly increased from 4.28 µmol g − 1 (0 µg L − 1 ) in the CK to 4.55 µmol g − 1 (0.2 µg L − 1 ), 4.50 µmol g − 1 (0.5 µg L − 1 ), 5.18 µmol g − 1 (1 µg L − 1 ), and 5.52 µmol g − 1 (5 µg L − 1 ) in the experimental group. Within the 3 days of exposure, the concentrations of MDA were significantly increased with exposure to higher concentrations of MCLR. Similar result has also been observed in previous study(Li, Gu et al. 2020 ). Notably, the MDA contents in low MC-LR concentration (< 1 µg L − 1 ) treatment group were significantly increased. Compared with one day of exposure, MDA contents of 3 days of exposure increased by 2.3 µmol g − 1 , 1.6 µmol g − 1 , and 1.9 µmol g − 1 respectively. The concentrations of MDA were significantly increased with exposure to longer time and higher concentration of MC-LR, which indicated that the interactions between exposure concentration and exposure time for MC-LR all showed significant for MDA contents. The contents of MDA in Hydrocharis dubia (Bl.) backer significantly decreased 4.48 µmol g − 1 and 4.83 µmol g − 1 after 3 days under MC-LR treatment with a high concentration of 1 µg L − 1 and 5 µg L − 1 , meaning that high concentration of MC-LR would hurt the organism of Hydrocharis dubia (Bl.) backer and makes the Hydrocharis dubia (Bl.) backer have a weak resistance to adverse environment. However, the Fig. 4 displays that low concentration of MC-LR (0.2 and 0.5 µg L − 1 ) active the role of ROS molecules in Hydrocharis dubia (Bl.) backer leaves, which can maintain the normal physiological functions and avoid the damage of oxidative reaction for Hydrocharis dubia (Bl.) backer . The metabolic effect of ROS is the aquatic plants self-protection response to the external stress(Zhang, Wang et al. 2018 ). In contrast, MC-LR at the concentration of 1 µg L − 1 and 5 µg L − 1 could cause long-term ROS accumulation in Hydrocharis dubia (Bl.) backer leaves, resulting an oxidative cell damage. 3.5 Effects of MC-LR on antioxidative enzymes The antioxidant enzymes system can help plants to resist adverse stress and reduce the organism structure damage. Among the enzymes, the antioxidative enzymes of SOD, POD and CAT play significant roles in elimination ROS and keep the plants homeostasis. The SOD can catalyze ·O 2 − disproportionation to produce H 2 O 2 and O 2 to reduce oxidative damage(Boldaji, Khavari-Nejad et al. 2012 ). And the POD and CAT are important enzymes that catalyze the conversion of H 2 O 2 to H 2 O(Gu and Liang 2020 ). In this study, the oxidative stress responses of Hydrocharis dubia (Bl.) backer to MC-LR were analyzed via the activities of the three enzymes of the seedlings under different MC-LR concentrations. The Fig. 5 shows that the different concentration of MC-LR induced oxidative stress on the Hydrocharis dubia (Bl.) backer seedlings. Commonly, an increase in antioxidant enzyme activities is recognised as one of the responses for oxidative stress in plants. Figure 5 (a) displays that the SOD contents increased from 105.3 µ g − 1 FW to 139.7 µ g − 1 FW with MC-LR concentration increasing (0.2-5 µg L − 1 ), but the contents of SOD are less than the CK group, indicating that the antioxidant properties of Hydrocharis dubia (Bl.) backer decreased to some extent. Figure 5 (b) shows that POD activities decreased with the MC-LR concentration increases, and CAT activities reversed under the MC-LR concentration of 0.2–0.5 µg L − 1 . With the concentration of MC-LR is more than 1 µg L − 1 , the POD and CAT activities all decreased. Compared with the CK, the Fig. 5 (b) and (c) show that the max amount of POD increased by 55.5% under 0.2 µg L − 1 MC-LR, and the max amount of CAT increased by 100.4% at 0.5 µg L − 1 MC-LR, suggesting that low concentration MC-LR greatly promoted the activity of POD and CAT. As we know that the POD and CAT are two of the most vital enzymes involved in eliminating redundant ROS. These two antioxidant enzymes would be promoted when the H 2 O 2 increased in plants, and removed excess H 2 O 2 from the plants to protect themselves from damage. The enzymatic activity results indicate that increasing reactive oxygen species (ROS) produced by MC-LR led to oxidative stress (Yang and Wang, 2019 ). If such substances are not removed in plants, the accumulation of hydrogen peroxide will occur in plants, which will destroy proteins, cellular nucleic acids, and cell membranes. In order to avoid the damage caused by oxidative stress, aquatic plants will exert oxidative stress, and the active oxygen will be eliminated by the antioxidant enzyme system. However, it should be noted that antioxidant enzymes played different roles for Hydrocharis dubia (Bl.) backer in different concentrations of MC-LR. For example, SOD responded to different concentrations of MC-LR and continued to rise, reaching the highest activity under the MC-LR concentration of 5 µg L − 1 . POD almost immediately responded to 0.2 µg L − 1 of MC-LR and reached the highest activity, while the highest level of CAT activity occurred at 0.5 µg L − 1 MC-LR. In summary, MC-LR is harmful to Hydrocharis dubia (Bl.) backer in the process of purifying eutrophic water, but Hydrocharis dubia (Bl.) backer can resist the possible damage caused by MC-LR by increasing activities of CAT, POD, and SOD. MC-LR with a low concentration (< 1 µg L − 1 ) can improve the antioxidant enzyme activity and chlorophyll contents of Hydrocharis dubia (Bl.) backer , enhanced its ability to absorb nutrients. In contrast, when the MC-LR concentration is above 1 µg L − 1 , chlorophyll contents, MDA contents, and antioxidant enzyme activities decreased, generating antioxidant injury, subsequently the abilities to absorb nutrients weakened. Overall, these physiological responses are consistent with the results of the effects rule of MC-LR on the water purification. 