Comparative pharmacokinetics of oxytetracycline in tilapia (Oreochromis spp.) maintained at three different salinities

Aquaculture 495 (2018) 675–681

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Comparative pharmacokinetics of oxytetracycline in tilapia (Oreochromis spp.) maintained at three different salinities

T

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Pritam K. Sidhua, , Stephen A. Smithb, Corinne Mayerb, Geraldine Magnina, David D. Kuhnc, Majid Jaberi-Dourakia,d, Johann F. Coetzeea a

College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA c Department of Food Science and Technology, Virginia Tech, Blacksburg, VA, USA d College of Art and Sciences, Kansas State University, Manhattan, KS, USA. b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tilapia Oxytetracycline Pharmacokinetics Fresh water Brackish water Salt water

Environmental factors, such as temperature, pH, and salinity of water may affect the pharmacokinetics (PK) of a drug in aquatic animals and in most instances water salinity is ignored in PK studies. This study compared PK profiles of oxytetracycline (OTC) following a single oral dosage of 50 mg/kg in tilapia (Oreochromis spp.) maintained in three aquatic environments: freshwater (FW: 0 ppt salinity), brackish water (BW: 15 ppt salinity) and salt water (SW: 30 ppt salinity). Water quality parameters between the three systems were similar except salinity. Following OTC administration, blood samples were collected at 23 time points: 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24 h, and 2, 4, 6, 8, 10, 12, 14, 18, 22, 26, 30, 34, 38 and 42 days. At each sampling time, six fish from each group were netted, sedated with buffered MS-222, bled and then euthanized. The OTC was extracted from plasma by Solid Phase Extraction (SPE) and analyzed by Ultra High Pressure Liquid Chromatography coupled by as tandem quadrupole mass spectrometer. The plasma concentration versus time data of OTC for the FW, BW and SW tilapia were subjected to PK analysis using non-compartment methods. Pharmacokinetics of OTC was characterized by rapid absorption and slow excretion in the FW and BW tilapia. Compared to the FW and BW groups, absorption and elimination of OTC was faster in the SW tilapia. The AUC0-∞ of OTC was in order of FW (165 h.μg/mL) > BW (145 h.μg/mL) > SW (55.5 h.μg/mL) group. In SW tilapia, terminal half-life (69 h) of OTC was > 2 times shorter than FW (177 h) and BW (155 h) groups. However, AUCs and terminal half-lives of the FW and BW groups were not significantly different. The study indicated that rise in water salinity level increases clearance of OTC in tilapia. It is suggested that OTC residues in tissues will not be the same in tilapia maintained at different water salinity levels. The results confirmed that infectious diseases associated with bacteria having a MIC of 0.5–1.0 μg/mL can be treated with the 50 mg/kg dosage of OTC in the FW and BW group, but the same dosage in the SW tilapia may lead to therapeutic failure and increased risk of resistance emergence.

1. Introduction Aquaculture is one of the largest growing sectors around the world to supply food, with an average annual growth rate of 6.3% since 2000 (average 8.8% per year between 1980 and 2010) and currently accounts for about 47% of the world's fish supply (FAO, 2012). Fish living in the wild as well as reared in the aquaculture facilities are susceptible to infectious diseases. Bacterial diseases are a major limitation to productivity leading to significant economic losses by causing mortality up to 100% in the aquaculture industry (Tran et al., 2013). Threat of infectious diseases is on the rise, occurrence of infectious disease

⁎

outbreaks wiping out entire stocks have been reported in farmed fish (Pulkkinen et al., 2010; Leung and Bates, 2013). For instance, bacterial infections in the United States caused 60% mortalities in catfish, while 30% of mortalities were a result of parasitic infestations, 9% of mortalities from fungal infections, and 1% of mortalities from viral etiologies (MSU, 2010). Bacteria causing infections in fish include; Gramnegative: Aeromonas hydrophila, Aeromonas salmonicida, Flavobacterium columnare, Vibrio spp., and Pseudomonas spp.; and Gram-positive: Streptococcus spp. and Mycobacterium spp. (Austin and Austin, 1999). Treating bacterial infections in fish is one of the toughest challenges due to limited choices of antimicrobials available for aquaculture use

Corresponding author at: P222A, Mosier Hall, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA. E-mail address: [email protected] (P.K. Sidhu).

https://doi.org/10.1016/j.aquaculture.2018.06.044 Received 16 May 2018; Received in revised form 11 June 2018; Accepted 17 June 2018 Available online 19 June 2018 0044-8486/ © 2018 Elsevier B.V. All rights reserved.

