Title Page

Contents

ABSTRACT 12

I. Introduction 15

II. Literature Review 19

1. Veterinary antibiotics 19

2. Usage of veterinary antibiotics 21

3. Entering routes of veterinary antibiotics in environment 24

4. Risks of veterinary antibiotics in environment 27

4.1. Antibiotic-resistant bacteria 27

4.2. Microbial toxicity 28

4.3. Phytotoxicity 28

4.4. Toxic effects on various aquatic organisms 29

5. Decomposition techniques of veterinary antibiotics 32

6. Electron ionizing energy 35

III. Materials and Methods 40

1. Materials 40

2. Methods 42

2.1. Preparation of veterinary antibiotics and irradiation with electron ionizing energy 42

2.2. Analysis of veterinary antibiotics 42

2.3. Analysis of total organic carbon (TOC), anion and radicals 45

2.4. Microbial susceptibility of veterinary antibiotics 46

2.5. GR50 values and phytotoxicity of veterinary antibiotics on Oryza sativa L.(이미지참조) 49

IV. Results and Discussion 52

1. Degradation of veterinary antibiotics in aqueous solutions by irradiation with electron ionizing energy 52

2. Changes of total organic carbon (TOC) in aqueous solutions of veterinary antibiotics by irradiation with electron ionizing energy 61

3. Production of reactive radicals from the aqueous solutions of veterinary antibiotics by irradiation with electron ionizing energy 66

3.1. Hydroxyl radical (·OH) 67

3.2. Hydrated electron(e-aq)(이미지참조) 69

3.3. Hydrogen peroxide (H₂O₂) 70

4. Decomposition mechanism of veterinary antibiotics by irradiation with electron ionizing energy 77

4.1. Chloramphenicol 77

4.2. Ciprofloxacin 85

4.3. Oxytetracycline 94

5. Changes of microbial susceptibility of veterinary antibiotics by irradiation with electron ionizing energy 102

6. GR50 values and phytotoxicity of veterinary antibiotics on Oryza sativa L.(이미지참조) 111

6.1. GR50 values of veterinary antibiotics on Oryza sativa L.(이미지참조) 111

6.2. Phytotoxicity of veterinary antibiotics on Oryza sativa L. 119

V. Conclusion 127

VI. References 130

국문초록 147

Table 1. Usage of veterinary antibiotics in Korea 23

Table 2. The effects of antimicrobial growth promoter in relation to some broad issues of animal production 31

Table 3. Product yield in water radiolysis 39

Table 4. The characteristics of veterinary antibiotics used in this study. 41

Table 5. The summary of LC/MS analytical methods. 44

Table 6. The composition of the nutrient solution for hydroponic culture 51

Fig. 1. Routes of veterinary antibiotics entering the aquatic environment 26

Fig. 2. Radiolysis of water molecule 38

Fig. 3. Manual disk placement template for five disks on the plate. 48

Fig. 4. The chromatograms of chloramphenicol after irradiation with electron ionizing energy. 55

Fig. 5. The degradation rates of chloramphenicol by irradiation with electron ionizing energy. 56

Fig. 6. The chromatograms of ciprofloxacin after irradiation with electron ionizing energy. 57

Fig. 7. The degradation rates of ciprofloxacin by irradiation with electron ionizing energy. 58

Fig. 8. The chromatograms of oxytetracycline after irradiation with electron ionizing energy. 59

Fig. 9. The degradation rates of oxytetracycline by irradiation with electron ionizing energy. 60

Fig. 10. The removal efficiencies of TOC in aqueous solution of chloramphenicol by irradiation with electron ionizing energy. 63

Fig. 11. The removal efficiencies of TOC in aqueous solution of ciprofloxacin by irradiation with electron ionizing energy. 64

Fig. 12. The removal efficiencies of TOC in aqueous solution of oxytetracycline by irradiation with electron ionizing energy. 65

Fig. 13. The variation of hydroxyl radical (·OH) in aqueous solutions of veterinary antibiotics after irradiation with electron ionizing energy. 72

Fig. 14. EPR spectra of 4-POBN spin adducts in aqueous solutions of veterinary antibiotics after irradiation with electron ionizing energy. 73

Fig. 15. The variation of nitrate ion (NO3-), nitrite ion (NO2-) and chloride ion (Cl-) in aqueous solution of chloramphenicol after irradiation with electron ionizing energy.(이미지참조) 74

Fig. 16. The variation of hydrated electron (e-aq) in aqueous solutions of veterinary antibiotics after irradiation with electron ionizing energy.(이미지참조) 75

Fig. 17. The variation of hydrogen peroxide (H₂O₂) in aqueous solution of veterinary antibiotics after irradiation with electron ionizing energy. 76

Fig. 18. The enlarged chromatogram of chloramphenicol after 5 kGy irradiation with electron ionizing energy. 79

Fig. 19. LC/MS spectra for major products (CAP1, CAP2 and CAP3) in aqueous solution of chloramphenicol after 5 kGy irradiation with electron ionizing energy. 80

