Pharmacokinetics and bioavailability of oxytetracyclinein vannamei shrimp (Penaeus vannamei) and the effectof processing on the residues in muscle and shell

Kazuaki Uno • Tidaporn Chaweepack • Lila Ruangpan

Received: 23 July 2009 / Accepted: 23 December 2009 / Published online: 23 January 2010� Springer Science+Business Media B.V. 2010

Abstract The present study examined the pharmacokinetics and bioavailability of oxy-

tetracycline (OTC) in vannamei shrimp (Penaeus vannamei) after intra-sinus (10 mg/kg)

and oral (10 and 50 mg/kg) administration and also investigated the net changes of OTC

residues in the shrimp after the thermal, acid and alkaline processing methods. The

hemolymph concentrations of OTC after intra-sinus dosing were best described by a two-

compartment open model. The oral bioavailability was found to be 48.2 and 43.6% at

doses of 10 and 50 mg OTC/kg, respectively. The peak hemolymph concentrations after 10

and 50 mg OTC/kg doses were 3.37 and 17.4 lg/ml; the times to peak hemolymph con-

centrations were 7 and 10 h. The elimination half-lives were found to be 15.0 and 11.5 h

for the low and high dose, respectively. The residual OTC was rapidly eliminated from

muscle with the elimination half-life value of 19.4 and 15.4 h, respectively, for the groups

treated with doses of 10 and 50 mg/kg. The residual OTC levels in the muscle fell below

the MRL (0.2 lg/g) at 72 and 96-h post-dosing at dose levels of 10 or 50 mg/kg,

respectively. Residual OTC levels in muscle and shell were approximately 20–50% lower

in the thermal treatment such as boiling, baking and frying. By the acid treatment, OTC

residues were reduced to [80%, while those were reduced to around 30% by alkaline

treatment.

Keywords Penaeus vannamei � Oxytetracycline � Pharmacokinetics �Bioavailability � Processing

K. Uno (&)Laboratory of Food Safety, Department of Sciences for Living, Aichi Konan College,Konan, Aichi 483-8086, Japane-mail: uno@konan.ac.jp

T. ChaweepackChanthaburi Coastal Fisheries Research and Development Center, Bangkaja,Muang Chanthaburi 22000, Thailand

L. RuangpanDepartment of Fisheries, Ministry of Agriculture and Cooperative, Chatuchak,Bangkok 10900, Thailand

123

Aquacult Int (2010) 18:1003–1015DOI 10.1007/s10499-009-9318-7

Introduction

Recently, shrimp farming in Southeast Asia including Thailand has been undergoing a

dramatic transformation. The main farmed species has rapidly been switching from black

tiger shrimp (Penaeus monodon) to vannamei shrimp (Penaeus (Litopenaeus) vannamei).In Thailand, P. vannamei has been the dominant cultivated species (FAO 2009). It is an

economically important export, and the greater part of the production is consumed by the

USA, Japan and the EU. In 2007, the production of this species reached 490,000 metric

tons (equal to approximately 1.7 billion US dollars) (FAO 2009). With the significant

increase in the production, P. vannamei has become susceptible to a variety of viral and

bacterial diseases under intensive farming conditions. Disease outbreak is a serious

problem causing economic damage to the Thai shrimp industry. To reduce bacterial dis-

ease problems, a wide range of chemotherapeutics has been used. The use of chemo-

therapeutic drugs in shrimp farming can have an impact on the aquatic environment and

potentially can cause human health risks, such as allergic reactions in hypersensitive

individuals due to drug residues in shrimp sold for consumption (Holmstrom et al. 2003).

Therefore, the proper use of drugs is strongly recommended to avoid such problems.

Knowledge of pharmacokinetics, bioavailability and residue depletion is important in order

to minimize the human risk connected to drug residues and the environmental impact of

the drugs.

Oxytetracycline (OTC) is the most commonly used antibiotic in Thai shrimp culture

farms. It has potential for use in farm-raised shrimp for the treatment of vibriosis like

Vibrio alginolyticus and Vibrio harveyi. There are many studies on the pharmacokinetics

and residues of OTC in marine and freshwater shrimp (Mohney et al. 1997; Bermudez-

Almada et al. 1999; Brillantes et al. 2001; Uno 2002, 2004; Sangrungruang et al. 2004;

Reed et al. 2004, 2006; Weifen et al. 2004; Uno et al. 2006; Poapolathep et al. 2008a, b),

but limited information of OTC dispositions in P. vannamei is available (Chiayvareesajja

et al. 2006; Nogueira-Lima et al. 2006; Faroongsarng et al. 2007; Gomez-Jimenez et al.

