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Abstract [Objectives] This study was conducted on the effects of veterinary antibiotics on pasture growth.
[Methods] With alfalfa （Medicago sativa L.） as an experimental material and oxytetracycline， chlortetracycline and enrofloxacin as experimental contaminants， the effects of the three veterinary antibiotics on the germination percentage， germination energy， germination index and vigor index of alfalfa seeds and on the plant height and root length of alfalfa seedlings were study at the concentration gradient of 50， 100， 150， 200 and 250 mg/L， respectively.
[Results] The oxytetracycline treatments had no significant effect on the germination percentage of alfalfa seeds， but promoted its germination index. The chlortetracycline treatment solutions and enrofloxacin treatment solutions promoted the germination percentage， germination energy and germination index of alfalfa. The three antibiotics significantly inhibited the vigor index of alfalfa seeds， and had significant inhibitory effects on root length and plant height.
[Conclusions] These results provide an important basis for studying the ecotoxicity of veterinary antibiotics on pasture.
Key words Veterinary antibiotics; Alfalfa; Seed germination; Seedling growth; Ecotoxicity
With the development of animal cultivation industry and compound feed industry， veterinary antibiotics are used as animal feed additives， which are increasingly used in various animal cultivation industries. According to research， these antibiotics cannot be completely absorbed by animals， of which 40%-90% will be excreted in the form of feces， which will cause the unprocessed manure to carry residual antibiotics into the ecological environment to cause environmental pollution[1-3]. In addition， plant roots can enrich antibiotics in the soil， which can be then transported to the stems， leaves and fruits of plants， and eventually enter the food chain to bring about an negative impact that cannot be neglected on the health of humans and other organisms[3-4].
Alfalfa （Medicago sativa L.） is a leguminous plant known as the "king of the pasture". It is rich in crude protein， vitamins and other nutritive substances. It has high yield and excellent quality and is a high quality forage widely used for grazing[2]. Seed germination is an important stage of plant life history and the starting point of plant growth and development. The residual veterinary antibiotics in the soil directly affect the quality of seed germination and seedling growth. Therefore， it is important to study the effects of veterinary antibiotics on the seed germination and seedling growth of forage grass. In this study， 50， 100， 150， 200 and 250 mg/L oxytetracycline， chlortetracycline and enrofloxacin were used to treat the seeds of alfalfa， while the seeds were cultured in distilled water as a control， so as to investigate the effects of the three veterinary antibiotics on the seed germination and seedling growth of alfalfa. This study provides a scientific basis for studying the ecotoxicity of veterinary antibiotics and improving the quality of pasture in black land. Materials and Methods
Test materials
Forage seeds
The pasture seeds were alfalfa seeds.
Drugs
Oxytetracycline （purity 98%， Nanyang Veterinary Drug Factory）; chlortetracycline （purity 99%， Xionghui Pharmaceutical）; enrofloxacin （purity 98%， Beiliu Tongzhou Pharmaceutical Co.， Ltd.）.
Test instrument
Artificial climate box （Medical Equipment Factory， Shanghai Boxun Industry & Commerce Co.， Ltd.）.
Test methods
Test design
Full alfalfa seeds of the same size and normal color were disinfected with 75% ethanol solution for 10 min and rinsed three times with distilled water. The seeds were placed in disposable dishes laid with three layers of filter paper wetted with an equal volume of 0， 50， 100， 150， 200 and 250 mg/L of oxytetracycline， chlortetracycline and enrofloxacin solutions， respectively， according to 30 seeds per dish， which meant that the seeds were subjected to stress treatment， and each treatment was repeated 4 times. The seeds of different treatments were finally germinated in an incubator under the conditions of 14 h of illumination/25 ℃ and 10 h of darkness/20 ℃. The treatment solutions were changed every 12 h to ensure that the solution concentrations did not change. The germination of the seeds was observed and recorded every day， and it was determined that the seeds were germinated when the germ was just seen. After the germination， 5 seedlings were randomly selected from each dish to determine the root length and plant height.
Determination of parameters
Germination percentage GP=（Number of seeds that were normally germinated at the end of germination/Total number of seeds tested）×100%[5]
Germination energy GE=（Number of seeds germinated in the first 3 d/Total number of seeds）×100%
Germination index GI=∑（GT/Dt）
Vigor index VI=S×GI
Wherein GT is the number of seeds germinated in time t; Dt is the number of germination days; S is the growth amount of seedlings.
