Separate and Combined Effects of Chlortetracycline and Oxytetracycline on Growth and Antioxidant Enzymes in Myriophyllum Aquaticum

As two typical tetracyclines, chlortetracycline (CTC) and oxytetracycline (OTC) coexist widely in water. In the experiment, Myriophyllum aquaticum (Vell.) Verdc was exposed to the orthogonal hydroponic environment with the concentrations of CTC and OTC at 1, 10, and 50 mg/L for 7 days (7 D) and 14 days (14 D). The results showed that the plant height, relative growth rates (RGR), and photosynthetic pigment contents of M. aquaticum decreased signicantly after 14 D of CTC/ OTC, which was stronger than that after 7 D, and the toxicity of CTC was stronger than OTC. The combined toxicity of the two on the RGR and total chlorophyll content was mainly synergistic or additive after 7 D. After 14 D, the interaction changed to antagonism or addition, and the response of total chlorophyll content was more obvious than that of RGR. After 7 D of combined treatments, the malondialdehyde (MDA) content decreased signicantly, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities responded to different combined concentrations, respectively, and CAT was the main protective enzyme of M. aquaticum. After 14 D of combined treatments, the hydrogen peroxide (H 2 O 2 ) content increased signicantly, the MDA content increased rst and then decreased, and POD was the main protective enzyme among the three antioxidant enzymes. Overall, this study provided the rst insight into the single and combined toxicity of CTC and OTC on the growth and physiology of M. aquaticum at different time periods, revealing the reversal of the combined toxicity with time of exposure. antioxidant accumulation ROS, but the results that the H 2 O 2 remained at the decomposition of ROS by CAT and POD. The decrease of CAT smaller SOD and POD, and it remained at the control level CTC high concentration CTC and OTC combined results 7 CAT single CTC and combined stress CTC and OTC. After 7 D MDA H 2 did not mg/L OTC. We speculated that the decrease of POD activity the potential enzyme toxicity of After 14 D of combined treatments with CTC the activities of three antioxidant enzymes showed different trends. The POD activity increased rst then returned to the control level, while the SOD activity decreased and the CAT activity remained results After 14 D, ROS M. stress CTC