2008).

An important factor in risk assessment of antibiotic residues in shrimp is that most

shrimp are usually consumed after a thermal treatment such as cooking. Shrimp shells are

not well digested by humans, but contain about 20% of chitin, poly-b(1 ? 4)-N-acetyl-D-

glucosamine, which has many applications in food and pharmaceutical industries (Percot

et al. 2003). Currently, commercial chitin is produced from shrimp shells as by-product of

the seafood industry. The extraction of chitin from shrimp shells involves acid and alkaline

processing. It is of interest to determine if drug residues are decreased by these (thermal,

acid and alkaline) processes.

The objectives of the present study were to investigate the pharmacokinetics and bio-

availability of OTC in P. vannamei after intra-sinus and oral administration, and examine

the net changes of OTC residues in the shrimp after the thermal, acid and alkaline pro-

cessing methods.

Materials and methods

Chemicals

Oxytetracycline hydrochloride (purity[95%) was purchased from Sigma (St. Louis, MO,

USA). Unless otherwise indicated, chemicals used were of analytical or HPLC grade.

1004 Aquacult Int (2010) 18:1003–1015

123

Shrimp

Healthy P. vannamei (n = 185, 24.7 ± 3.5 g) were obtained from a shrimp farm in

Chanthaburi, Thailand. The shrimps were analyzed to confirm the absence of OTC before

the experiment. The shrimps were kept in tanks with brackish water at a salinity of

13.0 ± 1.1 ppt. The water temperature was 27.4 ± 0.7�C, and aeration was constant. The

pH of the water was 7.7 ± 0.1. The shrimps were fed ad libitum with commercial shrimp

pellets (LEADER 104 Feed meal, Grobest Co., Bangkok, Thailand) before and after

administration. The shrimps were starved for 1 day before administration of the drug.

Intra-sinus administration

A solution of OTC was prepared by dissolving OTC hydrochloride in isotonic sterile

phosphate buffered saline (PBS). OTC concentration in the solution was 10 mg/ml. Intra-

sinus administration of the drug occurred by injection into the ventral sinus with a 50-ll

Hamilton Microliter syringe 705 LT (Hamilton, Reno, NV, USA) fitted with a 27-G needle.

The dose was 10 mg/kg bw, and the injection volume was 1.0 ml/kg bw. Five shrimps

were sampled at 0.5, 1, 2, 4, 7, 10, 24, 48, 72, 96 and 120-h post-dosing.

Oral administration

Oxytetracycline was mixed in a slurry of food, and the slurry was orally administered by

catheter to the shrimp. The doses were 10 and 50 mg OTC/kg bw. Five shrimps were

sampled at 1, 2, 4, 7, 10, 24, 48, 72, 96 and 120-h post-dosing.

Hemolymph and tissue sampling

Hemolymph was sampled from the ventral sinus cavity by using a 2.5-ml syringe con-

taining 10 mg of N-ethylmaleimide (Sigma), used as the anticoagulant, fitted with a 21-G

needle. The tail muscle and shell were collected from each shrimp. All these samples were

kept frozen at -40�C.

Analytical procedures

The analytical procedures were previously described (Uno et al., 2006). Briefly, hemo-

lymph samples were filtered through 0.45-lm disposable syringe filter units equipped with

cellulose acetate membranes (Advantec, Tokyo, Japan). The filtrate was diluted fourfold in

PBS and then it was directly injected into the HPLC. Tissue samples (muscle 1 g and shell

0.5 g) were homogenized with 30 ml of 5% trichloroacetic acid containing 0.5% EDTA

and centrifuged for 20 min at 12,000 rpm. To the supernatant, 5 ml of n-hexane was added

and vortexed. The aqueous layer was poured into a Sep-Pak C18 cartridge column (Waters

Corporation, Milford, MA, USA). After the aqueous layer passed through the column, the

column was washed with 30 ml of water. The retained OTC was eluted from the column

with 20 ml of methanol. The eluate was evaporated to dryness. The residue was dissolved

in 1 ml of the HPLC mobile phase, and the solution was injected into the HPLC. The

HPLC system consisted of a Jasco PU-980 pump, a fluorescence detector FP-2020 (Japan

Spectroscopic, Japan), a Rheodyne 7125 injector (Rheodyne, Cotati, CA, USA) with a

20-ll loop, and a Chromatopac C-R6A integrator (Shimadzu Seisakusho, Kyoto, Japan).