Data processing
Statistical analysis was performed using Excel 2007 and SPSS 13.0 software. One way ANOVA was used to analyze the differences between the parameters of different treatments， and multiple comparisons were performed by the Duncans method.
Results and Analysis
Effects of oxytetracycline treatment solutions on seed germination and seedling growth of alfalfa
It can be seen from Table 1 that compared with the control group， the oxytetracycline treatments were not significantly different in the germination percentage of alfalfa seeds， and promoted the germination index to different degrees， with the strongest promoting effect at the oxytetracycline concentration of 250 mg/L. Oxytetracycline showed significant inhibition on the vigor index of alfalfa seeds， with the strongest inhibitory effect at the oxytetracycline concentration of 200 mg/L， and the differences between different treatments were significant. The oxytetracycline treatments had inhibitory effects on the root length and plant height of alfalfa seedlings with significant differences， and the inhibition on roots was the highest when the concentration of oxytetracycline was 200 mg/L. Effects of chlortetracycline treatment solutions on seed germination and seedling growth of alfalfa
It can be seen from Table 2 that the germination percentage， germination energy and germination index of alfalfa seeds were promoted at low concentrations of chlortetracycline， and inhibited at high concentrations. With the increase of the concentration of chlortetracycline treatment， the germination percentage of alfalfa seeds increased first and then decreased. When the concentration of chlortetracycline was 50 mg/L， the promoting effect was the strongest and the germination percentage reached 100%. When the concentration of chlortetracycline reached 250 mg/L， the germination percentage of alfalfa seeds began to be inhibited， and the differences were obvious. Compared with the control group， the chlortetracycline treatment solution promoted the germination energy of alfalfa seeds at 50-200 mg/L， and began to inhibit at 250 mg/L. With the increase of chlortetracycline concentration， the germination index of alfalfa seeds increased first and then decreased， and reached the maximum value at 100 mg/L. Compared with the control group， the chlortetracycline treatment solutions had significant inhibitory effects on the seed vigor index of alfalfa， different degrees of promoting effect on the seedling height， and different degrees of significant inhibitory effect on the root length.
Effects of enrofloxacin treatment solutions on seed germination and seedling growth of alfalfa
As can be seen from Table 3， compared with the control group， the enrofloxacin treatment solutions promoted the germination percentage， germination energy and germination index of alfalfa seeds， and most promoted the germination percentage when the enrofloxacin concentration was 50 mg/L. With the increase of enrofloxacin concentration， the germination energy and germination index increased first and then decreased. The germination energy was most promoted at 50， 100 and 150 mg/L， and the promoting effect on the germination index was the strongest at 150 mg/L. Compared with the control group， the enrofloxacin treatment solutions had significant inhibitory effects on the seed vigor index and plant height of alfalfa， and the seed vigor index and seedling height of alfalfa were most inhibited at the enrofloxacin concentration of 150 mg/L. With the increase of enrofloxacin concentration， the root length of alfalfa seedlings gradually decrease， and compared with the control group， enrofloxacin inhibited its root length. Discussion and Conclusions
This study investigated the ecotoxicity of veterinary antibiotics against alfalfa by controlling the concentration of different antibiotics on alfalfa.
Different veterinary antibiotics had different effects on the germination of alfalfa seeds. Oxytetracycline had no significant effect on the germination percentage of alfalfa， while chlortetracycline and enrofloxacin could increase the germination percentage of alfalfa seeds within a certain concentration limit. The effect of chlortetracycline on the germination energy of alfalfa was promotion at low concentrations and inhibition at high concentrations; and the enrofloxacin treatment solutions increased the germination energy of alfalfa seeds.
The ecotoxic effects of the three veterinary antibiotics on alfalfa are mainly achieved by inhibiting seed vigor and plant growth. The root length， plant height and germination percentage of alfalfa are more sensitive to the ecotoxicity of antibiotics. The results showed that the oxytetracycline， chlortetracycline and enrofloxacin treatments reduced the vigor index of alfalfa seeds and the root length and plant height of the seedling plants. The 50 mg/L treatment solutions of the three antibiotics can make its vigor index， root length and plant height drop suddenly. It can be seen that the growth of roots and shoots of alfalfa is sensitive to antibiotics during germination， which might be related to the fact that antibiotics contact with roots and shoots once they break through the seed coat[6].