Introduction
As natural, semi-synthetic, or synthetic compounds, antibiotics have been widely used to prevent and treat human and animal diseases caused by microbial infections, as well as feed additives to promote the growth of livestock (Kovalakova et al. 2020;Siedlewicz et al. 2020). China has an annual production capacity of about 210,000 tons of antibiotics, 85 percent of which is used in animal husbandry and medicine (Daghrir and Drogui 2013). China, Brazil, India, the United States, and Indonesia are projected to be among the top ve countries in the world in the use of antibiotics in livestock by 2030 (Klein et al. environments such as surface water, groundwater and seawater. For example, the residual concentrations of CTC and OTC are 130.67 and 82.59 ug/L, respectively, in the wastewater of a pig farm in Tianjin, China (Zhi et al. 2018). The concentrations of OTC as high as 361.1 ug/L and 56.1 ug/L were detected in surface water in northern China and Colorado, USA, respectively (Jiang et al. 2014;Kovalakova et al. 2020). In shallow groundwater in northern and southwestern China, the maximum concentrations of CTC and OTC were 8 ng/L and 237 ng/L, respectively . In aquaculture ponds near the Yellow Sea in China, the OTC concentration was 7.29 ng/L and 13.12 ng/L in wet and dry seasons, respectively (Lu et al. 2019). Studies have found that in the tetracyclines manufacturing industry, the concentrations of OTC and CTC in the water inlet are both high, which are 334.3 mg/L and 1.8 mg/L, respectively. Even after sewage treatment, about 20 mg/L of OTC was released into the water body (Hou et al. 2016). More generally, concentrations of CTC and OTC in aquatic environments are recorded in the range ng/L to ug/L ).
Tetracyclines exist widely in water and have been proved to have toxic effects on untargeted organisms.
In general, tetracyclines are more toxic to microalgae than crustaceans and sh (Daghrir and Drogui 2013). The semi-inhibitory and semi-lethal concentrations of CTC to Microcystis aeruginosa were 0.05 mg/L and 15.2 mg/L, respectively (Miazek and Brozek-Pluska 2019). The median inhibitory concentrations of OTC to Ankistrodesmus fusiformis and Chlorella vulgaris were 4.17 and 7 mg/L, respectively (Ando et al. 2007;Carusso et al. 2018). Different kinds of tetracyclines are absorbed and transported differently in plants. The EC50 of CTC and OTC to Medicago sativa were 7.2 ug/L and 6730 ug/L, respectively (Scaria et al. 2021). It is worth noting that there is often more than one pollutant in the actual aquatic environment, and the toxic substances in the mixture cause more complex toxicity mechanisms to organisms (Jijie et al. 2021). There are not many studies on the combined toxicity of antibiotics, mainly focusing on the combination of antibiotics and heavy metals (such as Cu 2+ , Zn 2+ ), the combination between different kinds of antibiotics, and the combined toxicity of antibiotics and other substances (such as phthalate esters and phenolic) (Wei et al. 2018;Qiu et al. 2020;Jijie et al. 2021). In addition, the joint experiments tend to ignore the multi-period studies, and the tested organisms tend to choose algae or sh, while aquatic plants are rarely studied. The combined toxicity of the same antibiotics to aquatic plant over multiple periods of time is not known.
Myriophyllum aquaticum (Vell.) Verdc is a perennial aquatic or emergent plant, widely distributed in tropical and subtropical regions . It is a promising phytoremediation agent that can absorb nutrients, heavy metals and organic pollutants in water with fast growth and strong environmental adaptability (Liu et al. 2016;Guo et al. 2019). This experiment was conducted to investigate the growth and physiological responses of M. aquaticum to individual and combined levels of CTC and OTC at different concentrations (1, 10, 50 mg/L) after 7 days (7 D) and 14 days (14 D). The measurement of plant growth indexes included relative growth rate (RGR) and plant height. The physiological indexes included photosynthetic pigment contents (chlorophyll a, chlorophyll b, total chlorophyll content, and carotenoid), malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), soluble protein contents, and antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD). The objectives of this study were: (1) to investigate the single and combined toxicity of CTC and OTC to M. aquaticum,(2) to research the response of antioxidant enzymes of M. aquaticum, (3) to discuss the effect of exposure time on the combined toxicity of CTC and OTC.

Study design
All the plants used in this experiment were collected from East Lake (30° 32' N; 114° 25' E), Wuhan, China. Each plant was washed with running tap water and rinsed with double steaming water to remove attachments, and then placed in 10% Hoagland nutrient solution for 7 D of acclimation. After acclimatization, the plant attachments, yellow leaves, and adventitious roots were removed again. Plants of the same size and growth state were selected as experimental material after 15 cm of the front end of M. aquaticum. CTC and OTC were obtained from chlortetracycline hydrochloride (USP grade, CAS NO. 64-72-2) and oxytetracycline hydrochloride (USP grade, 95% purity, CAS NO. 2058-46-0), all purchased from Wuhan Xinshentest Chemical Technology Co., Ltd., China. CTC and OTC concentrations were set at 1, 10, and 50 mg/L, respectively. A total of 16 treatment groups were included, including 9 different combinations of CTC and OTC, 6 CTC and OTC alone, and 1 control group with only 10% Hoagland nutrient solution (Table 1). In the experiment, each treatment group was set with 3 replicates, and each replicate contained 4 plants of the same size. The labeled plants were transplanted to 5 L 10% Hoagland nutrient solution with CTC and OTC at treatment concentrations. Change the solution every 48 hours (48 h). The temperature of the experimental greenhouse was controlled at 25 ± 0.5 ℃, the light/dark cycle was 16-h/8-h, the relative air humidity was 60%, and the light intensity was 10,000 lx. After 7 D and 14 D, the plants were harvested respectively. After rinsing with double steam water to remove impurities, the surface moisture of the plants was dried, and the indexes were determined. The plant growth was evaluated by the relative growth rate (RGR) and plant height. The plant height was measured directly with a ruler, and the fresh weight of the plant was measured with an electronic analytical balance (accuracy 0.1 g). RGR was calculated according to Liu et al. (Liu et al. 2020). RGR = [ ln ( nal weight) -ln (initial weight) ]/ △t (g g −1 day −1 ), where weight is the plant fresh weight over △t days.