Fig. 20. LC/MS/MS spectra and proposed bond cleavages of CAP1 ([M+H] m/z 307.1). 81

Fig. 21. LC/MS/MS spectra and proposed bond cleavages of CAP2 ([M+H] m/z 291.1). 82

Fig. 22. LC/MS/MS spectra and proposed bond cleavages of CAP3 ([M+H] m/z 321.1). 83

Fig. 23. Proposed degradation products of chloramphenicol by irradiation with electron ionizing energy. 84

Fig. 24. The enlarged chromatogram of ciprofloxacin after 5 kGy irradiation with electron ionizing energy. 87

Fig. 25. LC/MS spectra for major products (CFX1, CFX2 and CFX3) in aqueous solution of ciprofloxacin after 5 kGy irradiation with electron ionizing energy. 88

Fig. 26. LC/MS/MS spectra and proposed bond cleavages of CFX1 ([M+H] m/z 330.1). 89

Fig. 27. The variation of fluoride ion (F-) in aqueous solution of ciprofloxacin after irradiation with electron ionizing energy.(이미지참조) 90

Fig. 28. LC/MS/MS spectra and proposed bond cleavages of CFX2 ([M+H] m/z 314.1). 91

Fig. 29. LC/MS/MS spectra and proposed bond cleavages of CFX3 ([M+H] m/z 263.1). 92

Fig. 30. Proposed degradation products of ciprofloxacin by irradiation with electron ionizing energy. 93

Fig. 31. The enlarged chromatogram of oxytetracycline after 1 kGy irradiation with electron ionizing energy. 96

Fig. 32. LC/MS spectra for major products (OTC1, OTC2 and OTC3) in aqueous solution of oxytetracycline after 1 kGy irradiation with electron ionizing energy. 97

Fig. 33. LC/MS/MS spectra and proposed bond cleavages of OTC1 ([M+H] m/z 447.1). 98

Fig. 34. LC/MS/MS spectra and proposed bond cleavages of OTC2 ([M+H] m/z 433.2). 99

Fig. 35. LC/MS/MS spectra and proposed bond cleavages of OTC3 ([M+H] m/z 415.2). 100

Fig. 36. Proposed degradation products of oxyetracycline by irradiation with electron ionizing energy. 101

Fig. 37. The inhibition rates of microbial activity on chloramphenicol by irradiation with electron ionizing energy. 105

Fig. 38. The comparison of microbial sensitivities on chloramphenicol by irradiation with electron ionizing energy. 106

Fig. 39. The inhibition rates of microbial activity on ciprofloxacin by irradiation with electron ionizing energy. 107

Fig. 40. The comparison of microbial sensitivities on ciprofloxacin by irradiation with electron ionizing energy. 108

Fig. 41. The inhibition rates of microbial activity on oxyteracycline by irradiation with electron ionizing energy. 109

Fig. 42. The comparison of microbial sensitivities on oxytetracycline by irradiation with electron ionizing energy. 110

Fig. 43. The changes on shoot length of Oryza sativa L. after 0, 3 and 7 days treatment with chloramphenicol. 113

Fig. 44. The variation on shoot length of Oryza sativa L. after 7 days treatment with chloramphenicol. 114

Fig. 45. The changes on shoot length of Oryza sativa L. after 0, 3 and 7 days treatment with ciprofloxacin. 115

Fig. 46. The variation on shoot length of Oryza sativa L. after 7 days treatment with ciprofloxacin. 116

Fig. 47. The changes on shoot length of Oryza sativa L. after 0, 3 and 7 treatment with oxytetracycline. 117

Fig. 48. The variation on shoot length of Oryza sativa L. after 7 days treatment with oxytetracycline. 118

Fig. 49. The changes on shoot length of Oryza sativa L. after 0, 3 and 7 days treatment with chloramphenicol GR50 (1,000㎎ L-1) irradiated electron ionizing energy.(이미지참조) 121

Fig. 50. The variation on shoot length of Oryza sativa L. after 7 days treatment with chloramphenicol GR50 (1,000㎎ L-1) irradiated electron ionizing energy.(이미지참조) 122

Fig. 51. The changes on shoot length of Oryza sativa L. after 0, 3 and 7 days treatment with ciprofloxacin GR50 (250 ㎎ L-1) irradiated electron ionizing energy.(이미지참조) 123

Fig. 52. The variation on shoot length of Oryza sativa L. after 7 days treatment with ciprofloxacin GR50 (250 ㎎ L-1) irradiated electron ionizing energy.(이미지참조) 124

Fig. 53. The changes on shoot length of Oryza sativa L. after 0, 3 and 7 days treatment with oxytetracycline GR50 (350 ㎎ L-1) irradiated electron ionizing energy.(이미지참조) 125

Fig. 54. The variation on shoot length of Oryza sativa L. after 7 days treatment with oxytetracycline GR50 (350 ㎎ L-1) irradiated electron ionizing energy.(이미지참조) 126

본 연구에서는 가축에 대한 사용량이 많거나 위해성이 높은 동물용 의약품 중 인체 발암 가능 물질로 사용 금지되었으나 환경 중에 검출되고 있는 chloramphenicol, 인간과 동물에 널리 사용되고 있는 ciprofloxacin 및 동물용 의약품 중 사용량이 많고 배출계수가 높은 oxytetracycline 을 제거하기 위하여 전자이온화에너지를 조사한 후 분해율과 분해산물을 확인하였다. 또한 동물용 의약품의 분해산물이 미생물과 식물에 미치는 영향을 살펴보고, 전자이온화에너지 조사기술이 수중 동물용 의약품 제거에 활용 가능성을 평가하고자 하였다.