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Institutional Animal Care and Use Committee of Virginia Tech. Four hundred and fifty juvenile tilapia (Oreochromis spp.) were obtained from a commercial aquaculture facility (Blue Ridge Aquaculture, Martinsville, VA, USA) and maintained at the Conservation Aquaculture Facility at Center Woods, Virginia Tech. The fish were placed in a 500gal recirculation system and acclimated for 2 weeks. The system was fitted with a mechanical filter for solids removal, a bio-filter for nitrification, and air stones for aeration and carbon dioxide removal. During this time, water quality parameters monitored on a daily basis were dissolved oxygen and temperature; while ammonia, nitrite, nitrate, alkalinity and pH were monitored three times a week using a commercial aquaculture kit (HACH Company, Loveland, CO, USA). A standard pelleted tilapia diet containing 36% protein and 6% fat (Zeigler Brothers, Inc., Gardners, PA, USA) was fed to fish at a rate of 3% body weight per day. The feed was delivered continuously over a period of 12 h using an automated 24 h belt feeder (Pentair AES, Apopka, FL, USA). After 2 weeks of acclimation, fish were arbitrarily divided into three groups of 138 fish and placed into three separate recirculation systems each of which was fitted with a mechanical filter for solids removal, a bio-filter for nitrification, and air stones for aeration and carbon dioxide removal (Table 1). As demonstrated in the past, activated carbon filter material was also installed in the filter of each system to bind any free OTC or OTC metabolites excreted into the water column by the fish (Hughes, 2003). Total fish acquired were 450 (138 × 3groups = 414 + 36 = 450) because an additional 36 fish were included to account for any transportation mortality and/or unacceptable small sized fish.

and no chemotherapeutic agent is approved specifically for tilapia. Antibacterial drugs approved by the US Food and Drug Administration for use in food fish include oxytetracycline, sulfadimethpoxine/ormetoprim, sulfamerazine and florfenicol (Smith et al., 2008). Oxytetracycline (OTC) is one of the most widely used antimicrobials in the livestock industry due to its broad-spectrum activity, good penetration into body fluids and tissues, low cost and low toxicity risk. It binds to the 30S ribosomal subunit of susceptible microbes to produce antimicrobial activity. Upon binding, the OTC interferes with transfer RNA's ability to bind with messenger RNA, thereby preventing bacterial protein synthesis (Rivere and Spoo, 1995). In the US, OTC has been approved to treat bacterial hemorrhagic septicemia (Aeromonas liquefaciens) and pseudomonas (Pseudomonas spp.) infection in catfish, ulcer disease (Hemophilus piscium), furunculosis (Aeromonas salmonicida), bacterial hemorrhagic septicemia (A. liquefaciens), and pseudomonas disease (Pseudomonas spp.) in salmonids, cold-water disease (Flavobacterium psychrophilum) in freshwater reared salmonids, columnaris disease (F. columnare) in rainbow trout, and gaffkemia (Aerococcus viridians) in lobsters (FDA Approved Aquaculture Drugs, 2018). Limited approval of OTC is partly due to the lack of data pertaining to elimination kinetics, target animal efficacy and safety in species other than salmonids and catfish. Off-label use of OTC in feed has historically been used to treat infections such as flavobacteriosis in common carp (Cypinus carpio) and grass carp (Ctenopharyngodon idella); furunculosis in coho salmon (Oncorhynchus kisutch); and columnaris and streptococcosis in rainbow trout (Oncorhynchus mykiss) (Treves-Brown, 2000). Despite this widespread use, information on the pharmacokinetics (PK) and pharmacodynamics (PD) of OTC is very limited for farmed fish species. Pharmacokinetics of OTC have been studied in African catfish (Grondel et al., 1989), Atlantic salmon (Elema et al., 1996), chinook salmon (Abedini et al., 1998; Namdari et al., 1998), carp (Grondel et al., 1987), rainbow trout (Black et al., 1991; Bjorklund and Bylund, Björklund and Bylund, 1991; Abedini et al., 1998), red pacu (Dio et al., 1998), summer flounder (Hughes, 2003), tench (Reja et al., 1996) and yellow perch (Bowden, 2001). Tilapia is a popular farmed fish species in the US and > 75% of the annual production is supplied by recirculating systems (Zajdband, 2012). Despite this, no studies on the PK of OTC have been conducted in tilapia (Oreochromis spp.) except tissue depletion studies in which residues of OTC at a limited number of time points were determined in serum of tilapia following in feed drug administration (Chen et al., 2004; Paschoal et al., 2012). Thus, for off-label usage in tilapia dosing regimens are often extrapolated from other species that may lead to therapeutic failure and antibiotic resistance. In fish, extrapolation is not advised because of excessive PK variability between species, route of administration and formulation of drugs. Moreover, in aquatic animals, the PK of a drug can be affected by environmental factors, such as temperature, pH, and salinity of water in which the animals are raised (Rigos and Smith, 2013). The tilapia can be and are grown in freshwater, brackish water and seawater around the world, but therapeutic compounds are currently approved for a particular species of fish, regardless of the salinity in which they are maintained. Our hypothesis is that a single therapeutic recommended dose of OTC may not be appropriate for the different environmental salinities and no study comparing the PK of OTC at different water salinity levels in the same species of fish has been found in literature. Therefore, the purpose of this study was to compare PK profile of OTC following oral administration in tilapia (Oreochromis spp.) maintained in water with different salinity levels to determine the effect of salinity on the metabolism of the drug.