In summary， the effects of the three veterinary antibiotics on the germination percentage， germination energy and germination index of alfalfa seeds were relatively small. However， they had significant inhibitory effects on the root length and shoot growth of alfalfa seedlings， which is consistent with the effects of the stress of three antibiotics on the germination of cucumber， zucchini and melon seeds studied by Feng et al.[7].
Agricultural Biotechnology2019
References
[1] LI ZJ， YAO ZP， ZHANG J， et al. A review on fate and ecological toxicity of veterinary antibiotics in soil environments[J]. Asian Journal of Ecotoxicolog， 2008， 3（1）： 15-20. （in Chinese）
[2] CHAI JP. The value of alfalfa （Medicago sativa L.） and its planting method[J]. Modern Animal Husbandry Science & Technology， 2019（3）： 44-45. （in Chinese）
[3] ZHOU ML. Rational use of veterinary antibiotics to reduce public health hazards[J]. The Chinese Livestock and Poultry Breeding， 2013， 9（5）： 23-24. （in Chinese）
[4] HUANG YQ， CHEN Y， QIN X， et al. Pollution of residual drugs in the environment and its treatment[J]. Guangzhou Chemical Industry， 2019， 47（4）： 105-108， 153. （in Chinese）
[5] XIN SQ， GAO Y， ZHAO JM， et al. Effect of Seed Soaking in Spermidine（SPD）under Salt Stress on Rice Seed Germination[J]. ？North Rice， 2010， 40（6）： 23-25， 30. （in Chinese）
[6] XU QT， BAO CY， ZHANG MK. Effects of soil antibiotics pollution on seed germination and root elongation of three kinds of vegetables[J]. Acta Agriculturae Jiangxi， 2014， 26（9）： 37-43. （in Chinese）
[7] FENG JJ， WEN HY， MENG YQ， et al. Effects of three kinds of antibiotic stress on seed germination of cucumber， squash and wax gourd[J]. Seed， 2018， 37（2）： 86-89. （in Chinese）
[Methods] With alfalfa （Medicago sativa L.） as an experimental material and oxytetracycline， chlortetracycline and enrofloxacin as experimental contaminants， the effects of the three veterinary antibiotics on the germination percentage， germination energy， germination index and vigor index of alfalfa seeds and on the plant height and root length of alfalfa seedlings were study at the concentration gradient of 50， 100， 150， 200 and 250 mg/L， respectively.
[Results] The oxytetracycline treatments had no significant effect on the germination percentage of alfalfa seeds， but promoted its germination index. The chlortetracycline treatment solutions and enrofloxacin treatment solutions promoted the germination percentage， germination energy and germination index of alfalfa. The three antibiotics significantly inhibited the vigor index of alfalfa seeds， and had significant inhibitory effects on root length and plant height.
[Conclusions] These results provide an important basis for studying the ecotoxicity of veterinary antibiotics on pasture.
Key words Veterinary antibiotics; Alfalfa; Seed germination; Seedling growth; Ecotoxicity
With the development of animal cultivation industry and compound feed industry， veterinary antibiotics are used as animal feed additives， which are increasingly used in various animal cultivation industries. According to research， these antibiotics cannot be completely absorbed by animals， of which 40%-90% will be excreted in the form of feces， which will cause the unprocessed manure to carry residual antibiotics into the ecological environment to cause environmental pollution[1-3]. In addition， plant roots can enrich antibiotics in the soil， which can be then transported to the stems， leaves and fruits of plants， and eventually enter the food chain to bring about an negative impact that cannot be neglected on the health of humans and other organisms[3-4].
Alfalfa （Medicago sativa L.） is a leguminous plant known as the "king of the pasture". It is rich in crude protein， vitamins and other nutritive substances. It has high yield and excellent quality and is a high quality forage widely used for grazing[2]. Seed germination is an important stage of plant life history and the starting point of plant growth and development. The residual veterinary antibiotics in the soil directly affect the quality of seed germination and seedling growth. Therefore， it is important to study the effects of veterinary antibiotics on the seed germination and seedling growth of forage grass. In this study， 50， 100， 150， 200 and 250 mg/L oxytetracycline， chlortetracycline and enrofloxacin were used to treat the seeds of alfalfa， while the seeds were cultured in distilled water as a control， so as to investigate the effects of the three veterinary antibiotics on the seed germination and seedling growth of alfalfa. This study provides a scientific basis for studying the ecotoxicity of veterinary antibiotics and improving the quality of pasture in black land. Materials and Methods
Test materials
Forage seeds
The pasture seeds were alfalfa seeds.
