Effects of combined pollution of tetracycline and sulfamethazine on tomato growth and antibiotic absorption

doi:10.21203/rs.3.rs-2128513/v1

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Effects of combined pollution of tetracycline and sulfamethazine on tomato growth and antibiotic absorption

https://doi.org/10.21203/rs.3.rs-2128513/v1

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Food safety has raised immense concern globally. Due to the improper use of chemical fertilizers and pesticides, antibiotic pollution in vegetable land is becoming increasingly serious. In this study, the effects of single and combined pollution of tetracycline and sulfamethazine on tomato plants were studied. The results showed that tomato plants simultaneously absorbed tetracycline and sulfamethazine from soil and transferred them to leaves, stems and fruits. The contents of tetracycline and sulfamethazine in plant tissues elevated with the increase of soil antibiotics, which were higher in the growth stage than that in the late growth stage. Antibiotics exhibited the highest concentration in roots, followed by leaves, stems and fruits. At the concentrations of 25 and 50 mg/kg, both tetracycline and sulfamethazine hampered the growth and physiology of tomato in terms of photosynthetic rate, chlorophyll content, plant height and biomass. High concentration of antibiotic contamination led to the accumulation of nitrogen, phosphorus and potassium in leaves, reduced the carbon/nitrogen ratio in leaves, and reduced the vitamin C and sugar contents in fruits. Overall, our results indicated that the combined pollution of antibiotics can strengthen the impact on plant growth, physiological indicators and biomass.

Antibiotics

Combined pollution

Tomato plants

Physiological effect

With the rapid development of animal husbandry, the use of antibiotics as veterinary drugs and feed additives has increased year by year. It is estimated that the total amount of antibiotics used worldwide (including veterinary antibiotics and medical antibiotics) has reached 100,000 to 200,000 tons¹. Among them, veterinary antibiotics accounted for the vast majority of the use. Most antibiotics fed to animals are poorly absorbed in the intestines of animals, and most of them can be excreted as organic fertilizer through urine or feces^2–4. Therefore, veterinary antibiotics can be directly excreted on grassland by grazing livestock or transferred to farmland soil by feces^5–7. It is reported that more than 70% of veterinary antibiotics are released into the environment through various channels, and soil is the main pool of antibiotics in the environment^8–10. Antibiotics remaining in soil may be ingested into human body through the food chain or drinking water, and even lead to the formation of super bacteria with antibiotic resistance, which pose a serious threat to human health^11,12. The exposure and adverse reactions of antibiotics in terrestrial ecosystems have attracted great attention in the past 20 years¹³. The effects of antibiotic residues in the environment can include the evolution of resistance through long-term exposure to pathogens and bacteria, the transfer of antibiotic resistance genes, and the effects on human health through the ingestion of animal or plant foods with antibiotic residues^12,14,15. Eco-toxicity effects on non-target organisms and ecological impacts on agroecosystem¹⁶. The sources, fate and ecological risks of antibiotics in the environment have been carried out extensively, including antibiotic residue levels in the environment, environmental exposure assessment, adsorption, migration and degradation, and ecological risks to ecosystems^17–19. Tetracycline and sulfamethazine are widely used in farm animals to control intestinal and respiratory infections, and they are the most common antibiotics in pigs, beef and pig manure²⁰. Tetracycline and sulfonamides are widely distributed worldwide, which come from the septic tanks or feces of livestock farms². The concentration of antibiotic residues ranges from a few micrograms to grams per kilogram¹⁴. Chen et al. found that tetracycline, oxytetracycline, chlortetracycline and doxycycline were the main pollutants in the feces of eastern China, with the highest concentrations of 98.2, 354.0, 139.4, 37.2 and 7.1 mg/kg, respectively²¹. Loke et al. and Brambilla et al. showed that the concentration of oxytetracycline in soil was as high as 50 mg/kg^22,23. Hamscher et al. detected that the concentration of tetracycline in agricultural soil with liquid fertilizer was as high as 0.3 g/kg²⁴. Many crops can absorb tetracycline and sulfamethazine from contaminated or artificial soils²⁵. It has been found that tetracycline can inhibit the growth of microorganisms in soil, reduce soil enzyme activity, and significantly reduce the growth of roots, stems and leaves of crops²⁶. However, until recently, research on the use of antibiotics has mainly focused on the beneficial and adverse effects of single antibiotic on human and animals. Most studies have focused on phytotoxicity to young plants, especially seed germination, root and stem development²⁷. There are few studies on the accumulation of antibiotics in edible parts of vegetable crops such as tubers, rhizome crops, fruit and vegetable crops. At present, few studies have been conducted on the toxic effects of combined contamination of several antibiotics on crops²⁸. Therefore, the response of plants to the concentration of two or more combined antibiotics in soil is still unclear.

