Toxicity of chlortetracycline and oxytetracycline on Vallisneria natans (Lour.) Hare

Tetracyclines are frequently detected in water bodies due to their widespread use in aquaculture and animal husbandry. A hydroponic experiment was conducted to explore the phytotoxic effects of Vallisneria natans (Lour.) Hare exposed to various concentrations of chlortetracycline (CTC) and oxytetracycline (OTC) (0, 0.1, 1, 10, 30, 50, and 100 mg/L) for 7 days (7 D) and 14 days (14 D), respectively. The results showed that similar to OTC treatment for 7 D, the relative growth rates (RGR) and catalase (CAT) activity of V. natans, after 7 D of CTC exposure, decreased significantly at 10 mg/L and 30 mg/L, respectively. The content of soluble protein notably decreased when CTC ≥ 10 mg/L and OTC ≥ 30 mg/L. The hydrogen peroxide (H2O2) content was significantly stimulated when OTC ≥ 10 mg/L, while it hardly changed when exposed to CTC. After 14 D, the malondialdehyde (MDA) and H2O2 contents of V. natans were significantly higher than those of the control group under a high concentration of OTC (≥ 30 mg/L), but they did not change significantly under a high concentration of CTC. The activity of polyphenol oxidase (PPO), under CTC treatment after 14 D, showed first a significant increase then decreases; the maximum value (125% of the control) was noticed at 10 mg/L CTC, while it remained unchanged when exposed to OTC. The soluble protein content significantly decreased at 10 mg/L CTC and 0.1 mg/L OTC, respectively. The RGR, CAT, and peroxidase (POD) activities, similar to OTC treatment after 14 D, decreased evidently when CTC was 10 mg/L, 30 mg/L, and 0.1 mg/L, respectively. CTC and OTC harm the chlorophyll content of V. natans after 14 D, and the reductions of chlorophyll a and carotenoid were more pronounced than chlorophyll b. The results suggest that CTC and OTC both have a negative effect on the growth of V. natans, and OTC can cause oxidative damage in V. natans but CTC harms the metabolism process without inducing oxidative damage. Overall, the toxicity of OTC to V. natans is stronger than that of CTC.