The operating conditions were as follows: column, Hisep column (150 9 4.6 mm I.D.,

Aquacult Int (2010) 18:1003–1015 1005

123

Spelco, Bellefonte, PA, USA); mobile phase, 1 M imidazole buffer (pH 7.2) containing

50 mM magnesium acetate and 1 mM EDTA-methanol (90:10, v/v); flow-rate, 0.8 ml/

min; column temperature, 35�C; and detection, FL (Ex 380 nm/Em 520 nm).

The recoveries of the methods were 99.4 ± 0.5% for hemolymph, 82.7 ± 2.8% for

muscle and 85.1 ± 3.3% for shell. The limits of quantitation were 0.02 lg/ml for hemo-

lymph, 0.008 lg/g for muscle and 0.01 lg/g for shell.

Hemolymph protein binding

Hemolymph samples were taken from orally dosed shrimp at 1, 2, 4, 7, 10, 24, 48 and 72 h.

The binding of the drugs to hemolymph proteins was determined by ultrafiltration using a

Microcon YM-10 (Millipore, Bedford, MA, USA) with a 10,000 nominal molecular weight

cut-off limit. A 200-ll aliquot of hemolymph was used and centrifuged at 7,7409g for

15 min at room temperature. The total drug concentration and free drug fraction, ultra-

filtrates of hemolymph samples, were determined by HPLC as described earlier. Bound

drugs were calculated as the difference between total and free components. The drug bound

to the filter was determined by ultrafiltering 2 lg/ml of drug in PBS, and comparing the

drug level in the filtrates and the unfiltered sample. The recovery of OTC in the ultrafil-

tration procedure was 98.5 ± 0.3% (n = 4).

Pharmacokinetic and statistical analysis

All processes were assumed to follow first-order kinetics. Pharmacokinetic analysis was

performed by the computer program WinNonlin (version 1.1, Scientific Consulting, Apex,

NC, USA). Models were selected in accordance with Akaike’s information criterion

(Yamaoka et al. 1978). The area under the concentration–time curve (AUC) was calculated

using the trapezoid rule, including the terminal portion. The bioavailabilities were deter-

mined by calculating the AUC for the oral and intra-sinus experiments and utilizing the

following equation:

Bioavailability %ð Þ ¼ AUCoral � doseISð Þ= AUCIS � doseoralð Þ � 100

The average steady-state concentration (Css) during multiple oral dosing was calculated

using the following equation:

Css ¼ F � dose=CLb � s

where s is the dosing interval.

Student’s t-test was used for statistical comparisons; the level of significance was

chosen at P \ 0.05.

Thermal, acid and alkaline processing methods

Tissue samples (muscle and shell) were taken from orally dosed (50 mg/kg) shrimp at 24-h

post-dosing. One matched tissue from each shrimp was analyzed raw for OTC residue and

the other was processed and analyzed. The thermal (cooking), acid and alkaline processing

methods were as follows:

Boiling: Samples were wrapped in laboratory wipes (Kimwipes� S200, Kimberly-Clark,

Co., USA), soaked in boiling water for 4 min, removed and cooled.

1006 Aquacult Int (2010) 18:1003–1015

123

Baking: Samples were placed on a metal tray and baked in an electric oven at 200�C for

4 min, removed and allowed to cool.

Frying: Samples were fried in canola oil at 180�C for 1 min, removed and allowed to

cool.

Acid treatment: Shell samples were soaked in 1 M HCl at room temperature for 1 h and

rinsed with distilled water. Excess water was removed.

Alkaline treatment: Shell samples were soaked in 1 M NaOH solution at room

temperature for 1 h and rinsed with distilled water. Excess water was removed.

Results

Figure 1 shows hemolymph concentrations of OTC after intra-sinus administration. The

hemolymph concentrations of OTC could be described best by a two-compartment open

model. The time course of hemolymph levels (Ct) was described by the following equation:

Ct ¼ 29:2� expð�3:249tÞ þ 10:9� expð�0:0588tÞ:The distribution and elimination half-lives (t1/2a and t1/2b) were found to be 0.21 and

11.8 h, respectively. The apparent volume of distribution at a steady state (Vss) and total

body clearance (CLb) were estimated to be 839 ml/kg and 51.6 ml/kg/h, respectively. The

volume of the peripheral compartment (Vp) was obtained Vss - Vc (the volume of the

central compartment). The Vc and Vp of OTC in the shrimp were 250 and 589 ml/kg,

respectively. The pharmacokinetic parameters calculated from this model are given in

Table 1.