4. Conclusion This study mainly investigated the effect of the MC-LR on the purification of the drinking water source by Hydrocharis dubia (Bl.) backer and discussed the effect of MC-LR on plant tissue growth and physiological response. The purifying effect of Hydrocharis dubia (Bl.) backer on contaminated water could be promoted at low concentration of MC-LR (< 1 µg L − 1) ) and inhibited at high concentration (1–10 µg L − 1 ). The physiological characteristics of Hydrocharis dubia (Bl.) backer reveal some stress and injury induced by MC-LR level. In low concentration of MC-LR, the growth of roots enhanced, and subsequently improved the ability of Hydrocharis dubia (Bl.) backer to absorb nutrients. While high concentration MC-LR affects the plant tolerance and inhibited the root growth. As response to that Hydrocharis dubia (Bl.) backer displays enzymatic defense to tolerate relatively low concentration of MC-LR exposure. The activities of POD, SOD, and CAT were enhanced under stimulation of low concentration MC-LR. The relative activities of these three enzymes remove the excess of H 2 O 2 from the plant and protect themselves from damage. These results provide guidance for further revealing the role of MC-LR in aquatic ecosystems, when the cyanobacterial occur but not in a large scale in water bodies used as the drinking water source, low concentrations of MCs (< 1 µg L − 1 ) exert a positive effect on contamination removal by Hydrocharis dubia (Bl.) backer . Declarations Ethics approval and consent to participate : Not applicable Consent for publication : All authors have read and approved the final manuscript for publication. Availability of data and materials : All data are available from the corresponding author. Competing interests : The authors declare no competing interests. Funding: This work was financially supported by the Fundamental Research Funds for the Central Universities (FRF-TP-22-084A1), Guangdong Basic and Applied Basic Research Foundation (2022A1515111178), and Major Science and Technology Program for Water Pollution Control and Treatment of China (No. 2017ZX07101004). Authors' contributions: Junfei Liu: Conceptualization, Writing-original draft, Investigation, Formal analysis, Validation, Resources, Writing, review&editing, Formal analysis，Funding. Yingbo Dong: Supervision, Writing-review& editing, Funding 404 acquisition, Project administration. Hai Lin: Conceptualization, Supervision, Validation, Resources, Writing, review&editing, Formal analysis. References Alzandi, A. A. and D. M. Naguib (2020). "Effect of hydropriming on Trigonella foenum callus growth, biochemical traits and phytochemical components under PEG treatment." Plant Cell, Tissue and Organ Culture 141(1): 179-190. Amrani, A., Nasri, H., Azzouz, A., Kadi, Y., Bouaicha, B., 2014. Variation in cyanobacterial hepatotoxin (microcystin) content of water samples and two species of fishes collected from a shallow lake in Algeria. Archives of Environmental Contamination & Toxicology. 66(3), 379-389. Aravind, P., Prasad, M., 2003. 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Interactions between Microcystis aeruginosa and coexisting bisphenol A at different phosphorus levels. Science of the Total Environment. 658, 439-448. Zhang, F., Wang, Y., Lou, Z., Dong, J.D., 2007. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemosphere. 67(1), 44-50. Zhang, Q., Huber, H., Beljaars, S.J.M., Birnbaum, D., de Best, S., de Kroon, H., Visser, E.J.W., 2017. Benefits of flooding-induced aquatic adventitious roots depend on the duration of submergence: linking plant performance to root functioning. Annals of Botany. 120(1), 171-180. Zhong, G., Wu, Z., Liu, N., Yin, J., 2018. Phosphate alleviation of glyphosate-induced toxicity inHydrocharis dubia (Bl.) backer. Aquatic Toxicology. 2201, 91-98. Zhang, J., L. Wang, Q. Zhou and X. Huang (2018). "Reactive oxygen species initiate a protective response in plant roots to stress induced by environmental bisphenol A." Ecotoxicology and Environmental Safety 154: 197-205. Zhang, Y., J. K. Whalen and S. Sauvé (2021). "Phytotoxicity and bioconcentration of microcystins in agricultural plants: Meta-analysis and risk assessment." Environmental Pollution 272: 115966. Zhao, J., Shi, G., Yuan, Q., 2008. Polyamines content and physiological and biochemical responses to ladder concentration of nickel stress inHydrocharis dubia (Bl.) backer leaves. Biometals. 21(6), 665-674. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3066142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":220971537,"identity":"2a6966dc-e6f7-4ddf-85d9-ef7d43753e3a","order_by":0,"name":"Junfei Liu","email":"","orcid":"","institution":"University of Science and Technology Beijing","correspondingAuthor":false,"prefix":"","firstName":"Junfei","middleName":"","lastName":"Liu","suffix":""},{"id":220971538,"identity":"06a1d27b-b057-4d84-b3eb-a1f14c521c99","order_by":1,"name":"Yingbo Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDACCQYDIGkDxDxAzEa8ljSgahK1HCZBi8Ht5o2PC36dT5w/v/cAw4eywwz8sxsIaLlzrNh4Zt/txA3H+BIYZ5w7zCBx5wABLTdyzKR5e4Ba2HgMmHnbDjMYSCQQpeVc4vw2oJa/RGvh+XEgseEYUAsjMVokQX7hbUg23nAsL+Fgz7l0HokbBLTwgUKM54+d7Pzmswcf/CizluOfQUCLwgEgwdgG4YDYPPjVA4F8A4j8Q1DdKBgFo2AUjGQAAJDCRrnqZgbbAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Environmental Engineering, University of Science and Technology Beijing","correspondingAuthor":true,"prefix":"","firstName":"Yingbo","middleName":"","lastName":"Dong","suffix":""},{"id":220971539,"identity":"a5db9401-5a6b-4c68-9aa3-1d1a1408a298","order_by":2,"name":"Hai Lin","email":"","orcid":"","institution":"University of Science and Technology Beijing","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2023-06-15 06:58:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3066142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3066142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":40664018,"identity":"bbc0df27-fd3f-4a57-a3bf-030449adfce0","added_by":"auto","created_at":"2023-07-27 14:16:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95742,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MC-LR concentration on pollutants removal rate (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, COD).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3066142/v1/8ece75cef25e8a4edb5c0519.jpg"},{"id":40664016,"identity":"207912d8-6f30-40eb-8789-94d1211ac864","added_by":"auto","created_at":"2023-07-27 14:16:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":456991,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MC-LR concentration on root morphology of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e (CK (a); 0.2 μg L\u003csup\u003e-1\u003c/sup\u003e (b); 0.5 μg L\u003csup\u003e-1\u003c/sup\u003e (c); 1 μg L\u003csup\u003e-1\u003c/sup\u003e (d); 5 μg L\u003csup\u003e-1\u003c/sup\u003e (e))\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3066142/v1/53a2ae64805eff84d03983e6.jpg"},{"id":40665140,"identity":"212d5ed9-3931-4b97-bb7e-ad715e1a958e","added_by":"auto","created_at":"2023-07-27 14:24:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":182923,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MC-LR concentration on chlorophyll content (Trends of relative chlorophyll content in \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e treated with different MC-LR treatments, (a); Reduction of relative chlorophyll content in \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e treated with different MC-LR treatments, (b)).