Introduction
Antibiotics are mainly used to treat microbial infections in humans and animals and as feed additives to promote the growth of livestock (Anchordoquy et al. 2019). According to the survey, the annual consumption of antibiotics worldwide is estimated to be between 100,000 and 200,000 tons, and the global use of antibiotics is expected to be 200% higher by 2030 than in 2015 (Dutta and Mala 2020). China is the world's largest producer and user of antibiotics, with 75-85% used in animal husbandry (Cheng et al. 2014). In China, the use of antibiotics for livestock and medicines was about 162,000 tons in 2013, compared with 150 times the consumption of antibiotics in the UK and 10 times that of the USA . Tetracyclines, including tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and so on, are broad-spectrum bacteriostatic compounds and widely used due to their favorable properties such as low cost and high antimicrobial activity (Maged et al. 2020), and also as veterinary drugs to prevent and treat a variety of animal infectious diseases or as feed additives to promote animal growth (Dai et al. 2020). According to the survey, tetracyclines are the second most commonly used antibiotics in the world , and CTC and OTC are two of the ten growth promoters approved in the USA (Jeong et al. 2010). In 2012, about 38,500 tons of antibiotics was used in veterinary medicine in China, of which more than 60% was used for tetracyclines, sulfonamides, and penicillin drugs (Lin et al. 2015). In Japan, tetracyclines for animal use account for about 43% of the total antibiotics (Fukushima et al. 2019).
Tetracyclines are frequently detected in water, with the highest reported residual concentrations at 2796.6 ng/L in the water of a high-intensity aquaculture lake . The detection ranges of TC and OTC in surface water of the Yellow River Delta were 3.65-64.89 ng/L and 4.60-83.54 ng/ L, respectively (Zhao et al. 2016). Chen et al. (2018) found that the average detected concentrations of OTC, CTC, and TC in the surface water in China were 8.21, 7.71, and 3.57 ng/ mL, respectively. In fact, the concentration of tetracyclines may increase further due to the accumulation of time and space (Carusso et al. 2018). In addition, Wang et al. (2017) found that the concentration of tetracyclines detected in water environment was affected by season. One reason for this is that the use of tetracyclines for preventing most respiratory infections increases during autumn and even winter in China, when animals are most susceptible to these diseases (Matsui et al. 2008). Another reason may be the longer half-life of tetracyclines in winter. The half-life of tetracyclines in low-temperature seasons (e.g., 51-59 D at 13°i n November) was longer than that in high-temperature seasons (e.g., 24°21-40 D in May) . What is more, tetracyclines can be introduced into the water environment through different ways. Firstly, due to incomplete metabolism of organisms, most antibiotics are discharged from the body in the form of mother or its metabolites, which are directly released or indirectly discharged into the water environment through rainwater runoff (Christou et al. 2017). Secondly, the incomplete removal of sewage treatment plants. Studies have shown that concentrations of tetracyclines in downstream river are higher than upstream and midstream, which may be due to the discharge of untreated livestock wastewater or incomplete treatment of sewage treatment plants to the downstream sampling points of the connected rivers (Krzeminski et al. 2019). Finally, antibiotics are added directly to the aquaculture water as feed additives in aquaculture (Pham et al. 2015). It is estimated that 80% of antibiotics used in aquaculture are released directly in the aquatic environment (Cabello et al. 2013). The high detection rate of tetracyclines (62.5~75%) in Poyang Lake, China, is also closely related to the use of antibiotics in fish and poultry farming (Ding et al. 2017). And the results of tetracyclines in Taihu Lake showed that their contents (44.0-68.6 ng/L) were related to aquaculture of aquatic plants (Nkoom et al. 2018).
A large number of studies have shown unintended biological activity of residual tetracyclines in the water on non-targeted organisms. Moro et al. (2020) found that 10 mg/L of OTC altered the chloroplast shape of Isochrysis galbana, reduced pigment contents, and inhibited its growth. Half of the growth inhibitions of Pseudokichneriella subcapitata and Selenastrum capricornutum at different concentrations of CTC were 1.2-3.1 mg/L (Carusso et al. 2018). The toxic mechanisms of tetracyclines to aquatic animals (including Elliptio complanata, Danio rerio, Oncorhynchus mykiss, etc.) are mainly to damage the genetic material and nerve of organisms (Zargar et al. 2020). In addition, Ye et al. (2020) found that after 10 days of exposure, TC, CTC, and OTC showed different toxicity to Microcystis aeruginosa. When M. aeruginosa was exposed to TC, it showed excitation effect; inhibition effect was observed when exposed to OTC, but no significant effect was observed when exposed to CTC. The ecological toxicity of tetracyclines is mainly focused on algae, animals, and terrestrial plants, while very limited information is available about the toxicity of CTC and OTC on aquatic plants.
Vallisneria natans (Lour.) Hare, one of the most common submerged plants, plays an important role in maintaining ecosystem stability and water health . In addition, V. natans can absorb a variety of organic pollutants (such as pesticides, pharmaceuticals, and personal care products), and has become one of the important aquatic plants for the evaluation of aquatic pollution and ecological remediation (Olette et al. 2008;Wang et al. 2011). In this study, the growth and the physiological responses of V. natans in different CTC/ OTC concentrations were investigated. The resulting impacts include biomass, chloroplast pigment, soluble protein content, membrane lipid peroxidation, hydrogen peroxide (H 2 O 2 ), and antioxidant enzymes activity, including catalase (CAT), guaiacol peroxidase (POD), and polyphenol oxidase (PPO). The present work attempted to understand the toxicity of CTC and OTC on V. natans and to provide theoretical guidance for safety assessment of them.