2.2. Treatment groups The three separate recirculation systems were arbitrarily assigned to one of the three experimental aquatic environments: freshwater (FW: 0 ppt salinity), brackish water (BW: 15 ppt salinity) and salt water (SW: 30 ppt salinity) (Table 1). The fish in the BW and SW systems initially started in freshwater (0 ppt) followed by a slow increase in water salinity adjusting to a final salinity of their respective group (15 ppt or 30 ppt) over 2 weeks by adding synthetic marine salts (Instant Ocean, Blacksburg, VA, USA) to each system. The BW fish arrived at their final salinity in 5 days, while 11 days elapsed for the SW fish to arrive at salinity of 30 ppt as salinities were not increased > 3 ppt over a 24 h period. In each of the three systems fish were allowed to acclimate to their respective final salinities for an additional 3 weeks prior to initiation of the PK study. Table 1 Experimental design of oxytetracycline pharmacokinetic study in FW, BW and SW tilapia. Treatment groups

Water salinity (ppt) Water temperature (°F) - mean (SD) Number of tilapia per group (n) Number of sample times collected Weight (g) of tilapia (n = 138) Dose of OTC by oral gavage Mortality during experiment

2. Material and methods 2.1. Animals The experiments were conducted according to the guidelines of

Freshwater (FW)

Brackish water (BW)

Salt water (SW)

0 78.8 (1.2)

15 78.9 (1.1)

30 78.3 (1.3)

23 × 6 = 138

23 × 6 = 138

23 × 6 = 138

23

23

23

Mean = 123.6 Min = 86 Max = 171 50 mg/kg

Mean = 123.7 Min = 91 Max = 168 50 mg/kg

Mean = 118.7 Min = 87 Max = 178 50 mg/kg

0

0

0

Mean = Mean weight; Min = minimum weight; Max = maximum weight. 676

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1.2 min A (90% for 0.8 min) making total run time of 2 min. The flow rate was set at 0.6 mL/min and the injection volume was 2 μL. The autosampler temperature was maintained at 4 °C and the column temperature at 40 °C.

2.3. Dosing and sampling Oxytetracycline (Bio-Mycin 200; 200 mg/mL; Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO, USA) was administered to fish by oral gavage at the dosage of 50 mg/kg after a 24 h fasting period prior to dosing. The drug was administered using a curved stainless steel 20gauge 3″ gavage tube (Popper and Sons, Inc., New Hyde Park, NY, USA) attached to a 100 μL Hamilton syringe. Gavage tube placement in the stomach was confirmed manually in the posterior portion of the stomach. At the start of the study (0 time), 6 fish from each group were netted, sedated with sodium bicarbonate buffered (1:1) tricaine methanesulfonate (150 μg/L, MS-222, Sigma Scientific, St. Louis, MO, USA) and bled. Blood was collected from the caudal tail vessels using a 23-gauge needle and syringe, and after sample collection fish were euthanized with buffered MS-222 (250 μg/L) followed by cervical separation to insure death. Following OTC administration, blood samples (~1 mL) were collected at 23 time points: 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24 h, and 2, 4, 6, 8, 10, 12, 14, 18, 22, 26, 30, 34, 38 and 42 days. At each sampling time, six fish from each group were netted, sedated with buffered MS-222, bled and then euthanized. Blood samples were immediately placed in individual plasma separator tubes containing lithium heparin (BD Microtainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), mixed by inversion several times and kept on ice until centrifugation at 3000 ×g for 10 min at 12 °C. Plasma samples were separated and placed in individual micro-centrifuge tubes and stored at –80 °C until analyzed for OTC concentration.

2.4.3. Mass spectrometry Detection was performed using a quadrupole mass spectrometer Waters TQD equipped with an electrospray ionization (ESI) source operated in positive ion mode. The optimum MS conditions included the following parameters: source temperature at 150 °C, desolvation temperature at 250 °C and capillary voltage of 4 keV. Nitrogen was used as the desolvation gas at a flow of 900 L/h. The cone energy was set at 30 V for OTC and CTC. The OTC quantification was performed using selected reaction monitoring (SRM) for the transitions m/z 461 > 426 (quantifier), 461 > 443 and 461 > 201 (qualifiers). Transition m/z 479 > 462 was used to quantify CTC. 2.4.4. Standard calibration curve Linear regression was used with a weighing factor of 1/x2. The calibration curve was linear between 0.025 and 10 μg/mL and accepted if the correlation coefficient was at least 0.99 and measured concentrations were within 15% of actual concentrations. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) for OTC were 10 ng/mL and 25 ng/mL, respectively. Intra-day and inter-day variabilities were 6–11% and 16–21%, respectively. The accuracies were measured from QC samples and were, respectively, 105%, 94.4% and 97.0% at concentrations of 0.05, 0.10, 0.50 μg/mL. The extraction recoveries ranged between 80.7% and 116%.

2.4. Oxytetracycline determination in plasma Oxytetracycline was extracted from fish plasma by Solid Phase Extraction (SPE) and analyzed by Ultra High Performance Liquid Chromatography (UPLC) coupled by as tandem quadrupole mass spectrometer (Mokh et al., 2017; Reinholds et al., 2016).