Tomato is a typical fruit and vegetable crop. In this study, tomato plants (Lycopersicon esculentum Mill.) were planted in silt loam with different concentrations of tetracycline and sulfamethazine (0, 25 and 50 mg/kg) separately or at the same time to investigate the effects of tetracycline and sulfamethazine on the absorption of antibiotics. To investigate the adverse effects of combined contamination of two antibiotics on plants.

Collection and preparation of soil samples

The studied soils were collected from Keqiao District, Shaoxing City, Zhejiang Province, China(29°54′36″N, 121°29′36″E). It has a north subtropical monsoon climate. The average annual temperature is 16.9°C, and there is 1500 mm of precipitation on average per year. The soil type is Fluventic Eutrochrept (USDA soil taxonomic classification), and the soil samples were collected at 0-15cm using a bamboo shove land mixed thoroughly. The texture was silt loam, with no tetracycline and sulfamethazine detected. The collected soils were air-dried before analyses, mixed and screened by a 5-mm mesh sieve for experiment. Some soil samples were further sieved through a 2-mm and 0.25-mm soil mesh sieves for soil physical and chemical properties analyses. The soil properties were as follows: pH 5.67, soil organic carbon (SOC) 9.23 g/kg, available phosphorus (AP) 7.89 mg/kg and available potassium (AK) 56 mg/kg. The tetracycline (96% purity) and sulfamethazine (99% purity) were purchased from Sigma, Inc., Germany and stored in an ice chamber at 4°C. Commercial seeds of tomato purchased from Chinese Academy of Agricultural Sciences were used for on-soil tests. They were sterilized and transferred to Petri dishes for germination at 25°C in the dark. According to the already used analysis method in Migliore et al.^29–31, after germination seedlings were transferred to multipurpose jars on solid Murashige and Skoog (MS) medium (Sigma, Milan,Italy).

Pot experiment design

The pot experiment was carried out in a greenhouse of Zhejiang University, China. The soil was air-dried and ground to pass a 2-mmsieve. The pot experiment was conducted with 7 antibiotic treatments and 6 repetitions (Table 1). The amount of soil used for each pot was 25 kg, 0.5 g NH₄NO₃ and 0.2 g KH₂PO₄ were added to each kg of soil as chemical fertilizer to ensure adequate nutrition during the experiment.In order to avoid the introduction of other antibiotics from organic fertilizers, animal manure was not applied during the experiment, and antibiotics were added artificially according to the dosage in Table 1. After the soil and additives are fully mixed, the soil moisture content was adjusted to 75%-80% of the field capacity with deionized water and put into a bowl with a height of 15 cm and an outer diameter of 30 cm. Transplant tomato seedlings at the age of 25 days in each pot, 4 plants in each pot. Soil and plant sampling and related observations were carried out within 100 days after the experiment. The plants were cultured in an artificial climate chamber, where the photo-period of the plants was 16h light/8h dark and the diurnal temperature was 25/20°C. During the culture period, water was added weekly to maintain the field capacity by weighing method. The experiment lasted from April to September with sufficient illumination.

Table 1

Dosage of antibiotics for different treatments
Treatment	Dose of tetracycline (mg/kg)	Dose of sulfamethazine (mg/kg)
T0S0	0	0
T25S0	25	0
T0S25	0	25
T25S25	25	25
T50S0	50	0
T0S50	0	50
T50S50	50	50

Soil samples were collected 6 times at 1, 5, 10, 20, 50 and 100 days respectively after antibiotic exposure. During the experiment, plant samples were collected at the same time for the determination of antibiotic residues, total N, P, K, nitrate, vitamin C and reducing sugars in plants. In order to facilitate sample collection, the 6 replicate treatments were divided into two groups (each with 3 replicates). On the 20th and 50th days of the experiment, plant sampling was carried out in the first group, and two tomato plants were taken each time. At the end of the experiment(100th day), plant sampling was carried out in the second group, and four tomato plants were collected from each treatment. The harvested plants were first rinsed thoroughly with tap water and deionized water, then dried with toilet paper, and then divided into roots, stems, leaves and fruits, and the fresh weight was recorded immediately. Plant samples were stored at -15°C before analyses.