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3066142/v1/0204726707e88c5ba6724015.jpg"},{"id":40664015,"identity":"813fa5dd-c881-498a-a497-1552f642670f","added_by":"auto","created_at":"2023-07-27 14:16:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116415,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MC-LR on MDA content. All values represent the mean of three replicates ± standard deviation.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3066142/v1/5ad816fbbfa9ba9e5c3003d8.jpg"},{"id":40665141,"identity":"616cafa8-f632-4a20-887b-da43dbeadd52","added_by":"auto","created_at":"2023-07-27 14:24:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217919,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MC-LR concentration on the antioxidant enzyme of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e (SOD (a); POD (b); CAT (c)).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3066142/v1/b7768657776452a570cada4a.jpg"},{"id":43171684,"identity":"e9415387-3d83-4985-bd2b-4f09aafbcea7","added_by":"auto","created_at":"2023-09-15 05:55:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":765752,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3066142/v1/3fd56e34-f9b4-401c-a96c-c67fecef872f.pdf"}],"financialInterests":"","formattedTitle":"Effects of microcystin-LR on purification of drinking water source and physiological response of Hydrocharis dubia (Bl.) backer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA widespread fertility of harmful cyanobacterial blooms in many marine and fresh-water ecosystems is serious causing by eutrophication and global climate change(Xu, Pang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies showed that the average days of cyanobacterial blooms in the United State increased from seven days per year under present climate to 18\u0026ndash;39 days in 2090 causing by climate change(Chapra, Boehlert et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The massive cyanobacterial existing in water environment can block the light and affect the growth of herbivorous zooplankton(Huisman, Sharples et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In addition, some cyanobacteria cell lysis can release toxins (like Microcystin) into water that can cause digestive and neurological diseases(Zhang, Whalen et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The Microcystin-LR (MC-LR), one of the most common Microcystin, is set to a guideline limit of 1.0 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in drinking water by the World Health Organization(Xue, Steinman et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). How to control the content of MC-LR in drinking water under guideline limit is an important work.\u003c/p\u003e \u003cp\u003eTo date, the studies show that most methods for algae removal would produce the harmful by-products, or the heavy metals accumulation such as Cu in ecosystems(Du, Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, there is an urgent need for an effective and eco-friendly way to impede the proliferation of upcoming cyanobacterial blooms. Phytoremediation technology, an effective method of decreasing cyanobacteria, has gradually attracted more attention due to its advantages of low cost and the continuable ability of restoring drinking water source ecosystem. There are many aquatic plants that can purify water efficiently, of which the Hydrocharis dubia (Bl.) backer has strong tolerance to waste water, developed roots, fast growth and reproduction. Moreover, \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e can play a pushing role in the stability of aquatic ecosystem and water quality purification(Liu, Pang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Previous studies have reported that \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e has many adventitious roots that can insert into shallow water matrix and reproduce quickly in the center of open water. It can absorb nutrients such as N, P, and C, and compete with algae for light and space to inhibit the excessive reproduction of algae(Li, Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, when cyanobacteria bloom in the drinking water source and release large amounts of MC-LR, \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e directly contact with them and vulnerable to invasion(Li, Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, there are few has focused on the effects of MC-LR on water purification of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMC-LR can affect physiological characteristics of aquatic plant, including root morphology, enzyme activities, malondialdehyde (MDA) content and photosynthesis parameters. It has been reported that at different exposure times or dosages, the effects of MC-LR on plants are varied(Li, Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). MC-LR usually exists for months or even years in eutrophic water, chronic exposure may occur, which requires investigating the chronic effect of MC-LR on aquatic plants. And it can influence the nutrient removal efficiency of aquatic plant by inhibit the plant growth. In addition, MC-LR also has negative effects on chlorophyll content, net photosynthetic rate, and root system of plants(He, Wei et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In many cases, the toxicity of MC-LR to aquatic plants is related to the induction of oxidative stress, which plays an important role in the mechanism of MC-LR biotoxicity. In recent years, MC-LR has passive effects on superoxide dismutase and peroxidase activities of aquatic plants, the oxidative damage caused by it to cells has attracted extensive attention to researchers. Additionally, MC-LR can induce cells to produce excessive reactive oxygen species (ROS). The oxidizing stress caused by MC-LR can change the contents of Glutathione (GSH) and other sulfhydryl-containing substances in cells, and induces changes in mitochondrial membrane permeability to make membrane potential depolarization. This process further result in cell damage, lipid peroxidation and even apoptosis, and increase the cytochrome release(Jiang, Zhang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Baranwal et al. indicated that the production and oxidative damage of ROS in organisms under pollutant stress may be a significant pathway of organisms poisoning(Baranwal, Kang et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, it is necessary to pay attention to the influence of algae toxin on aquatic plants.