2.5. Pharmacokinetic analysis The OTC plasma concentration versus time profile from fish in the FW, BW and SW groups were subjected to PK analysis using commercially available software (Phoenix® Win-Nonlin® 7.0, Certara, Inc. Princeton, NJ, USA). Data from each group were analyzed using noncompartment methods implemented in the software with Model Type Plasma (200−202) with uniform weighting using sparse data option available in the software. In this method, mean OTC concentration curve of the data was generated by taking the mean concentration value of the plasma data (n = 6) for each time point. The concentrations above the limit of quantitation (LOQ) were used. Standard error of mean plasma concentrations for each time point was calculated to measure inter-individual variability. The PK parameters determined were; slope of the terminal phase (λz), terminal half-life (λz-HL), maximum plasma concentration (Cmax); time to achieve peak concentration (Tmax), area under the concentration time (time from the first measurement to the last measurement of drug concentration) curve (AUC0- last), apparent systemic clearance (CL/F) and mean residence time (MRT). The rate constant (λz) of the terminal part of the curve was calculated using linear regression of the terminal part of the log plasma concentration versus time curve and a linear trapezoidal linear interpolation method was used to determine AUC0- last. The total area under the first moment curve (AUMC) was calculated by combining the trapezoid calculation of AUMC0-last and extrapolated area. The MRT was calculated as MRT = AUMC/AUC and CL/F was calculated as CL = dose/AUC. The Cmax represented the observed peak plasma concentration, and the time to peak plasma concentration (Tmax) was the time to reach Cmax. The PK parameters calculated were based on the mean profile for all the subjects in the data set. Therefore, in this analysis, standard error-mean (SEM) is calculated for Cmax and AUC and 95% confidence interval (CI) for all the parameters.

2.4.1. Extraction Oxytetracycline hydrochloride and chlortetracycline hydrochloride (CTC) were purchased from Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA). The purity of the OTC and CTC were 96.9% and 96.1%, respectively. The OTC calibration was obtained using matrixmatched calibration curve. The OTC calibration stock solutions were prepared in methanol at 0.1, 0.25, 0.50, 1, 2.5, 5.0, 10 mg/mL and kept at −20 °C. Working solutions were prepared daily in phosphoric acid 4% at the following concentrations (10 times dilution): 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 10 μg/mL. Quality controls were prepared in untreated fish plasma at 0.05, 0.10, 0.50 μg/mL. The CTC was used as an internal standard and prepared in phosphoric acid at a concentration of 200 ng/mL. A 50 μL sample of fish plasma (blank, QC or sample) was added in a 0.6 mL microcentrifuge tube, 100 μL of CTC 200 ppb in aqueous phosphoric 4% and 150 μL of aqueous phosphoric acid 4% (total volume: 300 μL). After mixing, the microcentrifuge tubes were spun down for 15 min at 17,000 ×g and the supernatant was cleanedup by solid-phase extraction (SPE) using Oasis HLB Prime μElution plates (Waters Co, Milford MA, USA). Calibrants, blank, QC and samples were loaded on the plate using a Positive pressure-96 processor (Waters, Milford MA, USA). Following a wash with 0.3 mL of water containing 5% methanol the OTC was eluted with 50 μL of acetonitrilemethanol (10:90, v/v). A 50 μL of water containing 2% of formic acid was added to each well before analysis. 2.4.2. Liquid chromatography The chromatographic separation was performed using a UPLC system, Acquity H (Waters Co., Milford MA) and a UPLC column Waters HSS T3, 1.8 μ, 2.1 × 50 mm (P/N 186003538). The mobile phase was composed of a mixture of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) and the following gradient was used: from 0 to 0.8 min A (90%), at 0.8 min A (10%), hold for 0.4 min and at

2.6. Statistical analysis Statistical analysis was performed using MATLAB® R2017b (The MathWorks, Inc. Natick, MA, USA). Data of OTC concentrations versus 677

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time for three groups of tilapia were collected in a spreadsheet (Microsoft Excel® 2016, Microsoft Corporation, Redmond, WA, USA) for subsequent calculation and manipulation. The mean ± SEM were estimated for OTC concentrations in tilapia at each time point. Group differences among the three groups, FW, BW and SW, were evaluated with the assumption that data were not normally distributed using Kruskal–Wallis ANOVA on ranks and Wilcoxon rank sum test for two independent unequal-sized data and then later Kruskal–Wallis ANOVA were applied to perform follow-up multiple comparison tests on three independent unequal-sized data. P-values for statistical significance were set to ≤0.05. 3. Results

Fig. 1. Semi-logarithmic mean ± SEM plasma concentration versus time profile (0 h–720 h) of OTC following oral administration at the dose rate of 50 mg/ kg in FW, BW and SW tilapia.