Photosynthetic pigment measurements
Photosynthetic pigments include chlorophyll a, chlorophyll b, carotenoid, and total chlorophyll, which are determined according to the method of Jampeetong and Brix (2009

Lipid peroxidation measurements
The degree of lipid peroxidation in plant leaves can be judged by the content of MDA (Cang and Zhao 2013). The determination method of MDA content was as follow: 0.05 g fresh plant leaves were weighted and evenly cut into pieces, and 3 mL of 10% trichloroacetic acid (TCA) was added for grinding. The homogenate after grinding was placed in a centrifuge at 4 ℃ and centrifuged at 12,000 g for 25 min. Take 2 mL of centrifuged supernatant, add 2 mL 0.6% 2-thiobarbituric acid (TBA), mix evenly and heat in water bath at 100 ℃ for 30 min, remove and cool quickly before centrifugation. The absorbance of the supernatant was measured at 450, 532, and 600 nm.

H 2 O 2 and soluble protein contents measurements
Refer to Shi (2016) for the determination method of H 2 O 2 content. Weight and grind 0.1 g fresh leaves with phosphate buffer solution (50 mM, pH 7.8, containing 1% (m/v) polyvinylpyrrolidone (PVP)) under ice bath, and centrifuge the grinding solution at 4 ℃ for 12,000 g for 25 min (Eppendorf Centrifuge 5417 R, Hamburg, Germany). After centrifugation, 1.5 mL sample extract was added to 1.5 mL 5% (W/V) titanium sulfate, placed for 10 min, and centrifuged again at 4 ℃ for 12,000 g for 15 min. The absorbance of supernatant was measured at 410 nm.
The soluble protein content of plants was determined by Coomassie bright blue G-250 staining (Bradford 1976).

Enzyme activities measurements
Weight 0.1 g fresh plant leaves, add phosphate buffer solution (50 mM, pH 7.8) and grind them in an ice bath, centrifuge them at 12,000 g for 25 min in a frozen centrifuge. The supernatant after centrifugation was used for determination of antioxidant enzymes.
The activity of SOD was determined by NBT photochemical reduction (Cang and Zhao 2013). The reaction mixture consisted of 1.5 mL phosphate buffer solution (50 mM, pH 7.8), 0.3 mL methionine solution (130 mM), 0.3 mL EDTA-Na 2 solution (3 uM), 0.3 mL ribo avin (20 uM), 0.1 mL crude enzyme solution and 0.5 mL distilled water. The SOD activity unit (U/g FW) was de ned as an enzyme activity unit that inhibited 50% of NBT photochemical reduction. The activity of CAT was measured at 240 nm by uv absorption method (Cang and Zhao 2013). The reaction mixture consisted of 1.5 mL PBS (0.2 M pH 7.8), 0.3 mL H 2 O 2 solution (0.1 M), 0.2 mL crude enzyme solution, and 1 mL distilled water. The activity of POD was determined by guaiacol method at 470 nm (Liu and Li 2007). The reaction mixture consisted of 2.9 mL PBS (0.1 M, pH 6.0), 1.0 mL guaiacol (0.05 mM), 1.0 mLH 2 O 2 (2%) and 0.1 mL crude enzyme solution.