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e has been widely applied in ecological restoration engineering, and their existence is often accompanied by the growth of algae. However, the effects of MC-LR released by algae on water purification and the physiological response of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e are still unclear. The present work aims to analyze the influence of MC-LR on the remediation of contaminated drinking water source by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e. And the physiological responses of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e, including plant root morphology and cell structure, chlorophyll and MDA content of leaves, and antioxidant enzyme system, will be revealed the influence mechanism of MC-LR on the drinking water source purification by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant and experimental design\u003c/h2\u003e \u003cp\u003e \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e was purchased from Jinhua Farm Company (Zhejiang, China), and cultured in 1/5 Hoagland solution for 14 days. \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e with similar growth were selected and pruned to the same root length and washed with water for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe purification experiment of contaminated drinking source water was conducted in an artificial simulation. According to the water quality standards of China, the specific concentration of contaminated drinking water source is as follows ammonia (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) 2.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, total phosphorous (TP) 0.4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and chemical oxygen demand (COD) 45 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In accordance with that, 1.80 g NH\u003csub\u003e4\u003c/sub\u003eCl, 0.42 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e3\u003c/sub\u003e, 0.48 g KNO\u003csub\u003e3\u003c/sub\u003e, and 12.00 g C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO were dissolved and mixed into a 1 L system, then diluted 300 times (the purity of the above reagents was analytical pure). The MC-LR were purchased from Beijing Puhuashi Technology Development Co., Ltd. In each group, the same weight of plants was added and exposed into simulated contaminated drinking water source under different MC-LR concentrations (0, 0.1, 0.2, 1, 2, 5, 10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); The removal efficiency of pollutants in test water was fast, so the experiment of MC-LR on water purification was carried out for 3 days, and aeration for 2 hours every 12 hours; Three replicates were prepared for all treatments. After the experiment, representative concentrations were selected and the following physiological reaction experiments were carried out.\u003c/p\u003e \u003cp\u003ePhysiologic response experiments were exposed to different concentrations of MC-LR and maintained in artificial contaminated water under the described laboratory conditions for a total exposure period of 14 days. Each treatment had three replicates, and each replicate contained two plants of similar size. A control treatment was set up using a culture medium and plants without MC-LR. The plants were harvested after 3 days and 14 days of exposure. The 3 days was set to study the sensitivity and adaptive response in plants to MC-LR stress. To assess plant physiological stress response, malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) activity, and chlorophyll content as markers of defense and phytotoxicity were analyzed and evaluated. As roots take time to establish deep root growth, the evaluation of their physical and chemical properties was carried on after 14 days. During the experiment, half of the water in the treatment system with the contaminated drinking water source is replaced every day. They were first rinsed with double distilled water to remove the adsorbed substance from their surface and then packed individually for the estimation of various parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Pollutants measurements\u003c/h2\u003e \u003cp\u003eNessler\u0026rsquo;s reagent colorimetric method and Ammonium Molybdate Spectrophotometry were adopted in the determination of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content and TP content respectively. COD content was determined by Fast Digestion Spectrophotometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Measurement of plant root growth\u003c/h2\u003e \u003cp\u003eIn order to evaluate plant response to different MC-LR exposure, root morphology and change in cell structure were measured after 14 days of exposure. The length, tips, surface area and average diameter of the root of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e were measured by root scanner (LC4800P) and analyzed by WinRHIZO Pro 2005b, manufactured by REGENT from Canada. Scanning electron microscopy (SEM/JSM-6510A, manufactured by Japanese Electronic Company) was used to observe the cell change within the cell structure of the roots before and after exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Photosynthetic pigment measurements\u003c/h2\u003e \u003cp\u003eThe chlorophyll Meter (TYS-A) was used to analyze the relative chlorophyll contents (SPAD). The average value of the leaves measured everyday was assessed as the chlorophyll contents of the current treatment group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Measurement of the antioxidant system\u003c/h2\u003e \u003cp\u003eTo assess plant physiological stress response, malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) activity, and chlorophyll content as markers of defense and phytotoxicity were analyzed and evaluated.\u003c/p\u003e \u003cp\u003eThe SOD enzyme activity was determined by the nitroblue tetrazolium photochemical reduction method (Badger and Andrews, 2002). The POD activity was analyzed by guaiacol method of Espie G S and Canvin D T with minor modifications (Espie and Canvin, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The CAT activity was measured by UV absorption method. As reference to Cavender-Bares K with minor modifications (Cavender-Bares et al., 1999). The MDA content of leaves was determined by thiobarbituric acid (TBA) colorimetry, which refers to Aravind and Prasad (Aravind and Prasad, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe antioxidant capacity of leaves in different treatment groups was tested. 0.2 g of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e was homogenized with 2 mL of 50 mM PBS (pH 7.8). The mixture was centrifuged at 12000 g and 4\u0026deg;C for 15 min, and the supernatants were used for enzyme assays (Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are the means of three independent replicates and are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A one-way analysis of variance (ANOVA) using SPSS statistical software version 22.0 was used to analyze the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Effects of MC-LR on the pollutant purification capacity of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn this study, the effects of different MC-LR concentration on the drinking source water purification by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e were revealed by measuring the removal efficiency of ammonia nitrogen, phosphorous and COD. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the low MC-LR concentrations (0.1-1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) increased the removal of pollutants by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in comparison with the controls, especially for COD. Within this concentration range, the removal rates of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, and COD under optimal condition increased from 78.9%, 70.0% and 46.0\u0026ndash;90.7%, 93.2% and 77.3%, respectively. The effluent concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, and COD were 0.2\u0026ndash;0.4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.02\u0026ndash;0.05 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 12.1\u0026ndash;28.8 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. When the MC-LR concentration was between 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the removal rates of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, and COD decreased respectively to 70.1%, 84.9%, and 28.7% with the increase of MC-LR concentration. The effluent concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, and COD were 0.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.06 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 26 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. It might because the toxicity of MC-LR at concentrations above 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e exceeds the tolerance threshold of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e, resulting in the tissue growth of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e was inhibited. In this case, the roots had difficulty absorbing and utilizing the nutrients in the water, and the TP, TN and COD removal efficiencies decreased. The similar results were found by other studies in the decreasing of fresh weight for \u003cem\u003eIris pseudacorus L\u003c/em\u003e(Wang and Wang \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), negative effect on the photosynthesis (Pflugmacher, Jung et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e) and restrain the development of root tissue of the aquatic plant under superfluous MC-LR. The results indicate that low contents (0.1-1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) are in favor of the purification effect of eutrophic water by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e and a passive influence for \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e remediation with the concentration of MC-LR between 1\u0026ndash;10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Among the concentration scope of 0.1-1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the groups of 0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration were representative. From Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the MC-LR concentration of 1\u0026ndash;10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e inhibited the purification of eutrophic water, of which 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration was representative. Thus, four representative concentrations (0.2, 0.5, 1 and 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were selected to study the physiological response of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Effects of MC-LR on the root growth\u003c/h2\u003e\n\u003cp\u003eThe root growth of aquatic plants is related to the nutrient absorption ability of the root system. The function of roots is main to absorb the water and dissolved inorganic salts and transfers them to the interior of the aquatic plant for growth(Li, Gu et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the roots also have the functions of supporting, breeding, and storing of synthesizing organic substances. Hence, the growth of roots directly affects the growth of plants. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the effect of MC-LR on the root growth of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e seedlings. As illustrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, chronic exposure to different concentrations of MC-LR induced an obvious difference on the root growth of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The low-concentration MC-LR can stimulate both root length and activity growth, and the degree of promotion is 0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026gt;0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026gt;1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Obviously, the root growth rate of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e was the fastest under 0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MC-LR treatment, the average root length, root tip, root surface area and average diameter were 5.62 cm plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 5 tips plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.667 cm\u003csup\u003e2\u003c/sup\u003e plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1.397 mm plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Compared with the CK, the average diameter and surface area of the root, root length and root tips of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e increased by 113.0%, 265.5%, 71.3% and 271.4%, respectively. The poisonousness of the hydroponic solution exceeds the tolerance of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e under the MC-LR concentration of 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which the growth of the root was severely inhibited (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the CK, the root length, root tips, root surface area and root average diameter decreased by 15.2%, 7.1%, 37.0% and 25.9%, respectively. The results indicate that a certain degree of self-protection of the root was established under the low-concentration MC-LR. The \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e seedlings can absorb more nutrients through root extension and maintain a normal growth and development under low toxin concentration environment than that of suitable environment. Therefore, the low-concentration MC-LR plays a positive role in the growth of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e roots and a promoted absorption of pollutants in eutrophic water.