In addition, the height of tomato plants in each treatment was measured on the 20th, 50th and 100th days of the experiment. On the 50th day of the experiment, the chlorophyll content (SPAD value) and photosynthetic parameters of the largest expanded leaf of each plant were measured, including stomatal conductance, transpiration rate, intercellular carbon dioxide concentration, and photosynthetic rate.SPAD-502 chlorophyll (KONICA MINOLTA, Inc., Tokyo, Japan) was used to detect the leaf SPAD value (chlorophyll content), and the photosynthetic parameters were measured using the LI-6400 portable photosynthesis apparatus (LI-COR, Inc., USA).The measurements were carried out under daylight conditions between 11 a.m. and 14 p.m.

Chemical analyses

All chemicals used in the experiments were of analytical grade. All glassware and vessels used in the analyses were soaked in 10% HNO₃overnight and rinsed thoroughly with deionized water before use. The accuracy of the data was checked using materials that met national standards (GBW-07404), and precision based on duplicate samples was found to be better than ± 5%.

The pH of soil was determined with a pH meter (FE20-FiveEasy™ pH, Mettler Toledo, German).Sand, silt and clay concentrations in the soil samples were evaluated by the micropipette method³². Soil organic carbon (SOC)was determined by Element Analyzer (Elementar Vario EL Cube, German)³².Soil available phosphorus (AP) was measured using the molybdenum blue method after being digested with HClO₄-H₂SO₄³³. Soil available potassium (AK)was extracted with 1M neutral ammonium acetate and then estimated with Atomic absorption spectrometry (AAS, Analytik Jena novAA 300, German)³⁴. After the fresh plant samples are ground with a small food grinder, 5g of each sample is weighed and extracted with 20ml EDTA-Mcilvine (0.05mol/L EDTA + 0.06mol/L Na₂HPO₄ + 0.08mol/L citric acid, pH4) for 5 minutes. After extraction, the supernatant was separated under a centrifugal force of 2000 g for antibiotic determination. After extraction, the supernatant was separated under a centrifugal force of 2000 g for antibiotic determination. The sample was purified by chromatography column and determined by UV detector (wate UV 2487370 nm). The chromatographic column is SHIM-PACK.VP-ODS (5 µm, 150 mm×4.6 mm ID), the mobile phase is 0.01 mol/L oxalic acid + acetonitrile + methanol (76 + 16 + 8), and the flow rate is 1ml/min. The detection limits of tetracycline and sulfamethazine were 2.5 and 5 µg/kg, and the results are expressed on the basis of fresh weight^{24, 29− 31,36}. Acetonitrile and methanol used in the test were HPLC grade, and citric acid, Na₂HPO₄ and EDTA were analytical reagent grade. The total carbon, total phosphorus and total nitrogen of plant leaves were measured with a CHN elemental analyzer (Flash EA 1112, Thermo Finnigan). After digesting 0.5g dry leaf sample with H₂O-HNO₃ solution, the concentrations of calcium(Ca) and potassium(K) were determined with Atomic absorption spectrometry (AAS, Analytik Jena novAA 300, German)³⁴.At the same time, the contents of nitrate, vitamin C and reducing sugar in fruits were determined by conventional methods, and the results were expressed on the basis of dry mass³⁵.

Ethics statement

The collection of plant material complied with the relevant regulations and ethical approval of Zhejiang University.

Statistical analysis

Results of experiments are shown with means and standard deviations. Statistics were carried out in the SPSS v26.0(SPSS Inc., Chicago, IL, USA). P values < 0.05 by Tukey's-HSD one-way analysis of variance (ANOVA) was used to analyze the significant differences(P < 0.05). Statistical analysis was performed by OriginPro software (regression analysis).