Joint toxicity assessment method
The Abott model was used to evaluate the combined toxicity of CTC and OTC to M. aquaticum after 7 D and 14 D. In this model, the observed inhibition of CTC and OTC combined pollution is represented by C obs (%), and the predicted inhibition of combined pollution is represented by C exp (%). The calculation method of C exp (%) is as follow: Where, A and B represent the inhibition levels of CTC or OTC alone on M. aquaticum.
The Abott model evaluated the effect of combined toxicity by comparing RI with 1. The calculation method of RI is as follow: RI = C obs / C exp RI > 1 indicates that the interaction between CTC and OTC is synergistic, RI = 1 indicates simple addition, and RI < 1 indicates antagonism between CTC and OTC.

Date analysis
Statistical analysis was performed using SPSS 23.0 for Windows (IBM Inc., Chicago, IL, USA). Homogeneity of variance was analyzed by Levene's test. When necessary, the date was transformed and normalized to reduce variance heterogeneity., and then signi cance analysis was performed for control and treatment groups using one-way ANOVA. P-values ≤ 0.05 were considered signi cant. The experimental data were expressed as mean ± standard deviation. graphs using SigmaPlot 12.5 for Windows (Systat Software, Inc., USA).

Results
Page 7/27 Under the combined treatment of low concentration (1+1 mg/L), the plant height was signi cantly lower than that of the control, which was 89.51% (p < 0.05). Under the combined treatment of high concentration (50+50 mg/L), the plant height reached the minimum value, which was 76.27% of the control (Fig. 1b).
After 7 D, the RGR of M. aquaticum signi cantly increased at 1 mg/L CTC, which was 27.27% higher than that of the control (p < 0.05), and then returned to the control level. However, there was no signi cant difference in the RGR of M. aquaticum compared with the control under OTC treatments (p > 0.05). After 7 D of CTC and OTC combined treatments, OTC supplementation reduced the RGR of M. aquaticum under all CTC concentrations, and the lowest RGR appeared in the (50+10 mg/L) treatment of CTC and OTC, which decreased by 68% compared with the control. At the highest concentration of CTC and OTC combined treatment (50+50 mg/L), the RGR slightly increased, but still signi cantly lower than the control group ( Fig. 2a). After 14 D, the RGR of M. aquaticum showed obvious concentration dependence, that is, the RGR decreased with the increase of treatment concentrations. The minimum values were reached at 50 mg/L CTC, 50 mg/L OTC, and (50+50) mg/L combined treatments of CTC and OTC, which decreased by 36.44%, 27.77%, and 63.44% compared with the control, respectively (Fig. 2b).

Photosynthetic pigments
After 7 D, the contents of chlorophyll a and chlorophyll b at 10 mg/L CTC were signi cantly lower than those in control group (p < 0.05). The contents of total chlorophyll and carotenoid had no signi cant changes under CTC treatments (p > 0.05). There was no signi cant difference in the photosynthetic pigment contents after 7 D of exposure to OTC (p > 0.05). Under (1+10) mg/L combined treatment of CTC and OTC, the contents of chlorophyll a, chlorophyll b, and total chlorophyll reached the lowest point, which decreased by 29.41%, 28.57%, and 29.21%, respectively, compare with the control (p < 0.05). All treatment groups had no signi cant effect on the carotenoid content of M. aquaticum (p > 0.05) ( Table  2). After 14 D, chlorophyll a, chlorophyll b, and total chlorophyll contents of M. aquaticum showed the same trend, which showed obvious concentration dependence, that is, decreased with the increase of CTC and OTC concentrations. The three indexes of M. aquaticum signi cantly decreased to the minimum value at 50 mg/L CTC, which were 33.78%, 37.5%, and 35.35% lower than those of the control. respectively (p < 0.05). They reached the minimum value at 10 mg/L OTC, which were 22.97%, 25%, and 25.25% lower than the control group, respectively (p < 0.05). Under (10+50) mg/L of CTC and OTC combined treatment, they reached the minimum value, which were 58.11%, 57.11%, and 54.55% of the control group, respectively (p < 0.05). The carotenoid content of M. aquaticum increased signi cantly only under (50+50) mg/L combined treatment (p < 0.05) ( Table 3).  (Fig. 3a).
After 14 D, there was no signi cant difference in the MDA content when M. aquaticum plants were exposed to CTC/ OTC (p > 0.05). The MDA content of M. aquaticum increased rst and then decreased with the increase of CTC and OTC combined concentrations. Under the treatment of (1+50) mg/L of CTC and OTC, the MDA content signi cantly increased to the maximum, which was 1.89 times of that of the control (p < 0.05). At (50+50) mg/L, the MDA content decreased to the lowest value, which decreased by 76.88% compared with the control (p < 0.05) (Fig. 3b).