Mean initial weights of the FW, BW and SW tilapia were 123.6 g, 123.7 g and 118.7 g, respectively (Table 1). Water quality conditions for tilapia were within normal culture conditions for production tilapia as indicated by observed water quality parameters. Overall mean ± SD of water quality parameters of the systems were: temperature 25.9 ± 0.4 °C, dissolved oxygen > 6.50 mg/L, pH 8.38 ± 0.03, total ammonia-N 0.16 ± 0.02 mg/L, and nitrite-N 0.23 ± 0.15 mg/L. No significant differences were observed for water quality parameters between the three systems except for salinity (Table 1). The mean ± SEM plasma concentrations of OTC of the three fish groups at time intervals between 0 h–1008 h are given in Table 2. The time points included are those at which at least 3 of 6 sampled fish contained an OTC concentration above the LOQ. The comparative log OTC concentration versus time curves of the FW, BW and SW groups are shown in Fig. 1. The mean OTC profiles in the FW, BW and SW fish showed a very similar trend until 6 h post dosing (Fig. 2). No significant differences (P = 0.14–0.31) in mean OTC values of the three groups were observed up to 6 h after dosing when a gradual decline in drug concentrations began in the FW and BW after 8 h and 12 h, respectively (Fig. 2). In the

Fig. 2. Mean ± SEM plasma concentration versus time profile (0 h–48 h) of OTC following a single oral dose of 50 mg/kg in FW, BW and SW tilapia demonstrating the two separate peaks.

SW group, a rapid decline in the OTC concentrations occurred after 6 h showing significantly lower concentrations than either the FW or BW group (P value < 0.01–0.02). Lower OTC concentrations in SW indicated faster excretion of drug from the body compared to FW and BW (Figs. 1 and 2). The PK parameters derived from plasma concentration versus time data of the three groups are shown in Table 3. In all three groups, the OTC absorption was very quick as indicated by the first peak at 0.5 h and a second peak at 8 h, 12 h and 6 h in FW, BW and SW, respectively (Fig. 2). The OTC was detected in plasma up to 30 days, 22 days and 14 days, respectively, in the FW, BW and SW groups (Figs. 1 and 2). The Cmax (mean ± SEM) of the OTC were 1.221 ± 0.124 μg/mL, 1.343 ± 0.212 μg/mL and 1.220 ± 0.257 μg/ mL in the FW, BW and SW tilapia, respectively and corresponding Tmax values were 8 h, 12 h and 6 h. No significant differences were observed for mean plasma concentrations between FW and BW group (P = 0.31), however, plasma concentrations in SW tilapia were significantly lower than FW and BW fish at all time points after obtaining peak levels (P = 0.01–0.02). The AUC0-∞ (55.5 h.μg/mL) in SW tilapia was approximately 3 times lower than the values obtained for FW (165 h.μg/mL) and BW group (145 h.μg/mL). The AUC differences of the OTC between the SW: FW and the SW: BW groups were highly significant (P < 0.01–0.2) but there was no significant difference between the AUC of the FW and the BW group (Table 3). The CL/F of the FW, BW and SW groups were 0.293 L/h/kg, 0.330 L/h/kg and 0.879 L/h/kg, respectively, and the corresponding terminal half-lives (λz-HL) were 177 h, 154 h and 69 h.

Table 2 Mean ± SEM oxytetracycline concentration in plasma of FW, BW and SW tilapia after oral administration of a single dose of 50 mg/kg (n = 6). Time (h)

Concentration (ng/ml) FW

0.00 0.25 0.50 1.00 2.00 4.00 6.00 8.00 12.0 24.0 48.0 96.0 144 192 240 288 336 432 528 624 720 816 912 1008

BW

SW

Mean

SEM

Mean

SEM

Mean

SEM

0 530 1173 761.7 669.5 867.2 1018 1221 1203 1148 632.0 500.5 338.0 265.0 195.0 135.0 111.0 107.0 52.00 53.00 30.50 < LOQ < LOQ < LOQ

0 246.0 211.0 212.0 85.33 131.0 370.0 124.3 115.8 135.3 145.2 104.3 59.00 42.40 44.90 55.80 20.20 13.90 5.150 10.00 8.500 < LOQ < LOQ < LOQ

0 715.0 1260 639.0 783.0 949.0 1032 ⁎ 1155 ⁎ 1343 ⁎ 1095 ⁎ 744 ⁎ 451 ⁎ 304 ⁎ 251 ⁎ 110 ⁎ 98.2 ⁎ 88.6 56.20 52.20 < LOQ < LOQ < LOQ < LOQ < LOQ

0 236.0 176.0 169.0 126.0 152.0 86.80 98.00 212.0 164.0 131.0 91.00 77.90 18.50 11.30 30.50 21.40 6.330 13.50 < LOQ < LOQ < LOQ < LOQ < LOQ

0 450.0 949.0 655.0 706.0 925.0 1220 ¥ ⁎ , 724 ¥ ⁎ , 636 ¥ ⁎ , 471 ¥ ⁎ , 280 ¥ ⁎ , 140 ¥ ⁎ , 85.6 ¥ ⁎ , 72.5 ¥ ⁎ , 66.6 ¥ ⁎ , 59.9 ¥ ⁎ , 60.5 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

0 157.0 230.0 256.0 160.1 239.0 258.0 80.10 108.7 92.20 48.90 26.40 18.50 18.00 16.80 4.040 8.740 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

4. Discussion

< LOQ: below limit of quantification. ¥ values are significantly different between FW and SW (P < 0.01–0.02). ⁎ values are significantly different between BW and SW (P < 0.01–0.02).