Plant materials and experimental design
Healthy plants of V. natans were collected from East Lake (30°32' N; 114°25' E), Wuhan. Before the experiment, whole plants were thoroughly washed with running water to remove attachments and then cultivated for acclimatization in 5 L 10% Hoagland's solution (Hoagland and Arnon 1950). After 14 days (14 D), each plant was thoroughly washed with running tap water again, and then rinsed in redistilled water. The plants, with similar size and growth status, were chosen for experimental use. During acclimatization and experiment, the roots of the plants were fixed with sterilized pure white gravel and the plants were grown in nutrient solution in transparent plastic tanks at temperature 25±0.5°C, 16-h/8-h light/dark cycle, relative air humidity 60%, and light intensity of 10,000 lx. The antibiotics used in the experiment were obtained from chlortetracycline hydrochloride (USP grade, CAS NO.64-72-2) and oxytetracycline hydrochloride (USP grade, purity 95%, CAS NO.2058-46-0), which were purchased from Wuhan Xinshen Chemical Technology Co., Ltd., China.
To encompass both environmental realism and concentrations that would elicit measurable toxic responses (Brain et al. 2005), the concentrations of CTC and OTC were set at 0.1, 1, 10, 30, 50, and 100 mg/L, respectively. A control treatment was set up using culture medium and plants without CTC/ OTC. Each treatment concentration was replicated three times, and the solutions were replaced every 48 h. The plants were collected after 7 days (7 D) and 14 D of exposure. They were first rinsed with double-distilled water to remove the attachments on the surface of them and then packed individually for the estimation of various indexes.

Measurement of plant growth
The plant growth was presented as relative growth rates (RGR). After 7 D and 14 D of CTC/OTC exposure, plants were harvested and washed with double-distilled water. After being dried on filter paper, the fresh weight of each plant was weighted by an electronic analytical balance (accuracy 0.1 mg). RGR was calculated according to Liu et al. (2019).

Photosynthetic pigment measurements
The photosynthetic pigments were measured according to the method of Jampeetong and Brix (2009). Fresh weight (FW) plant leaves (0.05 g) were cut into pieces evenly. Then, 5 mL 95% (v/v) alcohol was added and flasks were placed in the dark for 48 h until the leaves turn white. The absorbance values of the extract at 470, 649, and 665 nm were determined by ultraviolet spectrophotometer (MAPADA UV-1200, Shanghai Meipuda Instrument Co. Ltd. China) with 95% ethanol as control. The contents of chlorophyll a, chlorophyll b, and carotenoids (mg/g FW) were calculated by the method of Lichtenthaler (1987).

Lipid peroxidation measurements and soluble protein content
The level of lipid peroxidation in the plant leaves was determined by the quantification of malondialdehyde (MDA) (Cang and Zhao 2013). Fresh leaves (0.05 g) were homogenized with 3 mL 10% trichloroacetic acid (TCA) at 12,000g for 25 min. The supernatant (2 mL) was mixed with 2 mL 0.6% 2-thiobarbituric acid (TBA), and the mixture was heated at 100°C for 15 min and then cooled quickly and centrifuged. The absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm.
Soluble protein content was determined following the method of Bradford (1976).

Enzyme activity measurements and H 2 O 2 content
Fresh leaves (0.1 g) were homogenized with a phosphate buffer solution (50 mM, pH 7.8), containing NaH 2 PO 4 , Na 2 HPO 4 , and 1% (m/v) polyvinylpyrrolidone (PVPP) at 4°C. The mixture was centrifuged in a centrifuge (Eppendorf Centrifuge 5417 R, Hamburg, Germany) at 4°C at 12,000g for 25 min. The supernatant was extracted and stored at 4°C and used for the measurement of antioxidant enzyme activities, including CAT, POD, and PPO activities.
The CAT activity was assayed according to Cang and Zhao (2013), where one enzyme activity unit (U/g·min FW) was defined as a decrease of 0.1 in absorbance at 240 nm in 1 min. The reaction mixture consisted of 0.2 M pH 7.8 PBS (1.5 mL), 0.1M H 2 O 2 solution (0.3 mL), crude enzyme solution (0.2 mL), and distilled water (1 mL). The activity of POD was measured according to Liu and Li (2007), with absorbance change of 0.01 per minute at 470 nm representing one unit of enzyme activity (U/g·min FW). The reaction mixture consisted of 0.1 M pH 6.0 PBS (2.9 mL), 0.05 mM guaiacol (1.0 mL), 2 % H 2 O 2 (1.0 mL), and crude enzyme solution (0.1 mL). The activity of PPO was estimated following the guaiacol method described by Shi (2016), and one unit of enzyme activity (U/g·min FW) corresponded to an absorbance change of 0.01/min at 398 nm. The content of H 2 O 2 (ug/g FW) was examined following the method described by Shi (2016). The reaction mixture contained the enzyme extraction and 5 % titanium sulfate in 20% sulfuric acid.