Changes of tetracycline and Sulfamethazine in soil

Tetracycline and sulfamethazine were not detected in the soil treated without tetracycline and sulfamethazine. The residues of tetracycline and sulfamethazine in the soil treated with tetracycline and sulfamethazine increased with the addition of antibiotics but decreased with the increase of culture time (Fig. 1). On the 100th day of the experiment, the residual rates of tetracycline in treatment T25S0 and treatment T25S25 (25mg/kg tetracycline treatment) were 22.04% and 23.88%, respectively. The residual rates of tetracycline in T50S0 and T50S50 (50 mg/kg tetracycline treatment) were 26.94% and 28.26%, respectively. Meanwhile, the residual rates of sulfamethazine in the soil of treatmentsT0S25 and treatment T25S25 with 25 mg/kg sulfamethazine were 11.87% and 12.52%, respectively. The residual rates of sulfamethazine in soil of treatment T0S50 and T50S50 with 50 mg/kg sulfamethazine were 16.48% and 14.42%, respectively. In general, the degradation rate of sulfamethazine in soil was higher than that of tetracycline. The residual percentage of the two antibiotics in soil decreased with the increase of the dosage of antibiotics, that is, the higher the dosage of antibiotics, the lower the degradation ratio of antibiotics. Compared with the treatment of single antibiotics, the combined contamination of two antibiotics had no significant effect on the degradation of antibiotics in soil.

Changes in tetracycline and Sulfamethazine in roots and shoots of tomato plants

Similar to the changes of tetracycline and sulfamethazine in soil content, tomato plants growing on soil without any antibiotic treatment were not detected either, while tetracycline and sulfamethazine were detected in plant tissues which grown on soil treated with antibiotics (Fig. 2). The absorption of tetracycline and sulfamethazine increased with the increase of antibiotic content in soil, indicating that tomato plants would absorb more antibiotics with the increase of antibiotic pollution level in soil. It is worth noting that the contents of tetracycline and sulfamethazine in tomato roots were significantly higher than those in the shoots (Fig. 2). The concentrations of tetracycline and sulfamethazine in tomato roots were 31.00-102.88 and 23.56-107.64 times higher than those in the shoots, respectively, indicating that the concentrations of tetracycline and sulfamethazine in tomato roots were significantly enriched. However, the migration of antibiotics in plants were relatively weak. The difference of antibiotics between roots and shoots decreased with the extension of the experiment time. From the 20th day to the 50th day of the experiment, the contents of tetracycline and sulfamethazine in shoots and roots showed a trend of increasing at first and then decreasing. After the 50th day of the experiment, the decrease of antibiotics in plants might be related to the dilution effect caused by the rapid increase of plant biomass, and the decrease of the availability of tetracycline in soil and reduced the absorption of antibiotics by plants due to the degradation of antibiotics in soil. There was no significant difference in the contents of antibiotics in roots and shoots between two kinds of antibiotic combined contamination and single antibiotic contamination treatments, indicating that antibiotic combined contamination had no significant impact on the absorption and migration of antibiotics in tomato plants. The results also showed that the absorption of sulfamethazine was slightly lower than that of tetracycline when antibiotics were added at the same concentration.

Residues of tetracycline and Sulfamethazine in different tissues of Tomato during harvest

Figure 3 shows the residues of two antibiotics in various organs of tomatoes at the time of harvest (100th day). The results showed that tetracycline and sulfamethazine can still be detected in the roots, leaves, stems and fruits of tomatoes grown in the soil treated with antibiotics during the harvest. The contents of tetracycline and sulfamethazine in plant tissues increased with the increase of antibiotic concentration in soil. The contents of tetracycline and sulfamethazine were the highest in roots, followed by leaves, stems and fruits. Most of the antibiotics remained in the roots and the concentrations of antibiotics in the roots were 42–101, 24–50 and 100–317 times more than that in stems, leaves and fruits, respectively.

Effects of antibiotic pollution on nutrient content in plant organs

Table2 showed the analysis results of C, N, P, K, Ca and other elements in the leaves of each treatment at the 50th and 100d of the experiment (vegetative growth period and fruit harvest period). When the concentration of antibiotics was 25 mg/kg, the content of C in leaves was similar to that in the control treatment (without antibiotic treatment), and there was no significant difference in the detection value of C in leaves. However, when the concentration of antibiotics was 50 mg/kg, there was a significant difference in the content of C in the leaves between the plants grown on the soil treated with antibiotics and the control. After the treatment with antibiotics, the content of C in the leaves of the plants grown on soil contaminated with tetracycline and sulfamethazine decreased significantly. Meanwhile, the N content in leaves increased after antibiotic treatment, but when the antibiotic dosage was 25 mg/kg, the N content in leaves was significantly higher than that in the control group at the vegetative stage, but the difference was not significant at the harvest stage. However, when the antibiotic dosage was 50 mg/kg, the N content in leaves was significantly higher than that of the control in both vegetative stage and the harvest stage.The results showed that the nitrogen content in the leaves of the plants under the combined pollution of the two antibiotics was higher than that under the single pollution, indicating that the combined pollution of the two antibiotics affected the N accumulation in the leaves of the plants.