\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy (SEM) images of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e roots are shown the same results in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The root epidermal cells of the CK were flat and the cells were no visible damage. The degree of epidermal damage of the roots increased by the MC-LR concentration. When \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e seedlings were exposed to 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MC-LR, the root epidermal cells began to collapse and the cell wall was distorted. It could be seen that high concentration of MC-LR has obvious inhibitory effect on root growth. In summary, when the concentration of MC-LR exceeds the tolerance of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e seedlings, root tissue structure would be destroyed. And the low concentration (0.1-1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) can stimulate the root tissue and the morphology structure of the root remains unchange.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffect of MC-LR on root growth of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMC-LR concentration (\u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot length\u003c/p\u003e\n\u003cp\u003e(cm plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot tips\u003c/p\u003e\n\u003cp\u003e(tips plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRoot surface area\u003c/p\u003e\n\u003cp\u003e(cm\u003csup\u003e2\u003c/sup\u003e plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAverage diameter\u003c/p\u003e\n\u003cp\u003e(mm)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.122\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.814\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.675\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.656\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.632\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2.467\u0026thinsp;\u0026plusmn;\u0026thinsp;1.646\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.397\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.449\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.765\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.586\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e3.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.073\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.229\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.393\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.358\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.064\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.816\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.425\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.486\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Effects of MC-LR on photosynthetic pigments\u003c/h2\u003e\n\u003cp\u003ePhotosynthetic pigment is one of the criteria for evaluating plant growth. According to the chlorophyll content of the CK in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(a), the photosynthesis of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e without MC-LR was significantly affected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). It could be seen from Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(a) that a significant increase in the chlorophyll content was observed in leaves of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e at 1 d after 0.2 and 0.5 \u0026micro;g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MC-LR treatment (p\u0026thinsp;\u0026gt;\u0026thinsp;0.01). With the exposure time under MC-LR increasing, chlorophyll contents of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e in each treatment group continued to decrease, but still increased in varying degrees compared with the CK. The results showed the reduction rates of chlorophyll contents under the influence of low-concentration MC-LR (\u0026lt;\u0026thinsp;1\u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and high-concentration MC-LR (\u0026ge;\u0026thinsp;1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were 42.0%-43.2% and 50.1%-56.2% respectively, while the reduction rate of chlorophyll content in the CK was 57.7% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(b)). Overall, chlorophyll contents under the influence of low-concentration MC-LR were higher than those of other treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and showed a 14.5%-15.7% promoting effect compared with the CK, which promoted the ability of plants to photosynthesize. This result was consistent with Section \u003cspan class=\"InternalRef\"\u003e3.2\u003c/span\u003e. Plants exposed to environmental stress can produce ROS such as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. Excessive ROS production may destroy cell membranes and lead to membrane lipid peroxidation and oxidative stress(Song, Yin et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e, Alzandi and Naguib \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). MC-LR induces oxidative stress and inhibits plant growth through the accumulation of reactive oxygen species (ROS) in plants(Cao, Rediske et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, when \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e was exposed to high concentrations of MC-LR, the decrease in chlorophyll content might be due to the accumulation of reactive oxygen species (ROS), leading to cell damage and accelerating the decomposition of chlorophyll(Li, Riaz et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Effects of MC-LR on MDA content\u003c/h2\u003e\n\u003cp\u003eMembrane lipid peroxidation of plant tissues is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. MDA content is often used to indicate the degree of oxidative damage in plants(Kumar and Prasad \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). After one day exposure, the MDA content in leaves of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e showed no significant changes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), which slightly increased from 4.28 \u0026micro;mol g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the CK to 4.55 \u0026micro;mol g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 4.50 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 5.18 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and 5.52 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the experimental group. Within the 3 days of exposure, the concentrations of MDA were significantly increased with exposure to higher concentrations of MCLR. Similar result has also been observed in previous study(Li, Gu et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, the MDA contents in low MC-LR concentration (\u0026lt;\u0026thinsp;1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) treatment group were significantly increased. Compared with one day of exposure, MDA contents of 3 days of exposure increased by 2.3 \u0026micro;mol g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.6 \u0026micro;mol g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1.9 \u0026micro;mol g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively. The concentrations of MDA were significantly increased with exposure to longer time and higher concentration of MC-LR, which indicated that the interactions between exposure concentration and exposure time for MC-LR all showed significant for MDA contents.