Aquaculture species can be susceptible to infectious disease outbreaks due to high stocking density of monocultures, stress and poor 678

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Table 3 Pharamcokinetic parameters of oxytetracycline after oral administration after a single dose of 50 mg/kg in FW, BW and SW tilapia. Parameter

Cmax

Units

μg/mL

FW

BW

Mean

95% CI CCCICI

1.225 (0.124) 8.000 0.0039 176.8 165.3 a (9.98) 170.8 208.0 292.8

0.75

a

Tmax λz λz-HL AUC0-last

h 1/h h h × μg/mL

AUC0-∞ MRT0-∞ Cl_F

h × μg/mL h mL/h/kg

SW

Mean

95% CI

1.343 (0.212) 12.00 0.0045 ⁎ 154.1 ⁎ 144.6 a (10.7) 151.5 162.4 330.1

0.870

a

7.45 0.0017 123.8 76.9 69.8 163.4 207.9

Mean

95% CI

1.220 (0.257) 6.000 0.0099 ⁎⁎ 69.35 ⁎⁎ 55.48 a (5.86) 56.90 100.0 878.7

0.960

a

11.35 0.0013 119.5 68.29 66.9 119.3 229.0

5.450 0.002 59.70 42.15 27 131.0 598.7

a

=SEM. Values of BW are different than SW tilapia (P < 0.02). ⁎⁎ Values of SW are different than FW tilapia (P < 0.01). ⁎

intravenous administration in tilapia. Two peaks for the OTC might be attributed to enterohepatic recycling. Enterohepatic cycling had been associated with double peaks and long apparent elimination half-life for doxycycline. Reabsorption from water also might be contributing to the second peak of the OTC because of its low oral bioavailability and large extent of excretion via feces (Yang et al., 2014; Rivere and Spoo, 1995). However, this is unlikely in our study as activated carbon was added to the recirculation filter to remove any free OTC or OTC metabolites that may have been excreted by the fish. To our knowledge, there is only one published study reporting depletion of the OTC from plasma and tissues of Nile tilapia following oral dosing (Chen et al., 2004). These authors used OTC top-coated feed at the dose rate of 82.8 mg/kg when fish were fed at 1% body weight per day for 10 days and plasma concentrations were determined only at three time points (0.04, 1, 6 days). They reported plasma concentration of 1.18 ± 0.18 μg/mL at 24 h which was similar to the plasma concentration obtained for our FW (1.15 ± 0.13 μg/mL) and BW (1.10 ± 0.16 μg/mL) group and 2.5 times higher than SW tilapia (0.471 ± 0.092 μg/mL). Compared to the FW group, absorption of OTC was not different in the BW group, but in SW tilapia the OTC adsorption was faster as indicated by an early Tmax of 6 h followed by a rapid decline on the drug concentrations. In another study, peak plasma concentration (2.27 ± 0.38 μg/mL) was obtained at 24 h after oral administration of doxycycline in tilapia (Yang et al., 2014). This suggests a faster absorption of OTC than doxycycline in tilapia. A critical period during our study was between 8 h and 336 h after dosing when mean OTC concentrations in SW fish were significantly (P < 0.01–0.02) lower than FW and BW group. Lower plasma OTC concentrations in SW tilapia indicated that drug distribution is influenced by water salinity due to changes in osmoregulatory functions in SW leading to an increased plasma volume. The findings were in agreement with previous work reporting effect of aquatic environment on the OTC tissue residue concentrations in a comparative study that included warm water and cool-water fish species (Chen et al., 2004). Similarly, plasma and tissue concentrations of praziquantel were lower in brackish water grass carp than freshwater grass carp (Xie et al., 2015). In FW tilapia, the λz-HL = 177 h of OTC was higher than the reported value of λz-HL for sunshine bass (74.2 h) and shorter than 243 h for walleye following oral OTC administration (Chen et al., 2004). Species differences in terminal half-lives of OTC following other routes of administration have been recorded in fish (Dio et al., 1998; Grondel et al., 1989; Grondel et al., 1987; Reja et al., 1996). A wide range (60.3 h–89.5 h) of terminal half-life has been reported within species (Björklund and Bylund, 1991; Grondel et al., 1989). These differences could be explained in part by species, weight or size of fish, experimental design and environmental factors such as pH, temperature and