Statistical analysis
The experiment utilized a randomized block design. All values were expressed as the mean ± standard deviation. Homogeneity of the variance was analyzed by performing Levene's test. When necessary, the date was transformed and normalized to reduce the heterogeneity of variance. ANOVAs were performed to assess the variability of data and validity of results. Post hoc Duncan tests were done to separate differences between pairs of treatments. p-values ≤ 0.05 were considered significant. The statistical analysis was performed by SPSS 23.0 for Windows (IBM Inc., Chicago, IL, USA), and graphs were generated in SigmaPlot 12.5 for Windows (Systat Software, Inc., USA).

Plant growth
As shown in Table 1, no significant effect on the RGR of V. natans was observed at up to 10 mg/L CTC/ OTC after 7 D, beyond which it declined evidently (p < 0.05). The 100 mg/ L CTC treatment yielded the minimum fresh weight growth (decreased by 32.76 % compared with the control), and the OTC concentration of 50 mg/L yielded the lowest fresh weight value (decreased by 35.56% of the control).
Similar to the RGR of V. natans after 7 D, the RGR of the plants declined markedly beyond 10 mg/L with increasing concentrations of CTC/ OTC after 14 D (p < 0.05). In addition, under 100 mg/L CTC and 50 mg/L OTC, the RGR of V. natans compared with the control were as low as 38.70% and 49.14%, respectively.

Photosynthetic pigments
As shown in Fig. 1, no significant differences were found for the photosynthetic pigments when V. natans plants were exposed to CTC after 7 D. When the concentration of OTC was between approximately 1.0 and 30 mg/L, the contents of photosynthetic pigments of V. natans decreased, then returned to the normal level. And the content of chlorophyll a and carotenoid of V. natans significantly decreased at 30 mg/L OTC.
Exposed to CTC treatment, the contents of chlorophyll a and total chlorophyll of V. natans showed a similar response to those of OTC treatment after 14 D, and both significantly decreased at 10 mg/L CTC/OTC ( Fig. 2a and d). After 14 D, no significant differences were observed in the content of chlorophyll b of V. natans under various CTC concentrations, and when the concentration of OTC exceeded 10 mg/L, the content of chlorophyll b was significantly suppressed (Fig.  2b). The carotenoid content of V. natans showed a concentration-dependent decrease, and 100 mg/L CTC and 50 mg/L OTC significantly reduced the carotenoid content to 40.30% and 39.87%, respectively (Fig. 2c).

MDA content
There was no significant difference in the MDA content of V. natans after 7 D of CTC/OTC treatment (Fig. 3a). After 14 D of CTC exposure, the maximum content of MDA was 11.00 ± 2.13 nmol/g FW obtained at 10 mg/L, and then returned to the normal level, while for the OTC exposure, the maximum value (14.79 ± 2.05 nmol/g FW) was noticed at 10 mg/L, beyond which the MDA content of V. natans still remained at a high level and was significantly higher than that of the control group (Fig. 3b).

Antioxidative enzymes
After 7 D, the CAT activity of V. natans hardly changed before the concentration of CTC/OTC reached 10 mg/L; when the concentration of CTC/OTC exceeded 30 mg/L, the CAT activity decreased significantly; and there were no remarkable differences between the high-concentration treatment groups (30~100 mg/L) (Fig. 4a). No significant differences were observed in the POD and PPO activities of V. natans compared with the control group (p > 0.05) (Fig. 4c and e).
After 14 D, the CAT activity of V. natans was more obvious than that of 7 D. The maximum value was noticed at 1.0 mg/L CTC/OTC, beyond which the CAT activity decreased significantly as the concentration of CTC/ OTC increased (Fig. 4b). The POD activity of V. natans significantly increased at 0.1 mg/L CTC/OTC, while the value returned to the normal level at higher concentrations of CTC/OTC (≥30 mg/L) (Fig. 4d). The PPO activity of V. natans showed first a significant increase and then a decreasing trend, and the maximum value (125% greater than the control) was noticed at 10 mg/L CTC. The effect of OTC on the PPO activity of V. natans was negligible (p > 0.05) (Fig. 4f).