Drugs
Oxytetracycline （purity 98%， Nanyang Veterinary Drug Factory）; chlortetracycline （purity 99%， Xionghui Pharmaceutical）; enrofloxacin （purity 98%， Beiliu Tongzhou Pharmaceutical Co.， Ltd.）.
Test instrument
Artificial climate box （Medical Equipment Factory， Shanghai Boxun Industry & Commerce Co.， Ltd.）.
Test methods
Test design
Full alfalfa seeds of the same size and normal color were disinfected with 75% ethanol solution for 10 min and rinsed three times with distilled water. The seeds were placed in disposable dishes laid with three layers of filter paper wetted with an equal volume of 0， 50， 100， 150， 200 and 250 mg/L of oxytetracycline， chlortetracycline and enrofloxacin solutions， respectively， according to 30 seeds per dish， which meant that the seeds were subjected to stress treatment， and each treatment was repeated 4 times. The seeds of different treatments were finally germinated in an incubator under the conditions of 14 h of illumination/25 ℃ and 10 h of darkness/20 ℃. The treatment solutions were changed every 12 h to ensure that the solution concentrations did not change. The germination of the seeds was observed and recorded every day， and it was determined that the seeds were germinated when the germ was just seen. After the germination， 5 seedlings were randomly selected from each dish to determine the root length and plant height.
Determination of parameters
Germination percentage GP=（Number of seeds that were normally germinated at the end of germination/Total number of seeds tested）×100%[5]
Germination energy GE=（Number of seeds germinated in the first 3 d/Total number of seeds）×100%
Germination index GI=∑（GT/Dt）
Vigor index VI=S×GI
Wherein GT is the number of seeds germinated in time t; Dt is the number of germination days; S is the growth amount of seedlings.
Data processing
Statistical analysis was performed using Excel 2007 and SPSS 13.0 software. One way ANOVA was used to analyze the differences between the parameters of different treatments， and multiple comparisons were performed by the Duncans method.
Results and Analysis
Effects of oxytetracycline treatment solutions on seed germination and seedling growth of alfalfa
It can be seen from Table 1 that compared with the control group， the oxytetracycline treatments were not significantly different in the germination percentage of alfalfa seeds， and promoted the germination index to different degrees， with the strongest promoting effect at the oxytetracycline concentration of 250 mg/L. Oxytetracycline showed significant inhibition on the vigor index of alfalfa seeds， with the strongest inhibitory effect at the oxytetracycline concentration of 200 mg/L， and the differences between different treatments were significant. The oxytetracycline treatments had inhibitory effects on the root length and plant height of alfalfa seedlings with significant differences， and the inhibition on roots was the highest when the concentration of oxytetracycline was 200 mg/L. Effects of chlortetracycline treatment solutions on seed germination and seedling growth of alfalfa
It can be seen from Table 2 that the germination percentage， germination energy and germination index of alfalfa seeds were promoted at low concentrations of chlortetracycline， and inhibited at high concentrations. With the increase of the concentration of chlortetracycline treatment， the germination percentage of alfalfa seeds increased first and then decreased. When the concentration of chlortetracycline was 50 mg/L， the promoting effect was the strongest and the germination percentage reached 100%. When the concentration of chlortetracycline reached 250 mg/L， the germination percentage of alfalfa seeds began to be inhibited， and the differences were obvious. Compared with the control group， the chlortetracycline treatment solution promoted the germination energy of alfalfa seeds at 50-200 mg/L， and began to inhibit at 250 mg/L. With the increase of chlortetracycline concentration， the germination index of alfalfa seeds increased first and then decreased， and reached the maximum value at 100 mg/L. Compared with the control group， the chlortetracycline treatment solutions had significant inhibitory effects on the seed vigor index of alfalfa， different degrees of promoting effect on the seedling height， and different degrees of significant inhibitory effect on the root length.