water quality (Leung and Bates, 2013). This necessitates availability of chemotherapeutic agents for effective treatment of infections. Antibiotic resistance to pathogens isolated from Atlantic salmon has been reported (Inglis et al., 1991). Thus, it is crucial to maintain the sensitivity of OTC because it is one of the four antimicrobials approved in the U.S. for food fish species. Pharmacokinetic and tissue depletion studies of OTC have been conducted in Atlantic salmon (Elema et al., 1996), eel (Ueno et al., 2004), rainbow trout (Björklund and Bylund, 1991; Namdari et al., 1999), freshwater rainbow trout (Grondel et al., 1989), Arctic charr (Haug and Hals, 2000), Africa catfish (Grondel et al., 1989), sea bass (Rigos et al., 2004a, 2004b), summer flounder (Hughes, 2003; Chen et al., 2004) and yellow perch (Bowden, 2001). These reports suggested that the PK of OTC is influenced by species, health condition, age and size, route of drug administration and environmental conditions such as water temperature and salinity. Excretion of oxolinic acid was slower in freshwater trout than in seawater trout (Ishida, 1992). Similarly, Abedini et al. (1998) found lower elimination and clearance rates of the OTC in freshwater trout than in seawater salmon. Feng et al. (2008) demonstrated that florfenicol tissue drug concentrations of seawater tilapia were lower than that in freshwater tilapia; and the elimination of florfenicol in seawater tilapia was more rapid than that in freshwater tilapia. This necessitates studying the effect of factors on PK of OTC in tilapia in order to determine optimum dosage regimen for achieving therapeutic success and minimizing the risk of resistance development. The present study was undertaken to determine the dosage implications of OTC due to differences in PK caused by water salinity levels in the tilapia following oral administration. The dose (50 mg/kg) of the OTC was chosen based on the established approved dose (2.5–3.75 g/100 lb. body weight) for treatment in food fish. Differences in food intake may lead to large variations in plasma/tissue concentrations of drugs within the same fish species when antibiotics are fed in feed (Chen et al., 2004). Keeping this in mind, an oral gavage route was used for the OTC administration in tilapia and lower inter- individual variation (10%–40%, Figs. 1 and 2) in plasma concentrations compared to earlier reports was observed (Chen et al., 2004). In their report, plasma and tissue concentrations among individuals of Nile tilapia and walleye varied to a great extent with OTC in the medicated feed. In the FW group, the plasma concentration–time profile showed a double peak phenomenon with an initial peak (1.173 ± 0.211 μg/mL) at 0.5 h, which started to decay, then raised again to a second peak (1.221 ± 0.124 μg/mL) at 8 h. The first peak seemed to be real as it occurred in both the BW and SW tilapia at 0.5 h with a second peak at 12 h and 6 h, respectively (Fig. 2). Yang and co-workers (2014) observed two peaks with doxycycline administration in tilapia reporting Cmax1 (1.99 ± 0.43 μg/mL) at 2.0 h and Cmax2 (2.27 ± 0.38 μg/mL) at 24 h. The authors also reported double peaks of doxycycline after 679

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and 6 h in the SW tilapia. This indicates that in FW and BW tilapia, oral administration of OTC at dosage of 50 mg/kg per day would be theoretically effective to treat bacterial diseases caused by bacteria having MIC ≤1.0 μg/mL, but in the SW group the drug would need to be administered after every 6 h. However, infections caused by bacteria with MIC > 1.0 μg/mL cannot be treated with oral administration of OTC in all three groups. The OTC residue depletion studies need to be carried out in tilapia maintained at different salinity levels and the withdrawal period should be reevaluated for a particular environmental condition in which they are maintained. Further PK/PD studies in clinically sick tilapia are required using multiple oral doses of OTC to validate these recommendations. In conclusion, water salinity influences the PK of OTC following oral administration in tilapia. Pharmacokinetics of the OTC was characterized by rapid absorption and slow systemic clearance in the FW and BW groups, while the SW tilapia showed faster absorption and elimination than the FW and BW tilapia. Following administration of OTC in tilapia maintained at three salinity levels, exposure of the OTC was in order of FW > BW > SW group. Plasma concentrations in the SW tilapia were significantly lower than the FW and BW tilapia at all time points after achieving peak concentration. Compared to the FW and BW tilapia, SW tilapia showed faster drug clearance and shorter terminal half-life. These findings indicate that water salinity alters the OTC clearance by influencing osmo-regulation in tilapia and result is faster excretion of OTC with increase in water salinity. Based on the results, an oral dose of 50 mg/kg of OTC may be used in FW and BW group, but a higher dosage is required in SW tilapia for treatment of infectious diseases caused by sensitive pathogens with MIC = 0.5–1.0 μg/mL. This study in tilapia will serve as a model for future investigations in examining our hypothesis that a single recommendation of a particular drug dose is not appropriate for a fish species grown at different water salinities. The findings also indicate that residue depletion time of the OTC will vary in tilapia depending upon the salinity level of water. To the authors' knowledge, this is the first report of the effect of water salinity on PK of OTC after oral administration in tilapia.