Soluble protein content
The effect of CTC on the soluble protein content of V. natans after 7 D was similar to that of 14 D, and both evidently decreased at 10 mg/L (p < 0.05). In addition, the 100 mg/L CTC treatment yielded the lowest soluble protein content, and decreased by 47.80% and 49.99%, respectively, compared with the control group of 7 D and 14 D ( Fig. 5a and b). There was no significant difference in the soluble protein content of V. natans under 0.1-10 mg/L OTC treatment compared with the control group after 7 D (p > 0.05). Beyond 10 mg/L with increasing concentration of OTC, the soluble protein content of V. natans declined significantly (p < 0.05) (Fig. 5a). After OTC treatment for 14 D, the soluble protein of V. natans showed a concentration-dependent decrease with increasing concentrations of OTC (Fig. 5b).

H 2 O 2 content
After 7 D, no significant differences in H 2 O 2 content were observed between the control and the other experimental groups after CTC treatment. The H 2 O 2 content did not change significantly when the OTC concentration was less than 10 mg/L, and when the OTC concentration was more than 10 mg/ L, it increased significantly with the increase of OTC concentration (p < 0.05) (Fig. 6a).
After 14 D, no significant differences were observed for the H 2 O 2 content when V. natans plants were exposed to CTC. The OTC treatment resulted in a significant decrease in the H 2 O 2 content at 0.1 mg/L, where a minimum decrease of 17.18% was observed, and when  the OTC concentration ≥ 1 mg/L, the H 2 O 2 content of V. natans increased significantly with the increasing concentration of OTC (Fig. 6b).