Table 2

Changes of C, N, P, K, Ca content in tomato leaves at different growth stages with tetracycline and sulfamethazine pollution
Treatment	Vegetative stage						Harvest stage
	C	N	C/N	P	K	Ca	C	N	C/N	P	K	Ca
T0S0	40.44a	3.04d	13.30a	0.34c	1.48c	5.32c	41.12ab	3.31c	12.42a	0.36c	1.21c	6.14bc
T25S0	39.76a	3.32c	11.98b	0.38bc	1.53bc	5.44bc	41.14ab	3.29c	12.50a	0.37c	1.20c	6.08c
T0S25	40.01a	3.43c	11.66b	0.39bc	1.57bc	5.29c	41.87a	3.39c	12.35a	0.39bc	1.26bc	5.92c
T25S25	38.67b	3.72b	10.40c	0.42b	1.62b	5.64b	40.23b	3.52b	11.43b	0.42b	1.36b	6.54b
T50S0	39.11b	3.59bc	10.89c	0.41b	1.59bc	5.58b	40.21b	3.47bc	11.59b	0.41b	1.32b	6.42b
T0S50	38.76b	3.63bc	10.68c	0.45b	1.65b	5.63b	40.18b	3.51b	11.45b	0.43b	1.35b	6.42b
T50S50	38.32b	4.17a	9.19d	0.51a	1.78a	6.78a	40.03b	3.91a	10.24c	0.54a	1.78a	8.08a
Notes: Different lowercase letters in the same column mean significant differences among different treatments(P < 0.05). T0S0: control, T25S0: tetracycline25mg/kg + sulfamethazine0mg/kg, T0S25: tetracycline0mg/kg + sulfamethazine25mg/kg, T25S25: tetracycline 25mg/kg + sulfamethazine25mg/kg, T50S0: tetracycline50mg/kg + sulfamethazine0mg/kg, T0S50: tetracycline0mg/kg + sulfamethazine50mg/kg, T50S50: tetracycline50mg/kg + sulfamethazine50mg/kg.

Effects of antibiotic pollution on Photosynthesis

Figure 4 showed the chlorophyll content (SPAD value) and photosynthetic parameters, photosynthetic rate, intercellular CO₂ concentration, transpiration rate and stomatal conductance of the largest expanded leaves in the upper part of the plants measured by LI-6400 portable photosynthetic system on the 50th day of the experiment. The results showed that antibiotic pollution could increase the concentration of intercellular CO₂ and decrease the photosynthetic rate, transpiration rate, stomatal conductance and chlorophyll content in the leaves of tomato plants growing on the soil. This effect increased with the increase of antibiotic pollution concentration, especially under the condition of combined pollution. Compared with the control, when the single addition concentration of tetracycline and sulfamethazine is 25 mg/kg, it can slightly reduce the photosynthetic rate, transpiration rate and chlorophyll content, but there was no significant difference from the control. However, the addition of tetracycline and sulfadiazine (25 mg/kg) can significantly increase the intercellular CO₂ concentration and reduce the stomatal conductance. Among them, the single addition of 25 mg/kg of tetracycline and sulfadiazine increased the intercellular CO₂ concentration by 10.32% and 7.94%and decreased the stomatal conductance by 11.84% and 9.87% compared with the control, respectively. However, under the combined pollution of tetracycline and sulfamethazine (25 mg/kg)at the same time, the intercellular CO₂ concentration was significantly increased by 13.10% compared with the control. Compared with the control, the chlorophyll decreased by 12.51%, the stomatal conductance decreased by 19.74%, the transpiration rate decreased by 8.77%, and the photosynthetic rate decreased by 17.77%.