H 2 O 2 and soluble protein contents
After 7 D, all treatment groups had no signi cant effects on the H 2 O 2 content of M. aquaticum (p > 0.05) (Fig. 4a). After 14 D, both CTC and OTC treatments showed no signi cant difference in the H 2 O 2 content, but it was signi cantly higher than that of control group at medium and high combined concentrations ((10+10) mg/L, (10+50) mg/L, (50+1) mg/L, (50+10) mg/L, and (50+50) mg/L). The content of H 2 O 2 in (50+50) mg/L of CTC and OTC mixture was 39.59 umol/g FW, which was 1.32 times of that in the control group (Fig. 4b).
After 7 D and 14 D, the soluble protein content of M. aquaticum was no signi cantly affected by all concentration treatment groups (p > 0.05) ( Fig. 5a and b).

Enzyme activities
After 7 D, the activities of SOD, CAT, and POD increased rst and then decreased at single CTC exposure. The SOD activity reached the maximum value at 1 mg/L CTC, while the CAT and POD activities reached the maximum value at 10 mg/L CTC, which were 1.18, 1.44, and 1.27 times of that of the control, respectively. The activities of SOD and CAT did not change signi cantly under single OTC treatments (p > 0.05). The POD activity decreased signi cantly by 27% compared with the control at 50 mg/L OTC (p < 0.05). The activities of the three enzymes also increased rst and then decreased signi cantly under the combined treatments of CTC and OTC. The activities of three enzymes reached the maximum values under the combined treatments (1+1) mg/L, (10+50) mg/L, and (50+1) mg/L of CTC and OTC, respectively, which were 1.13 times, 1.69 times and 1.58 times of that of the control. Under the combined treatment of (50+50) mg/L CTC and OTC, the activities of SOD and POD were only 33.08% and 65.11% of the control group, while the CAT activity returned to the control group level (Fig. 6a, c and e).
After 14 D, the activities of CAT and POD did not change signi cantly, and the SOD activity reached the lowest value at 50 mg/L CTC/ OTC, which decreased by 47.94% and 44.43% compared with the control, respectively. Under combined treatments, OTC did not signi cantly change the SOD activity of M. aquaticum at low concentration of CTC (1 mg/L) (p > 0.05). With the increase of CTC concentrations, OTC signi cantly reduced the SOD activity, which was lower than that of CTC alone. At the high concentration of CTC and OTC (50+50) mg/L, the SOD activity reached the lowest value (only 15.68% of the control). There was no signi cant change in the CAT activity of M. aquaticum (p > 0.05). After 14 D of CTC and OTC combined treatments, the POD activity changed similar to that of 7 D. With the increase of the combined treatments, the POD activity rst increased and then decreased. It reached the maximum value at the combined treatment of (1+50) mg/L, increased by 89.05% compared with the control group (p < 0.05), and recovered to the level of the control group at (50+50) mg/L combined concentration of CTC and OTC (Fig. 6b, d and f).