Discussion
After 7 D and 14 D of CTC/OTC exposure, no significant difference was observed in the RGR at low levels of CTC/ OTC (≤ 1.0 mg/L). The result showed that low concentrations of CTC/OTC did not lead to a "hormesis" effect of V. natans, which was similar to the results of Zhou et al. (2007) and Liu et al. (2017). Whether the plants produce hormesis may depend on a variety of factors, such as the type and concentration of antibiotic, the organism under test, and the duration of exposure. The RGR decreased significantly when the concentration of CTC/OTC exceeded 10 mg/L, which indicated that CTC and OTC can harm the growth of V. natans. Our study was consistent with the result of Cui et al. (2008), Guo et al. (2020), and Liu et al. (2020). A possible reason for the negative effect may be because they inhibit the absorptions of water and trace elements by plants which are of great significance for photosynthesis, respiration, and protein synthesis in plants (Cui et al. 2008;Munns 2002). Other studies have reported that tetracyclines can complex with metal ions such as Cu 2+ , Mn 2+ , Fe 2+ , Fe 3+ , and Zn 2+ , which may reduce the biomass and the absorption of trace elements by plants (Tongaree et al. 1999). When exposed to high concentrations of CTC/OTC (≥ 30 mg/L) after 14 D, the inhibition degree of OTC on the fresh weight of V. natans was always stronger than that of CTC treatment, which was similar to the result of Ye et al. (2017) but different from that found in Brassica campestris (Zhu et al. 2018). It has been suggested that a degree of species-specific properties leads to differences in susceptibility to poisons (Brain et al. 2005;Hanson et al. 2006). Although tetracyclines have a similar structure, subtle differences in structure can lead to differences in toxicity to plants (Dong et al. 2012).
Soluble proteins are important macromolecules in organisms, which play an important role in maintaining cell structure and regulating physiological metabolic activities in organisms . Tetracyclines can enter cells through active transfer and irreversibly bind to the cell ribosome 30s subunit, prevent ammonia radical transfer from binding to DNA, and inhibit the synthesis of cell proteins, thus inhibiting their growth (Halling-Sørensen 2000; Lu et al. 2015). After 7 D of CTC exposure, the soluble protein content of V. natans was significantly decreased at 10 mg/L, while it showed a marked decrease at 30 mg/L OTC. The chemical structure of CTC may be responsible for the result; the presence of halogens can enhance the molecular polarity and lipid solubility of CTC, making it easier to integrate with enzyme system in a short-term (Dong et al. 2012). However, 0.1 mg/L OTC observably reduced the content of soluble protein in V. natans after 14 D, while CTC still required 10 mg/L. Antibiotics can be absorbed and transported by plants through passive transport or active uptake, depending largely on the respective logarithm octanol/water partition coefficient (log Kow) (Krzeminski et al. 2019). Given the higher solubility (0.008 mol/L for CTC, 0.062 mol/L for OTC) and lower octanol/water coefficient (log Kow: − 0.62 and − 0.9 for CTC and OTC, respectively) than CTC, OTC is easier to transport to plant tissues driven by transpiration flow (Mathews and Reinhold 2013). In addition, the observation indicated that CTC/OTC could impede the synthesis of soluble proteins in V. natans, which was consistent with the results of Zhang et al. (2019) and Siedlewicz et al. (2020). The content of soluble protein in plants depends on the kinetic equilibrium of their catabolism, and the decrease in soluble protein content may be due to the increased proteolysis in the organism to compensate for the loss of energy, or to the formation of lipoproteins to repair cells, tissues, and organs (Singh et al. 2006). Analogously, other studies have reported that the synthesis rate of soluble protein in plants will slow down when exposed to adversity stress Zhu et al. 2020).
Photosynthetic pigment is a basic parameter for evaluating photosynthetic activity which is often used as an indicator of plant damage under adversity stress . Tetracyclines can significantly reduce chlorophyll contents, which has been reported in the literatures (Rydzyński et al. 2019;Siedlewicz et al. 2020). Tetracyclines mainly affect chlorophyll contents by inhibiting chloroplast translation activity and the activities of enzymes related to chlorophyll molecule synthesis (Kasai et al. 2004). Moro et al. (2020) reported that 10 mg/L OTC reduced photosynthetic pigment contents by causing changes of chloroplast structure in Isochrysis galbana Parke. In addition, Jiao et al. (2008) found that exposure to 20 mg/L of TC in V. natans resulted in the plasmolysis of mitochondria and chloroplasts and disordered interlamellar structure of chloroplasts. After 7 D of CTC/OTC, there was no significant difference or only a partial decrease in the chlorophyll contents. However, the chlorophyll contents of V. natans significantly decreased with the increase of CTC/ OTC concentration after 14 D. The results indicated that both antibiotics had a cumulative toxic effect, that is, the organism exposed to CTC/OTC may not or showed a lighter toxic effect in a short time, but the toxic effect would increase in the later period. Studies have shown that chlorophyll degradation is related to ·OH and MDA produced by O 2and H 2 O 2 (Dhindsa et al. 1981). The increase of H 2 O 2 content will cause a decline in chlorophyll contents, affect the integrity of thylakoid membrane, and inhibit the synthesis of PSII-related proteins (Kar and Choudhuri 1987;Takahashi and Murata 2008). In this experiment, the contents of MDA and H 2 O 2 in V. natans both maintained a higher level under higher concentrations of OTC treatment after 14 D, which may be the reason for that the more obvious decreasing trend of the chlorophyll contents in V. natans under OTC treatment than that of CTC treatment. In addition, the reductions of chlorophyll a and carotenoid in V. natans were more pronounced than chlorophyll b content after 14 D of CTC/OTC treatment, which was consistent with that found in Trapa bispinosa to TC stress  but different from the result of Guo et al. (2020). The different performance of photosynthetic pigments may indicate that the content of chlorophyll b in V. natans has a delayed response to CTC and OTC, and the effect of tetracyclines may be species-specific.
Pollutants in the environment can cause oxidative stress, leading to the accumulation of reactive oxygen species (ROS) in plants, and excessive ROS will cause membrane lipid peroxidation which leads to the accumulation of MDA (Zhong et al. 2018). No lipid peroxidation was observed in V. natans after 7 D of CTC/OTC treatment, which was similar to the findings in Hydrocharis dubia (Bl.) Backer . After 14 D, the content of MDA in V. natans significantly increased at 10mg/L CTC, and with the increase of CTC concentration, it showed no significant difference compared with the control. The results illustrated that the antioxidant system of V. natans worked well to scavenge free radicals and maintain the stability of cell membrane. Notably, the MDA content of V. natans decreased significantly at 1.0 mg/L CTC, contrary to what we expected because of oxidative stress in plants exposed to tetracyclines (Bártíková et al. 2016;Xie et al. 2019). The findings in Trapa bispinosa under TC exposure were consistent with ours .The decrease of MDA content may show an adaptation response of V. natans exposed to CTC. It is possible to hypothesize that given the similar chemical structure to the antioxidant vitamin E, the decline may be related to the potential antioxidant properties of CTC (Kraus et al. 2005;Nunes et al. 2015). However, the contents of MDA and H 2 O 2 in V. natans under high concentration of OTC were always significantly higher than those of the control, which confirmed the presence of oxidative stress in V. natans. In general, high levels of H 2 O 2 may induce lipid peroxidation. Many studies also demonstrated the increase in lipid peroxidation in tetracyclines-exposed plants associated with high levels of H 2 O 2 (Yonar 2012).
The antioxidant system composed of antioxidant enzymes and antioxidants in the body can remove free radicals produced by cell growth and metabolism under normal circumstances (Zhou et al. 2018). However, when an organism is stimulated by the outside, there will be excessive production of ROS, such as superoxide radicals, hydroxyl radicals, and H 2 O 2 (Wu et al. 2010 (Kouka et al. 2018;Liu and Wu 2018). After 7 D of CTC/OTC exposure, no significant differences were found for the POD and PPO activities, but the CAT activity of V. natans decreased significantly under high concentrations of CTC/ CTC (≥ 30 mg/L). Considering that the content of MDA did not change, the decrease of CAT content confirmed that the antioxidant enzyme system in V. natans responded well to the stress of CTC/ OTC. The CAT activity still reduced markedly at high concentrations (≥ 30 mg/L) after 14 D, which was a somewhat interesting result, since tetracycline stress could cause an increase in CAT activity (Dong et al. 2012;Xie et al. 2011). Chi et al. (2010 found that OTC can interact with a binding site of CAT through van der Waals interaction and hydrogen bonding, and the microenvironment of tryptophan residues and the secondary structure of CAT change after combining with OTC. Other studies have reported that the CAT activity of ryegrass and maize decreased under tetracycline stress which confirmed our result (Cui and Zhao 2011;Han et al. 2019). The work of POD activity at 0.1 mg/L CTC/OTC indicated that the antioxidant system of V. natans was activated to remove ROS with the increasing substrate levels. In addition, the changes in membrane lipid permeability induced by CTC/ OTC could promote the uptake of environmental nutrients by the plants. Iron in nutrient solution not only is involved in the synthesis of chlorophyll, but also is a component of POD, which may lead to the increase of POD activity . The contents of MDA and H 2 O 2 of V. natans under high concentrations of OTC were always significantly higher than the control level, which indicated that the antioxidant enzyme system of V. natans was not enough to eliminate the ROS persecuted cells. The different responses of PPO activity of V. natans under CTC and OTC stress may be one reason why the content of H 2 O 2 and MDA was still high at a high concentration of OTC. PPO can participate in the oxidation of phenols, and the oxidation products (such as quinones) formed by the oxidation of phenolic compounds can combine with the side chain of amino acids to reduce the protein content (Mayer 2006;Singh et al. 2008). These results suggest that the antioxidant defense system of plants is unstable and can change over time. Plants under external stress will constantly adjust the activity of antioxidant enzymes to strictly control the ROS level (Song et al. 2012).