When the concentration of tetracycline was added to 50 mg/kg, compared with the control, the chlorophyll, stomatal conductance, transpiration rate and photosynthetic rate decreased 15.98%, 25.00%, 7.12% and 15.90%, respectively. Accordingly, 50 mg/kg of sulfadiazine was added, the above four parameters decreased by 12.04%, 21.05%, 5.75% and 15.60%, respectively.

Effects of antibiotic pollution on tomato plant height and length

Figure 5 showed the variation of tomato plant height with test time under different antibiotic treatments. When 25 mg/kg of antibiotics were added alone, tetracycline and sulfamethazine had no significant effect on the plant height of the three growth stages, and the height was similar to that of the control.However, when 25 mg/kg of tetracycline and sulfamethazine were added at the same time, the plant height in each growth stage was significantly lower than that of the control treatment. The plant heights on the 20th, 50thand 100th days of the experiment were decreased by 13.17%, 11.16% and 8.64%, respectively.

When a single antibiotic was added, tetracycline and sulfamethazine (50 mg/kg) had significant effects on the plant height in three growth stages. Among them, 50 mg/kg tetracycline was added on the 20th, 50th and 100th days plant height were decreased by 16.96%, 14.19% and 9.81% respectively compared with the control.The plant height decreased by 12.72%, 10.05% and 7.68% respectively compared with the control, on the 20th, 50th and 100th days after adding 50 mg/kg sulfamethazine.At the same time, the plant height of 50 mg/kg tetracycline and sulfamethazine was significantly lower than that of the control, and decreased by 23.44%, 19.78% and 16.10% on the 20th, 50th and 100th days, respectively, compared with the control.In general, the effect of tetracycline was slightly greater than that of sulfamethazine, and the effect of combined pollution was greater than that of single pollution. The degree of influence decreased with the increase of tomato growth period.

Effects of antibiotic contamination on yield and quality

Figure 6 showed that soil antibiotic pollution can reduce tomato yield but has little impact on quality indicators. When 25 mg/kg antibiotics were added alone, tetracycline and sulfamethazine could reduce tomato yield by 13.35% and 10.45%, respectively, compared with the control. When 50 mg/kg of antibiotics were added alone, tetracycline and sulfamethazine could reduce tomato yield by 27.27% and 16.44%, respectively, compared with the control. Similar to the effect on plant height, the effect of tetracycline was slightly greater than that of sulfamethazine. The effect of combined pollution was greater than that of single pollution. When 25 mg/kg of tetracycline and sulfamethazine were added at the same time, the tomato yield w significantly decreased by 21.86% compared with the control. When 50mg/kg of tetracycline and sulfamethazine were added at the same time, The yield of tomato decreased obviously by 39.26% compared with the control.

The effects of antibiotic contamination on the accumulation of nitrate in tomatoes were only observed in three treatments: in the addition of 25 mg/kg sulfamethazine, 25 mg/kg tetracycline combined with sulfamethazine, 50 mg/kg tetracycline combined with sulfamethazine, which significantly increased the accumulation of nitrate by 2.97%, 7.20% and 3.38% compared with the control, respectively. The effect of antibiotic pollution on vitamin C content in tomatoes only appeared in the treatment of adding 50mg/kg of tetracycline and sulfamethazine at the same time, which significantly reduced vitamin C content by 6.10% compared with the control. The effect of each treatment on reducing sugar content in tomatoes was not obvious.

Our results indicate that high concentration of antibiotic pollution can affect the growth and photosynthesis of tomatoes, which should raise more concern in tomato production, especially in areas confronting combined antibiotic pollution.

The absorption of antibiotics in the environment by tomato plants is mainly through the root system, most of which are absorbed and accumulated by the roots. Only a small proportion of antibiotics are transported to the shoot of the plant^37,38. Compared with the shoot, the antibiotic concentration in the root is higher. Therefore, for soil antibiotic pollution, the roots are more sensitive and the response are more directly. Generally, plant resistance to soil pollutants such as antibiotics increases as crop biomass increases. Our results showed that the contents of tetracycline and sulfamethazine accumulated in tomato leaves increased first and then decreased with the added time of tetracycline and sulfamethazine, which may be related to the different growth stages of tomatoes and the change of the amount in tetracycline in soil^39,40. Similar to the combined pollution of heavy metals in soil, when two antibiotics coexist in soil, their inhibition effect on vegetable growth is significantly increased, which may be related to the cumulative effect of different antibiotics on plants^41,42. There are similarities in these mechanisms. For instance, photosynthesis is a fundamental process of plants, which can support growth, nutrient absorption and increase resistance to abiotic or biological stresses⁴³. The effect of antibiotics on the photosynthesis of tomato plants is similar to the stress of salt, alkali and heavy metals^43,44.