salinity that play an important role in drug absorption, distribution, metabolism and elimination in fish (Abedini et al., 1998; Feng et al., 2008; Ishida, 1992; Toutain et al., 2010; Xie et al., 2015). In our study, λz-HL (69.4 h) of the OTC decreased markedly (2.3–2.6 times) in SW tilapia compared to the FW and BW groups. This indicates that drug clearance was faster in tilapia maintained in higher salinity (SW) than tilapia raised in low salinity water. Similarly, slower elimination of florfenicol in fresh water tilapia compared to sea water tilapia was observed by Feng et al. (2008). An increased clearance of chemicals with rise in water salinity has been observed in other fish species as well (Abedini et al., 1998; Ishida, 1992; Xie et al., 2015). The effect of salinity on drug clearance was supported by highly significant (P < 0.01) reduction in AUC (55.5 h.μg/mL) of SW tilapia compared to the AUC of FW (165 h.μg/mL) and BW (145 h.μg/mL) groups. The decreased AUC value in the BW and SW fish may be either due to decreased bioavailability or increased systemic clearance of the OTC. Although bioavailability was not determined in this study, approximately similar concentrations in the initial phase of the plasma concentration-time curve (before Cmax) in the three groups negates the possibility of change in drug bioavailability with water salinity. Alternatively, decreased OTC concentrations in plasma and resultant AUC value in SW compared to FW and BW fish may be explained by increased systemic OTC clearance in SW due to rise in salinity. It is hypothesized that the change in water salinity from 0 to 30 ppt influenced the regulation of osmotic pressure in the SW tilapia resulting in an increase in the excretion of the OTC through osmoregulation process. It is known that FW fish are living in a hypo-osmotic environment with respect to their body fluids leading to volume loading and a tendency to lose salt. Contrary to this, the environment for SW fish is hyper-osmotic compared to their body fluids. As a result of this, SW fish face the challenge of dehydration and salt loading and to combat this SW fish drink vast amounts of water that may increase the excretion of a drug. Increased water drinking ability of teleosts with rise in water salinity levels has been observed (Greenwell et al., 2003). Clinical application of a PK study is to integrate in vivo PK data with PD data to establish appropriate dosage regimen for the target species and predict clinical significance. The PK/PD indices, such as Cmax/MIC, AUC/MIC, and T > MIC values, of the target organism are widely used as surrogate markers to predict drug effectiveness in man and other animal species (Lees et al., 2006; Sidhu et al., 2014; Toutain et al., 2010; Waraich et al., 2016). In fish, we are not able to apply the full power of the PK/PD approach due to a lack of recommended values of these indices, large PK and PD differences within species and effect of environmental factors on PK and PD of antimicrobials. However, for drugs that exhibit time-dependent activity such as OTC, efficacy is correlated with T > MIC. It has been suggested that plasma drug concentrations of these drugs should exceed the MIC of the pathogen for at least 50% of the dosing interval (Toutain et al., 2010; Waraich et al., 2016). It is important to remember that OTC exhibits bacteriostatic activity and it is the host immune system that is ultimately responsible for success in combating pathogens causing disease. It is pertinent to mention here that the MIC of antimicrobials varies between isolates and species of pathogens (Kirkan et al., 2006; Darwish et al., 2008). Kirkan and co-workers (2006) investigated tetracycline susceptibility to 39 pathogenic bacterial strains (Yersinia ruckeri, Enterococcus seriolicida, Aeromonas salmonicida) collected from clinical cases of fish and tetracycline MIC range was 0.13–32 μg/mL. A lower range of OTC MIC for Flavobacterium columnare isolated from channel catfish suffering from columnare outbreaks in USA was found to be 0.031–0.50 μg/mL (Darwish et al., 2008). Considering this variation, MIC90 of a target pathogen is used to establish the relevance of drug concentration in plasma to MIC. Unfortunately, MIC90 against OTC is not available for most fish pathogens associated with commonly occurring diseases. Therefore, assuming MIC = 0.5–1.0 μg/mL of the most sensitive pathogens of fish species as determined by Miller and Reimschuessel (2006), T > MIC was 24 h in the FW and BW groups,

Competing interests The authors declare that they have no conflicts of interest with respect to the authorship, research and publication of this article. Authors' contributions Stephen A. Smith initiated and supervised the study, developed the experimental design, and edited the manuscript. Manuscript was primarily written by Pritam K. Sidhu after compiling, analyzing and interpreting the data. Corinne Mayer maintained the animals, collected blood and water quality samples, and processed the samples after collection. Geraldine Magnin performed laboratory analysis of blood samples and generated the concentration vs. time profile of OTC. David Kuhn helped with experimental design, aquaculture system design, statistical analysis of water quality data and funding. Majid JaberiDouraki contributed to statistical analysis and presentation of data as graphs. Johann F. Coetzee provided the facilities to conduct laboratory analysis, coordinated the project with SAS and helped in writing the manuscript. All authors have read and approved the final manuscript. Acknowledgements The authors gratefully acknowledge Certara for providing the software Phoenix-winnonlin for PK data analysis, and Jess Jones for providing space in the VT Conservation Aquaculture Facility. References Abedini, S., Namdari, R., Law, F.C.P., 1998. Comparative pharmacokinetics and

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