1. 동물용 의약품 (100 ㎎ L-1)에 전자이온화에너지 1, 5 그리고 10 kGy 선량으로 조사한 결과, chloramphenicol 은 각각 32.5, 86.9 그리고 100%, ciprofloxacin 은 각각 38.0, 80.4 그리고 97.3%, oxytetracycline 은 각각 72.2, 99.2 그리고 100%가 제거되었다. Chloramphenicol 과 ciprofloxacin 은 선량에 따른 분해율이 비슷하였으며, oxytetracycline 은 1 kGy 에서도 분해율이 높았고 5 kGy 에서는 대부분 제거되었다. 따라서 전자이온화에너지는 수중 동물용 의약품을 제거하는데 효과적인 것으로 판단된다.

2. 전자이온화에너지 10 kGy 조사시 chloramphenicol, ciprofloxacin 및 oxytetracycline 은 대부분 제거된 반면, 총유기탄소는 각각 17.1, 53.1 그리고 28.3% 감소하는 것으로 나타났다. 이는 안정된 구조를 가지고 있는 항생물질이 전자이온화에너지에 의해 C0₂, H₂O 그리고 NH₃와 같은 저분자 물질로 무기화되기 보다는 다른 유기화합물로 전환되었음을 의미한다.

3. 동물용 의약품 수용액에 전자이온화에너지를 조사한 결과, hydrogen radical (·OH), hydrate electron (e-aq) 및 hydrogen peroxide (H₂O₂)이 생성되었다. 반응성이 가장 높은 radical 인 hydroxyl radical (·OH)과 분자생성물인 hydrogen peroxide (H₂O₂)는 chloramphenicol 에서 가장 많이 생성되었으며, hydrate electron (e-aq)는 ciprofloxacin 에서 높게 나타났다. 생성된 라디칼 모든 물질에서 유사한 수준이었다.

4. 전자이온화에너지를 조사한 후 LC/MS 와 LC/MS/MS 분석 결과, chloramphenicol 은 CAP1([M+H]m/z307.1), CAP2([M+H]m/z291.1), 및 CAP3([M+H]m/z321.1)의 분해산물을 생성하였다. Ciprofloxacin 은 CFX1([M+H]m/z330.2), CFX2([M+H]m/z314.2), 및 CFX3([M+H]m/z263.1)의 분해산물을, oxytetracycline 은 OTC1([M+H]m/z447.2), 0TC2([M+H]m/z433.2) 및 0TC3([M+H]m/z415.2)의 분해산물을 생성하였다.

5. 동물용 의약품 수용액에 전자이온화에너지를 조사한 후 Escherichia coli, Pseudomonas putida 그리고 Bacillus subtilis에 대한 활성을 비교한 결과, 미생물에 대한 활성 감소율은 각각의 의약품의 분해율과 유사하였다. 또한 동물용 의약품이 분해되면서 생성된 분해산물이 본 연구에서 대상으로 하는 미생물에 대해 독성은 나타나지 않았다.

6. 동물용 의약품이 벼의 지상부 길이 생장에 50% 영향을 끼치는 농도 (GR50)를 조사한 결과, chloramphenicol, ciprofloxacin 그리고 oxytetracylcine 에 대한 GR50 값은 각각 1,000, 250 그리고 350 ㎎ L-1로 나타났다. 또한 GR50 값을 기준으로 하여 전자이온화에너지를 조사한 후 벼의 생육을 조사한 결과, 전자이온화에너지가 조사된 GR50 값 처리구의 벼 지상부 길이 생장은 전자이온화에너지가 조사되지 않은 GR50 값 처리구보다 약간 증가하였다. 이것은 높은 농도의 동물용 의약품이 전자이온화에너지 10 kGy 에서 완전히 제거되지 않아 잔류된 동물용 의약품에 의하여 벼의 생장이 저해된 것으로 판단된다. 따라서 높은 농도의 동물용 의약품을 제거하기 위해서는 더 높은 선량의 전자이온화에너지가 필요할 것으로 판단된다.

본 연구 결과, 전자이온화에너지는 수중 동물용 의약품 제거에 효과적인 것으로 나타났다. 따라서 전저이온화에너지 조사기술은 수생태계에 존재하는 동물용 의약품 처리에 유용할 것으로 판단된다.