High concentrations of tetracycline and sulfamethazine significantly decreased leaf photosynthetic rate, transpiration rate and stomatal conductance, and significantly increased intercellular CO₂ concentration. In this study, the decrease of tomato biomass exposed to high concentrations of soil tetracycline and sulfamethazine may be related to the negative effects of tetracycline and sulfamethazine on photosynthesis.

In addition, the results of this study also showed that antibiotic pollution could also cause changes in plant nutrients, mainly manifested in the decrease of total carbon in leaf tissue and the increase of total nitrogen, Ca, P and K contents.

The decrease of total carbon may be related to the impact of antibiotics on the photosynthesis of plants, that is, with the increase of antibiotic pollution, the concentration of intercellular CO₂ in plant leaves increases (accumulates), reducing the photosynthetic rate, transpiration rate, stomatal conductance and chlorophyll content, resulting in the photosynthetic efficiency of plant leaves decreased and the accumulation of organic carbon decreased. This effect may also be the main reason for the decline in tomato plants height and yield caused by antibiotic pollution.

The increase of N, Ca, P and K content in leaves may also be related to changes in photosynthesis, that is, antibiotics hinder photosynthesis and plant biomass decreases, thereby relatively increasing the content of other nutrient elements in leaves. In addition, high concentrations of antibiotic pollution can affect the absorption of soil water by plants, which can also promote the relative concentration of nutrients in leaves and reduce the migration of nutrients to other organs to a certain extent, but it needs further experimental verification.

Under the condition of antibiotic combined pollution, tomatoes can simultaneously absorb tetracycline and sulfamethazine from soil and transfer them to leaves, stems, fruits and other organs. The contents of tetracycline and Sulfamethazine in plant tissues increased with the increase of antibiotic pollution levels in soil, and were higher at the early growth stage than at the late growth stage.The content of antibiotics in different organs was highest in roots, followed by leaves, stems and fruits. When the concentrations of tetracycline and Sulfamethazine in soil were 25 and 50 mg/kg, both tetracycline and sulfamethazine could affect tomato growth, reducing photosynthetic rate, chlorophyll content, plant height and biomass. High concentrations of antibiotic contamination could lead to the accumulation of N, P and K in plant leaves, reduce the C/N ratio in leaves, reduce the vitamin content in fruits, and slightly increase the accumulation of nitrate, but had no significant effect on reducing sugars.The results showed that the combined pollution of antibiotics in soil had no interaction with the accumulation of tetracycline and sulfamethazine in plants and the degradation of antibiotics in soil, but both of them could enhance the impact on plant growth and biomass.

Data availability

All data generated or analysed during this study are included in this published article.

The datasets used and analyses during the current study are available from the corresponding author on reasonable request.

Acknowledgements

The research supported by the Natural Science Foundation of Zhejiang Province, China (No. R306011), and the National Natural Science Foundation of China (No. 21177108). For the analysis part of this study, we would like to thank Prof. Xiangfeng Tan of Zhejiang Academy of Agricultural Sciences.

Ethics declarations

Competing interests

The authors declare no competing interests.

Authorship contribution

Qiutong Xu: Conceived, Experiment, Data curation, Writing - original draft.

Mingkui Zhang: Writing - Review & Editing, Supervision.

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No competing interests reported.

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Effects of combined pollution of tetracycline and sulfamethazine on tomato growth and antibiotic absorption

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Abstract

Figures

Introduction

Materials And Methods

Collection and preparation of soil samples

Pot experiment design

Chemical analyses

Ethics statement

Statistical analysis

Results And Discussion

Changes of tetracycline and Sulfamethazine in soil

Changes in tetracycline and Sulfamethazine in roots and shoots of tomato plants

Residues of tetracycline and Sulfamethazine in different tissues of Tomato during harvest

Effects of antibiotic pollution on nutrient content in plant organs

Effects of antibiotic pollution on Photosynthesis

Effects of antibiotic pollution on tomato plant height and length

Effects of antibiotic contamination on yield and quality

Discussion

Conclusions

Declarations

References

Additional Declarations

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