Combined toxicity assessment
In this study, the RGR and total chlorophyll content were used as indicators to evaluate the combined toxicity of CTC and OTC to M. aquaticum after 7 D and 14 D. The results showed that the interaction between CTC and OTC was different at treatment time and the combined concentrations of CTC and OTC.
For the RGR, RI value was close to or signi cantly greater than 1 at each combined concentration of CTC and OTC after 7 D, showing additive or synergistic effect. After 14 D, RI value of OTC mixed with high concentration of CTC (50 mg/L) was signi cantly less than 1, indicating that CTC and OTC had antagonistic effect on the leaves of M. aquaticum (Table 4). For total chlorophyll content, after 7 D, RI value was signi cantly lower than 1 only when the combined concentration of CTC and OTC was (10+50) mg/L and (50+1) mg/L, indicating that CTC and OTC showed antagonistic effect. In other combined treatments of CTC and OTC, RI value was close to or signi cantly great than 1, and CTC and OTC showed additive or synergistic effect. After 14 D, RI values were close to or less than 1, and all showed antagonism except (10+50) mg/L combined treatment (Table   5).

Discussion
The aerenchyma structure of M. aquaticum with stem and root connecting is of great ability to absorb nutrients in water. No matter under single CTC/ OTC or combined treatments, the plant height and RGR decreased signi cantly after 14 D. The toxic effects of tetracyclines on plant growth have been demonstrated (Mathews and Reinhold 2013;Liu et al. 2020;Han et al. 2021;Li et al. 2021). In the experiment, the RGR of M. aquaticumw signi cantly increased with low concentration of CTC (1 mg/L) after 7 D. This phenomenon was often called hormesis, where low concentration of CTC may be used as a plant-speci c nutrient source (Gomes et al. 2014;Liu et al. 2019). Low concentration of exogenous stress can stimulate the metabolic process of plants, which is also a self-protection mechanism of plants. Hormesis is usually the result of compensatory biological process after direct toxicity induction or initial interruption of homeostasis . It is worth noting that after 14 D of CTC, the toxic excitation effect disappeared. This may be due to the fact that after 14 D of continuous exposure, 1 mg/L CTC has been higher than the concentration level that can promote the growth of M. aquaticum. After 7 D, CTC and OTC had almost no inhibition on the plant height and fresh weight of M. aquaticum. However, after 14 D, they not only signi cantly inhibited these two indexes, but also the inhibition degree under CTC treatments was higher than that of OTC. These results also indicated that CTC and OTC had a cumulative toxic effect on M. aquaticum, and CTC was more toxic to M. aquaticum than that of OTC over time. The difference in toxicity between CTC and OTC to non-targeted organisms may be species-speci c. Under CTC and OTC combined treatments after 7 D, we found that the effect of (50+50) mg/L on the RGR was signi cantly greater than that of 14 D. According to Abott model, the combined treatments of CTC and OTC at different concentrations had an additive or synergistic effect on the RGR of M. aquaticum, indicating that CTC and OTC may increase the toxic effects of both parties during short exposure time.
Because both of them belong to tetracyclines, the same compound often has similar toxic sites and modes of action (Carusso et al. 2018). The potential additive and synergistic effects may be related to toxicity dynamics of antibiotics. During short-term exposure, the presence of one antibiotic isolates the metabolic enzymes that normally metabolize another antibiotic in the plant, allowing more of the antibiotic to reach its target site, and resulting in greater toxicity (Broderius et al. 2005;Yang et al. 2008). However, after 14 D, the addition of OTC made the mixture exhibit an antagonistic effect on the RGR of M. aquaticum under the treatment of 50 mg/L CTC, indicating that the presence of CTC and OTC will reduce the damage of both sides to the growth of M. aquaticum under long-term treatments. The possible reason is the competition of similar binding sites or the antagonism caused by mutual inhibition of toxicity in plant cells (Guo et al. 2015;Wang et al. 2018).
In plant cells, chloroplasts convert light energy into biological energy through high metabolic activity and electron transfer rate. Chlorophyll and carotenoid are pigments for photosynthesis in green plants and have been widely used as indicators of pollutant stress (Ma et al. 2016;Guo et al. 2020). After 14 D, the chlorophyll (chlorophyll a, chlorophyll b, and total chlorophyll contents) of M. aquaticum signi cantly decreased by either single or combined treatments. Likewise, the inhibitory effect of CTC on photosynthetic pigments was also stronger than that of OTC. The effects of tetracyclines on the contents of chlorophyll in plants have been reported in some literatures (Brain et al. 2005;Li et al. 2011;Rydzyński et al. 2017). During photosynthesis, plants need to absorb water for photodecomposition of water in chloroplasts. Therefore, CTC and OTC were likely to be absorbed into the plant along with water. The prokaryotic nature and bacterial structure of plant chloroplasts may make them potential target for tetracyclines ). According to the Abott model, the interaction between CTC and OTC on the total chlorophyll content of M. aquaticum was additive or synergistic after 7 D, and antagonistic effect was only observed at (10+50) mg/L of combined treatment. After 14 D, all the combined treatments showed antagonistic effect except for (10+50) mg/L of CTC and OTC combined treatment. In addition, the interaction between CTC and OTC exposure on the total chlorophyll of M. aquaticum was more obvious than that on the RGR, but the change trend was generally consistent, that is, with the increase of exposure time, the