Effects of enrofloxacin treatment solutions on seed germination and seedling growth of alfalfa
As can be seen from Table 3， compared with the control group， the enrofloxacin treatment solutions promoted the germination percentage， germination energy and germination index of alfalfa seeds， and most promoted the germination percentage when the enrofloxacin concentration was 50 mg/L. With the increase of enrofloxacin concentration， the germination energy and germination index increased first and then decreased. The germination energy was most promoted at 50， 100 and 150 mg/L， and the promoting effect on the germination index was the strongest at 150 mg/L. Compared with the control group， the enrofloxacin treatment solutions had significant inhibitory effects on the seed vigor index and plant height of alfalfa， and the seed vigor index and seedling height of alfalfa were most inhibited at the enrofloxacin concentration of 150 mg/L. With the increase of enrofloxacin concentration， the root length of alfalfa seedlings gradually decrease， and compared with the control group， enrofloxacin inhibited its root length. Discussion and Conclusions
This study investigated the ecotoxicity of veterinary antibiotics against alfalfa by controlling the concentration of different antibiotics on alfalfa.
Different veterinary antibiotics had different effects on the germination of alfalfa seeds. Oxytetracycline had no significant effect on the germination percentage of alfalfa， while chlortetracycline and enrofloxacin could increase the germination percentage of alfalfa seeds within a certain concentration limit. The effect of chlortetracycline on the germination energy of alfalfa was promotion at low concentrations and inhibition at high concentrations; and the enrofloxacin treatment solutions increased the germination energy of alfalfa seeds.
The ecotoxic effects of the three veterinary antibiotics on alfalfa are mainly achieved by inhibiting seed vigor and plant growth. The root length， plant height and germination percentage of alfalfa are more sensitive to the ecotoxicity of antibiotics. The results showed that the oxytetracycline， chlortetracycline and enrofloxacin treatments reduced the vigor index of alfalfa seeds and the root length and plant height of the seedling plants. The 50 mg/L treatment solutions of the three antibiotics can make its vigor index， root length and plant height drop suddenly. It can be seen that the growth of roots and shoots of alfalfa is sensitive to antibiotics during germination， which might be related to the fact that antibiotics contact with roots and shoots once they break through the seed coat[6].
In summary， the effects of the three veterinary antibiotics on the germination percentage， germination energy and germination index of alfalfa seeds were relatively small. However， they had significant inhibitory effects on the root length and shoot growth of alfalfa seedlings， which is consistent with the effects of the stress of three antibiotics on the germination of cucumber， zucchini and melon seeds studied by Feng et al.[7].
Agricultural Biotechnology2019
References
[1] LI ZJ， YAO ZP， ZHANG J， et al. A review on fate and ecological toxicity of veterinary antibiotics in soil environments[J]. Asian Journal of Ecotoxicolog， 2008， 3（1）： 15-20. （in Chinese）
[2] CHAI JP. The value of alfalfa （Medicago sativa L.） and its planting method[J]. Modern Animal Husbandry Science & Technology， 2019（3）： 44-45. （in Chinese）
[3] ZHOU ML. Rational use of veterinary antibiotics to reduce public health hazards[J]. The Chinese Livestock and Poultry Breeding， 2013， 9（5）： 23-24. （in Chinese）
[4] HUANG YQ， CHEN Y， QIN X， et al. Pollution of residual drugs in the environment and its treatment[J]. Guangzhou Chemical Industry， 2019， 47（4）： 105-108， 153. （in Chinese）
[5] XIN SQ， GAO Y， ZHAO JM， et al. Effect of Seed Soaking in Spermidine（SPD）under Salt Stress on Rice Seed Germination[J]. ？North Rice， 2010， 40（6）： 23-25， 30. （in Chinese）
[6] XU QT， BAO CY， ZHANG MK. Effects of soil antibiotics pollution on seed germination and root elongation of three kinds of vegetables[J]. Acta Agriculturae Jiangxi， 2014， 26（9）： 37-43. （in Chinese）
[7] FENG JJ， WEN HY， MENG YQ， et al. Effects of three kinds of antibiotic stress on seed germination of cucumber， squash and wax gourd[J]. Seed， 2018， 37（2）： 86-89. （in Chinese）