\u003c/p\u003e\n\u003cp\u003eThe contents of MDA in \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e significantly decreased 4.48 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 4.83 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after 3 days under MC-LR treatment with a high concentration of 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, meaning that high concentration of MC-LR would hurt the organism of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e and makes the \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e have a weak resistance to adverse environment. However, the Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e displays that low concentration of MC-LR (0.2 and 0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) active the role of ROS molecules in \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e leaves, which can maintain the normal physiological functions and avoid the damage of oxidative reaction for \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e. The metabolic effect of ROS is the aquatic plants self-protection response to the external stress(Zhang, Wang et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, MC-LR at the concentration of 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could cause long-term ROS accumulation in \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e leaves, resulting an oxidative cell damage.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Effects of MC-LR on antioxidative enzymes\u003c/h2\u003e\n\u003cp\u003eThe antioxidant enzymes system can help plants to resist adverse stress and reduce the organism structure damage. Among the enzymes, the antioxidative enzymes of SOD, POD and CAT play significant roles in elimination ROS and keep the plants homeostasis. The SOD can catalyze \u0026middot;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e disproportionation to produce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e to reduce oxidative damage(Boldaji, Khavari-Nejad et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). And the POD and CAT are important enzymes that catalyze the conversion of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO(Gu and Liang \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the oxidative stress responses of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e to MC-LR were analyzed via the activities of the three enzymes of the seedlings under different MC-LR concentrations. The Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows that the different concentration of MC-LR induced oxidative stress on the \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e seedlings. Commonly, an increase in antioxidant enzyme activities is recognised as one of the responses for oxidative stress in plants. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(a) displays that the SOD contents increased from 105.3 \u0026micro; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW to 139.7 \u0026micro; g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW with MC-LR concentration increasing (0.2-5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), but the contents of SOD are less than the CK group, indicating that the antioxidant properties of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e decreased to some extent. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(b) shows that POD activities decreased with the MC-LR concentration increases, and CAT activities reversed under the MC-LR concentration of 0.2\u0026ndash;0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. With the concentration of MC-LR is more than 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the POD and CAT activities all decreased. Compared with the CK, the Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(b) and (c) show that the max amount of POD increased by 55.5% under 0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MC-LR, and the max amount of CAT increased by 100.4% at 0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MC-LR, suggesting that low concentration MC-LR greatly promoted the activity of POD and CAT. As we know that the POD and CAT are two of the most vital enzymes involved in eliminating redundant ROS. These two antioxidant enzymes would be promoted when the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased in plants, and removed excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from the plants to protect themselves from damage.\u003c/p\u003e\n\u003cp\u003eThe enzymatic activity results indicate that increasing reactive oxygen species (ROS) produced by MC-LR led to oxidative stress (Yang and Wang, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). If such substances are not removed in plants, the accumulation of hydrogen peroxide will occur in plants, which will destroy proteins, cellular nucleic acids, and cell membranes. In order to avoid the damage caused by oxidative stress, aquatic plants will exert oxidative stress, and the active oxygen will be eliminated by the antioxidant enzyme system. However, it should be noted that antioxidant enzymes played different roles for \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e in different concentrations of MC-LR. For example, SOD responded to different concentrations of MC-LR and continued to rise, reaching the highest activity under the MC-LR concentration of 5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. POD almost immediately responded to 0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of MC-LR and reached the highest activity, while the highest level of CAT activity occurred at 0.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MC-LR.\u003c/p\u003e\n\u003cp\u003eIn summary, MC-LR is harmful to \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e in the process of purifying eutrophic water, but \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e can resist the possible damage caused by MC-LR by increasing activities of CAT, POD, and SOD. MC-LR with a low concentration (\u0026lt;\u0026thinsp;1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) can improve the antioxidant enzyme activity and chlorophyll contents of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e, enhanced its ability to absorb nutrients. In contrast, when the MC-LR concentration is above 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, chlorophyll contents, MDA contents, and antioxidant enzyme activities decreased, generating antioxidant injury, subsequently the abilities to absorb nutrients weakened. Overall, these physiological responses are consistent with the results of the effects rule of MC-LR on the water purification.