interaction changed from additive or synergistic to antagonistic. The cumulative toxicity effects of CTC and OTC lead to the gradual occupation of binding sites in plants, and the two antibiotic molecules cannot effectively bind to the target to produce stronger toxicity ). There was no signi cant change in the carotenoid content, except for a signi cant increase at (50+50) mg/L after 14 D. The contents of carotenoid generally decreased in previous studies, which was inconsistent with our results (Di Marco et al. 2014;Li et al. 2021). It should be noted that Liu et al. (2020) found that there was no signi cant difference in the carotenoid content between Hydrocharis dubia (Bl.) Backer and Trapa bispinosa Roxb, suggesting that the effects of tetracyclines on carotenoids in plants may be species-speci c. In addition, studies have shown that carotenoids are effective quenchers of triple photosensitizer, singlet oxygen and peroxy free radicals (Chen et al. 2012). ROS can regulate the carotenoid concentration in plants by activating potential biosynthetic enzymes or genes encoding related enzymes (Zhong et al. 2018a). It is possible that the carotenoid can eliminate ROS produced by stress and maintain plant homeostasis through compensatory synthesis of carotenoids, which explained why the carotenoid content of M. aquaticum increased signi cantly at (50+50) mg/L.
There is a dynamic balance between the production and removal of ROS in plants under normal circumstances. H 2 O 2 is a relatively stable ROS, which can usually measure the degree of plant stress (Wu et al. 2010 (Gunes et al. 2009). The decrease in photosynthetic pigments in the experiment was consistent with this explanation. The soluble protein content re ects the vitality of plant physiological activities and plays an important role in maintaining the stability of organisms. In the experiment, there was no signi cant difference in the content of soluble protein of M. aquaticum. This was different from the results of Li et al. (2021) and Zhou et al. (2020), but consistent with the results of Liu et al. (2020).
The reason for no change in the soluble protein of M. aquaticum may be that: (1) CTC and OTC had no effect on the protein content of M. aquaticum.
(2) The increase of ROS led to a compensatory increase in the protein content. Tukaj and Tukaj (2010) believed that the increase of ROS level might trigger signal transduction pathway, thus inducing a series of protein synthesis related to antioxidant response and protein repair in plants to maintain the stability of plants.
Once the accumulation of ROS in plants is excessive, membrane lipid peroxidation will occur. MDA is the main product of membrane lipid peroxidation and usually used to measure the damage degree of plant cell membrane (Zhong et al. 2018b). After 7 D, the MDA content of M. aquaticum signi cantly decreased in 50 mg/L CTC alone or combination, which was obviously unexpected. Most plants exposed to tetracyclines seem to be accompanied by oxidative damage, that is, a phase of increased MDA levels (Guo et al. 2020;Zhou et al. 2020). However, Li et al. (2021) found that the MDA content of V. natans also did not increase after 7 D exposure to CTC. The decrease of membrane lipid peroxidation may be an adaptive response to stress (Nunes et al. 2015 (Zhong et al. 2018c). After 7 D of CTC alone or combined treatments with OTC, the SOD, CAT, and POD activities of M. aquaticum showed a trend of increasing rst and then decreasing at different concentrations. The increase of antioxidant enzymes indicated that ROS can be eliminated by activating the antioxidant enzyme system. The decrease of SOD activity may be due to the decrease of enzyme synthesis or the change of its subunit assembly leading to enzyme inactivation. The inactivation of SOD would aggravate the accumulation of ROS, but the results showed that the H 2 O 2 content remained at the normal level, which might be due to the decomposition of ROS by CAT and POD. The decrease of CAT activity was much smaller than that of SOD and POD, and it remained at the control level under high concentration of CTC and high concentration of CTC and OTC combined treatments. The results indicated that after 7 D, the antioxidant system of M. aquaticum operated well, and CAT may be the main protective enzyme of M. aquaticum under single CTC and combined stress of CTC and OTC. After 7 D of OTC, the activities of SOD and CAT as well as the contents of MDA and H 2 O 2 did not change, the POD activity signi cantly decreased at 50 mg/L OTC. We speculated that the decrease of POD activity might be related to the potential enzyme toxicity of antibiotics. After 14 D of combined treatments with CTC and OTC, the activities of three antioxidant enzymes showed different trends. The POD activity increased rst and then returned to the control level, while the SOD activity decreased and the CAT activity remained unchanged. Our results were similar to those of Shang et al. (2015). After 14 D, the excessive ROS produced by M. aquaticum under combined stress of CTC and OTC may not be eliminated by CAT, but by POD, suggesting that POD may be the main protective enzyme under 14 D of combined stress. The decrease of SOD activity was the oxidative damage induced by high combined concentration, and SOD, as the rst line of defense of the antioxidant system, was destroyed. The inconsistency of enzyme activity indicated that the three enzymes were functionally independent. Peter et al. (1994) believed that H 2 O 2 produced by SOD was not the only source of H 2 O 2 consumed by CAT and POD. H 2 O 2 can also be produced from activation of amino acid or cytochrome P450 oxidase. This may also be one of the reasons why the H 2 O 2 content increased at high combined concentration of CTC and OTC. The sensitivity and action of antioxidant enzymes in M. aquaticum may vary according to the type of antibiotics and exposure time.