Figure 2 shows the hemolymph concentration–time profiles after oral administration at

doses of 10 and 50 mg OTC/kg. A non-compartment model best described data from the

low and high OTC–dosed groups. The obtained parameters are listed in Table 2.

Administration of 50 mg OTC/kg to shrimp resulted in five times higher maximum

Fig. 1 Hemolymph concentrations of OTC in vannamei shrimp (P. vannamei) following intra-sinusadministration at a dose of 10 mg/kg. Symbols indicate the mean and standard deviations for five shrimp.The line was obtained from model fitted results

Aquacult Int (2010) 18:1003–1015 1007

123

concentration value when compared with that of 10 mg OTC/kg. The oral bioavailability

was found to be 48.2% for the low dose and 43.6% for the high dose.

If OTC was given three consecutive daily dosings (s = 8 h) at dosages of 10 and

50 mg/kg, the average steady-state concentration (Css) was calculated to be 12 lg/ml for

the former and 53 lg/ml for the latter.

Table 1 Pharmacokinetic values for oxytetracycline in hemolymph after intra-sinus administration tovannamei shrimp (P. vannamei)

Parameter Value

Shrimp weight (g) 24.8 ± 3.2

Dose (mg/kg) 10

C0 (lg/ml) 40.0

A (lg/ml) 29.17

B (lg/ml) 10.85

a (h-1) 3.249

b (h-1) 0.0588

t1/2a (h) 0.21

t1/2b (h) 11.8

AUC (lg h/ml) 194

CLb (ml/kg/h) 51.6

Vc (ml/kg) 250

Vp (ml/kg) 589

Vss (ml/kg) 839

Vdb (ml/kg) 878

C0 hemolymph drug concentration at time 0, A, B zero-time hemolymph drug concentration intercepts ofbiphasic intra-sinus disposition curve. The coefficient B is based on the terminal exponential phase, a, bvalues related to the slopes of distribution and terminal phases, respectively, of the biexponential drugdisposition curve, t1/2a, t1/2b distribution half-life and elimination half-life, respectively, of the drug, AUCarea under the concentration–time curve from zero to infinity, CLb total body clearance, Vc apparent volumeof central compartment, Vp apparent volume of peripheral compartment, Vss apparent volume of distributionat a steady state, Vdb apparent volume of distribution

Fig. 2 Hemolymphconcentrations of OTC invannamei shrimp (P. vannamei)following oral administration atdoses of 10 (filled circle) and 50(filled triangle) mg/kg. Symbolsindicate the mean and standarddeviations for five shrimp

1008 Aquacult Int (2010) 18:1003–1015

123

The hemolymph protein binding in vivo of OTC was 31.6 ± 4.1 and 39.8 ± 8.0% at

doses of 10 and 50 mg OTC/kg, respectively. A significant difference was observed

between the two groups (P \ 0.05).

Figure 3 shows the muscle concentration–time curves of OTC after oral administration at

doses of 10 and 50 mg OTC/kg. The peak levels after the low and high doses were 0.96 and

3.00 lg/g; the times to reach these levels were 7 and 10 h. The residual OTC was rapidly

eliminated from muscle with the elimination half-life values of 19.4 and 15.4 h for the

groups treated with doses of 10 and 50 mg OTC/kg, respectively. The residual OTC levels in

the muscle fell below the maximum residue limit (MRL) in Japan (0.2 lg/g) at 72 and 96-h

post-dosing at dose levels of 10 and 50 mg/kg, respectively. Figure 4 shows the shell

concentration–time curves of OTC after oral administration. In shell, the OTC levels fol-

lowing the low and high dose peaked at 7 and 10-h post-dosing with peak levels of 1.25 and

3.50 lg/g, respectively. The residual OTC persisted at high levels (0.7–2.0 lg/g) in shell,

and no elimination phase could be recognized until the last sampling time in the two groups.

