歐索林酸在石斑魚、海鱺及甲魚之殘留研究

Abstract

本研究分三部分探討石斑魚、海鱺及甲魚之殘留停藥期。（1）石斑魚以口服單次投藥，歐索林酸( Oxoinic acid, OXO )在石斑魚血液、肌肉、肝臟及腎臟之半衰期，並以多重劑量投藥，探討OXO之殘留停藥期試驗。（2）海鱺以OXO口服30 mg/kg 及其倍數劑量（60 mg/kg）連續投藥5天後，探討OXO之殘留停藥期。（3）甲魚以OXO口服30 mg/kg 及其倍數劑量（60 mg/kg）連續投藥5天後，探討OXO之殘留停藥期。 石斑魚半衰期試驗係以單一劑量口服灌食投予OXO 60 mg/kg b.w.，投藥後第0.5小時開始在不同時間點陸續採樣，經由高效液相層析儀（high performance liquid chromatography, HPLC）搭配螢光偵測器檢測石斑魚組織中殘留量，本分析方法檢量線線性範圍為2.5~1000 ng/mL，線性迴歸方程式 Ŷ = 0.0704，其相關係數R2為 0.999。平均回收率為89.9  1.64 %。在本試驗的分析條件下，最低偵測極限（limit of detection, LOD）為3 ng/ mL（ppb），最低定量極限（limit of quantitation, LOQ）為5 ppb。分析的結果顯示，OXO在石斑魚血液、肌肉、肝臟及腎臟中的濃度在口服後4小時皆達到最高峰，其平均濃度分別為 0.59、1.76、2.18及3.61 μg/mL（ppm），在魚組織中檢測出來的濃度與時間的趨勢，在腎臟濃度為最高，其次分別為肝臟及肌肉，而血液中濃度最低。在石斑魚之血液、肌肉、肝臟及腎臟中的半衰期則為 136小時、131小時、158小時及110 小時。石斑魚殘留試驗則以60 mg/kg b.w. OXO，連續5天多劑量投予後，第1小時開始在不同時間點採樣，經由高效液相層析儀分析結果顯示，OXO在石斑魚之血液、肌肉、肝臟及腎臟在口服投藥結束後依序分別在第 4 天、6天、10天、10天，其平均濃度皆已低於歐盟動物用藥殘留標準的殘留容許量（0.1 ppm），其停藥期訂為15天。 海鱺以30 mg/kg 及 60 mg/kg之OXO劑量分成2組添加至飼料，連續5天，每天一次投予後停藥，且於停藥後第1、3、5、7、10、14、21天採樣，以液相層析串聯質譜儀 ( LC-MS/MS )分析組織中殘留量，本分析方法以OXO添加至肌肉及肝臟之平均回收率分別為95.8 %及86.55 %、肌肉及肝臟之線性迴歸方程式分別為Ŷ = 115139.57 X + 455100.54（R2=0.9989）及 Ŷ = 25239.75X — 37040.26（R2= 0.9992）。海鱺在低劑量組（30 mg/kg）與高劑量組（60 mg/kg）之帶皮肌肉及肝臟在投藥後第24小時平均濃度均達高峰依低、高劑量組海鱺之前揭組織順序分別為62  11.7、64  20.6 ng/g（ppb）與202  44.5、264  95.4 ppb。低劑量組不論帶皮肌肉或肝臟在投藥後第5天之濃度均低於殘留容許標準0.05 ppm，然而高劑量組之帶皮肌肉則於投藥後第7天及肝臟在投藥後第10天，其殘留濃度始低於殘留容許標準。因此建議停藥期為15天（殘留期間10天加0.5倍安全期間）。 甲魚以30 mg/kg 及 60 mg/kg之OXO劑量分成2組添加至飼料，連續5天，每天一次投予後停藥，且於停藥後第1、2、4、8、12、16、20、24、28、32、36、40、48天採樣，以高效液相層析儀（HPLC）搭配光二極體陣列檢出器分析組織中殘留量，本分析方法檢量線線性範圍0.01-10 ppm，在帶皮肌肉及肝臟之平均回收率分別為75.04% 及80.80%，線性迴歸方程式Ŷ=22233X–3495，其相關係數R2=0.997。在本試驗的分析條件下，LOD為0.03 ppm。甲魚在低劑量組（30 mg/kg）與高劑量組（60 mg/kg）之帶皮肌肉及肝臟在投藥後第1天平均濃度均達高峰，依低、高劑量組甲魚之前揭組織順序分別為3.01±0.15、3.550.27 μg/g (ppm)與5.710.58、5.210.72 ppm。低劑量組不論帶皮肌肉或肝臟在投藥後第36天之濃度均低於殘留容許標準0.1 ppm，然而高劑量組之帶皮肌肉則於投藥後第44天及肝臟在投藥後第48天，其殘留濃度始低於殘留容許標準。因此建議停藥期為72天（殘留期間48天加0.5倍安全期間）。 總之，本試驗研究結果顯示建議石斑魚、海鱺及甲魚之殘留停藥期，分別為15天、15天及72天。提供給政府制定停藥期參考依據。有鑒於水產動物係變溫動物生理代謝特殊，且受水溫、水質與鹽度及給藥途徑等多重因素影響，投藥時獸醫師應依專業調整用法、用量及停藥期，以確保水產動物安全。