Conclusion
Based on the results of the present study, it can be concluded that: 1) After 14 D of CTC/ OTC, the inhibition degree on the plant height, RGR, and photosynthetic pigments of M. aquaticum was signi cantly strong than that after 7 D, showing obvious cumulative toxicity effect, and the toxicity of CTC was signi cantly stronger than that of OTC.
2) The combined effect of CTC and OTC were different at different concentration ratio and exposure time on M. aquaticum. After 7 D, the interaction between CTC and OTC on the RGR and total chlorophyll was mainly synergistic or additive. After 14 D, the interaction changed to antagonism or addition. The content of total chlorophyll was more sensitive to mixed stress than the RGR of M. aquaticum.
3) The combined presence of CTC and OTC can induce a certain degree of oxidative stress in leaf tissue, and stimulate the response of antioxidant system of M. aquaticum. The antioxidant defense system of M. aquaticum is not static, but varies with exposure time and stress intensity. After 7 D of combined treatments, CAT was the main protective enzyme of M. aquaticum, while POD was the main protective enzyme after 14 D.

Declarations Ethical Approval
This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to participate
No applicable.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of date and materials
All data generated or analysed during this study are included in this published article.  All values are the mean of three replicates ± standard deviation. Bars with different letters are signi cantly different between treatments (p ≤ 0.05, Duncan test).