Conclusion
The toxicity of CTC and OTC on V. natans was different. After 7 D, the content of H 2 O 2 , at higher concentrations of OTC treatment (≥ 10 mg/L), was always significantly higher than the control group, while it hardly changed when exposed to CTC. High concentration of CTC and OTC both had significant damage to the RGR, soluble protein content, and CAT activity of V. natans, which were consistent with the trend after 14 D. After 14 D, the activity of PPO increased significantly in the CTC concentration range from 1 to 30 mg/L and then decreased, while it remained unchanged when exposed to OTC. The soluble protein content significantly decreased at 10 mg/L CTC and 0.1 mg/L OTC, respectively. Different from the MDA and H 2 O 2 contents of V. natans of CTC exposure, they were significantly higher than the control under a high concentration of OTC after 14 D. Overall, the toxicity of OTC to V. natans is stronger than that of CTC.
Author contribution Jing Li: conceptualization, methodology, resources, investigation, writing-original draft Lu Yang: resources, validation Zhonghua Wu: Supervision, project administration, funding acquisition Funding The National Science Foundation of China (No. 31270410, No. 30970303) The Scientific Research Project of Hubei Province Environmental Protection Department (2014HB07) Data availability All data generated or analyzed during this study are included in this published article.

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.

Competing interests
The authors declare no competing interests.