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study mainly investigated the effect of the MC-LR on the purification of the drinking water source by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e and discussed the effect of MC-LR on plant tissue growth and physiological response. The purifying effect of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e on contaminated water could be promoted at low concentration of MC-LR (\u0026lt;\u0026thinsp;1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1)\u003c/sup\u003e) and inhibited at high concentration (1\u0026ndash;10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The physiological characteristics of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e reveal some stress and injury induced by MC-LR level. In low concentration of MC-LR, the growth of roots enhanced, and subsequently improved the ability of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e to absorb nutrients. While high concentration MC-LR affects the plant tolerance and inhibited the root growth. As response to that \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e displays enzymatic defense to tolerate relatively low concentration of MC-LR exposure. The activities of POD, SOD, and CAT were enhanced under stimulation of low concentration MC-LR. The relative activities of these three enzymes remove the excess of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from the plant and protect themselves from damage. These results provide guidance for further revealing the role of MC-LR in aquatic ecosystems, when the cyanobacterial occur but not in a large scale in water bodies used as the drinking water source, low concentrations of MCs (\u0026lt;\u0026thinsp;1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) exert a positive effect on contamination removal by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: All authors have read and approved the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e: All data are available from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was financially supported by the Fundamental Research Funds for the Central Universities (FRF-TP-22-084A1), Guangdong Basic and Applied Basic Research Foundation (2022A1515111178), and Major Science and Technology Program for Water Pollution Control and Treatment of China (No. 2017ZX07101004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJunfei Liu:\u003c/strong\u003e Conceptualization, Writing-original draft, Investigation, Formal analysis, Validation, Resources, Writing, review\u0026amp;editing, Formal analysis，Funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYingbo Dong:\u003c/strong\u003e Supervision, Writing-review\u0026amp; editing, Funding 404 acquisition, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHai Lin:\u003c/strong\u003e Conceptualization, Supervision, Validation, Resources, Writing, review\u0026amp;editing, Formal analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlzandi, A. 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Biometals. 21(6), 665-674.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aquatic plant, Microcystin-LR, Drinking water source, Hydrocharis dubia (Bl.) backer, Phytoremediation","lastPublishedDoi":"10.21203/rs.3.rs-3066142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3066142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe safety of drinking water source directly affects human health. Ensuring the safety of drinking water is critical. Microcystin-LR (MC-LR), a toxic and common pollutant, releases by algae into drinking water source and can impede the in-situ remediation effect of aquatic plant on drinking water source purification. However, the effect mechanism of the MC-LR on the purification of the drinking water by aquatic plant remains unclear. This study aims to explore the effects mechanism of MC-LR (0\u0026ndash;10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on drinking water source purification by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e. After an exposure experiment, the removal rate of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, and COD promoted significantly at low concentrations of MC-LR (\u0026lt;\u0026thinsp;1\u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). And the removal rate of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, TP, and COD increased from 78.9\u0026ndash;90.7%, 70.0\u0026ndash;93.2%, and 46.0\u0026ndash;77.3%, respectively. With the increase of MC-LR concentration, the pollutant removal rate was obviously inhibited causing by concentration-dependent. Furthermore, the growth and development of the \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e roots were significantly promoted at the concentration of 0.2 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The length, tips, surface area, and average diameter of the root increased by 71.3%, 271.4%, 265.5%, and 113.0%, respectively. Chlorophyll contents under low-concentration MC-LR shows a 14.5%-15.7% promoting effect compared with the control group (CK). The activities of POD and CAT were also stimulated with the MC-LR increasing at low concentration. Notably, the MDA contents increased with increasing MC-LR concentration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Therefore, the presence of MC-LR could affect the purification efficiency of \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e in drinking water source. This study indicates the effect mechanism of MC-LR on drinking source water purification by \u003cem\u003eHydrocharis dubia (Bl.) backer\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Effects of microcystin-LR on purification of drinking water source and physiological response of Hydrocharis dubia (Bl.) backer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-27 14:16:48","doi":"10.21203/rs.3.rs-3066142/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"288f8a1f-f725-471c-9c55-a5ccb54e2fce","owner":[],"postedDate":"July 27th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-15T05:47:10+00:00","versionOfRecord":[],"versionCreatedAt":"2023-07-27 14:16:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3066142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3066142","identity":"rs-3066142","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}