This study was conducted to evaluate the depletion of residues of the antibiotics oxolinic acid (OXO) after administration to three cultured species, orange-spotted grouper Epinephelus coioides, cobia (Rachycentron canadum), and softshell turtle (Pelodiscus sinensis). The elimination half-life and residue study of OXO in grouper was assessed in single-oral at dose of 60 mg/kg bw and was conducted in multi-oral administration at the same dose treated once daily after 5 consecutive days. The other two studies were established an adequate depletion period of OXO in cobia and softshell turtle by determining the residues of OXO post feeding with multiple doses. The dosages of OXO were applied via oral administration at the dose of 30 and 60 mg/kg bw for 5 consecutive days. Samples for the elimination half-life study and for the residue study were collected at various time points after cessation of treatments by determining OXO concentrations with a validated high-performance liquid chromatography-fluorescence detection method (HPLC-FLD) in grouper, liquid chromatography tandem mass spectrometry (LC-MS/MS) in cobia and HPLC coupled with photodiode array detector (HPLC-DAD) in softshell turtle. The methodology showed very good linear relationship and linear regression equation R2˃ 0.998, and the mean recovery rate ˃ 75%. Also, methods showed that the detection of limit (LOD), the quantitation limit (LOQ) in HPLC-FLD, LC-MS/MS and HPLC-DAD were 3 and 5 ng/mL（ppb）, 0.006 and 0.01 μg/mL（ppm）, 0.03 and 0.1 ppm , respectively. In elimination half-life study in grouper, the results showed that the concentration of OXO in blood, muscle, liver and kidney of grouper reached the highest peak after 4 hours of oral administration, with average concentrations of 0.59, 1.76, 2.18 and 3.61 μg/mL, respectively. The trend of concentration and time detected in fish tissue was the highest in the kidney, followed by the liver and muscle, and the lowest in the blood. The elimination half-lives of grouper blood, muscle, liver and kidney were 136, 131, 158 and 110 hours. The grouper residue study showed that OXO in grouper blood, muscle, liver and kidney after the oral administration were completed on the 4, 10, 10, and 10th days, respectively. The mean concentration were lower than the residue tolerance of the EU residual standard (0.1 ppm), the conclusive approach would then result in a withdrawal period of 15 days as in the present study. In cobia residue study after the 30 and 60 mg/kg dose, results showed muscle with skin and liver samples at 24 h following termination of medication had a mean peak (n = 5) concentrations of 62  11.7, 64  20.6 ng/g (ppb) (30 mg/kg), and 202  44.5, 264  95.4 ppb (60 mg/kg), respectively. The residue OXO concentrations decreased to below the maximum residue limits (MRLs) of 0.05 ppm on the 5th day in muscle with skin and liver after the 30 mg/kg dose, while it occurred on the 10th day in muscle with skin and the 10th day in liver, respectively, after last 60 mg/kg dose. The results from this study suggested that the recommended withdrawal period was 15 days. In softshell turtle residue study after the 30 and 60 mg/kg dose, results showed muscle with skin and liver samples at 24h following termination of medication had a mean peak (n=6) concentrations of 3.01  0.15, 3.55  0.27 μg/g (30 mg/kg), and 5.71  0.58, 5.21  0.72 μg/g (60 mg/kg), respectively. The residue OXO concentrations decreased to below the MRLs of 0.1 ppm on the 36th day in muscle with skin and liver after the 30 mg/kg dose, while it occurred on the 44th day in muscle with skin and the 48th day in liver, respectively, after last 60 mg/kg dose. The results from this study suggested that the recommended withdrawal period was 72 days. Residue depletion of OXO in reptiles is much more slowly than other aquatic animals studied. Therefore, the depletion behavior of OXO in softshell turtle should be used on destined for human consumption, and they should be used cautiously and under veterinary medicines guidance. In conclusion, depletion studies were conducted the time for OXO concentrations to fall below 100 ppb (the current tolerance set by the European Medicines Agency (EMA) among fish species establishing an adequate depletion period), and in further considering the MRLs of 50 ppb established by the Taiwan FDA for OXO in muscle with skin in natural proportions. These results provide information on tissue depletions of OXO residues in farmed fish and turtles, the withdrawal period of 15 days in grouper and cobia and 72 days in softshell turtle were calculated on the basis of a safety span (corresponds to 50% of the time point when at which residues fall below the MRL) added to the depletion time. The depletion behavior we have detailed in this work suggests withdrawal times with reference to human consumption of treated fish, to establish policy guidelines and basic principles with regard to the use of OXO for fish farming, and to fish-farmers for the proper handling to ensure safe fish that the consumer will not be at risk.