Oxytetracycline residues were lower in all processing samples than in the corresponding

raw samples. The residues in the processing samples were compared to that in the raw

Table 2 Pharmacokinetic valuesfor oxytetracycline in hemo-lymph after oral administration tovannamei shrimp (P. vannamei)

Cmax maximum concentration,tmax time when maximumconcentration was obtained, t1/2b

elimination half-life of the drug,AUC area under theconcentration–time curve fromzero to infinity, MRT meanresidence time, F bioavailability

Parameter Values

Dose (mg/kg) 10 50

Shrimp weight (g) 24.8 ± 3.9 24.5 ± 3.3

Cmax (lg/ml) 3.37 17.39

tmax (h) 7.0 10.0

t1/2b (h) 14.7 11.5

AUC (lg h/ml) 93.0 421.9

MRT (h) 26.4 22.9

F (%) 47.9 43.6

Protein binding (%) 31.6 ± 4.1 39.8 ± 8.0

Fig. 3 OTC concentrations in muscle tissues of vannamei shrimp (P. vannamei) following oraladministration at doses of 10 (filled circle) and 50 (filled triangle) mg/kg. Symbols indicate the mean andstandard deviations for five shrimp

Aquacult Int (2010) 18:1003–1015 1009

123

samples to evaluate the percent reduction. Figure 5 shows the substantial net reductions in

OTC by each of the processing methods. Residual OTC in muscle was reduced to 40–50%

by boiling for 4 min, whereas it was reduced to 25–40% either by baking at 200�C for

4 min or frying at 180�C for 1 min. Residual OTC levels in shell were approximately 20–

30% lower in the thermal treatment. By the acid treatment, OTC residues were reduced to

[80%, while those were reduced to around 30% by alkaline treatment. The released OTC

was detected in HCl solutions after the treatment. The solutions contained 76.6 ± 13.5%

of the reducing OTC residues. The acid treatment was the most effective treatment on

reducing OTC residues in shells.

Discussion

The time course of OTC hemolymph concentrations in P. vannamei after intra-sinus

administration could be described best by a two-compartment open model with bolus

injection and first-order elimination. Previous pharmacokinetic studies of OTC after intra-

sinus administration have been also analyzed by a two-compartment model in white shrimp

(Penaeus (Litopenaeus) setiferus), kuruma shrimp (Penaeus (Marsupenaeus) japonicus)

and P. monodon (Reed et al. 2004; Sangrungruang et al. 2004; Uno 2004; Uno et al. 2006).

In P. vannamei, the target species in this study, Chiayvareesajja et al. (2006) have dem-

onstrated both a two- and three-compartment models for OTC levels in hemolymph after

intra-sinus dosing and made use of a two-compartment model for simplicity. On the other

hand, Faroongsarng et al. (2007) argued that OTC pharmacokinetics in shrimp may be

modeled as a three-compartment system, including central (hemolymph), digestive

(hepatopancreas) and peripheral compartments, rather than two. However, Gomez-Jimenez

et al. (2008) suggested that the hepatopancreas may be an important peripheral tissue in

P. vannamei that accumulates OTC regardless of two-compartment or three-compartment

models proposed for pharmacokinetics studies. Consequently, it is still difficult to deduce

the factor affecting the type of model applicable.

Calculated pharmacokinetic parameters of intra-sinus injection in the present study were

in general agreement with those reported by Chiayvareesajja et al. (2006) (Table 3). The

distribution half-life of OTC in P. vannamei (0.21 h) was similar to that in P. monodonreported as 0.20 h (Uno et al. 2006), while it is much shorter than that in P. japonicus

Fig. 4 OTC concentrations inshell tissues of vannamei shrimp(P. vannamei) following oraladministration at doses of 10(filled circle) and 50 (filledtriangle) mg/kg. Symbols indicatethe mean and standard deviationsfor five shrimp

1010 Aquacult Int (2010) 18:1003–1015

123

(0.45 h; Uno 2004) and P. setiferus (2.05 h; Reed et al. 2004). This indicates that OTC is

initially distributed rapidly to the tissues from the hemolymph compartment as well as

P. monodon. A steady-state volume of distribution (Vss: 839 ml/kg) was found for OTC in

P. vannamei. The large Vp (589 ml/kg) suggests a wide distribution and concentration of

OTC into tissues outside the hemolymph. The extent of distribution (Vss and Vdb) was

similar to that in P. japonicus (Uno 2004) and P. monodon (Uno et al. 2006). Reed et al.

(2004) estimated large distribution volume in P. setiferus (2,304 ml/kg). The protein

binding of a drug is related to the volume of distribution and influences the concentration

of active drug in the blood. The inverse relationship of hemolymph protein binding to the

volume of distribution was demonstrated in other shrimp species (Park et al. 1995; Reed

et al. 2004). The shrimp hemolymph protein binding (44–48%) is similar to that in

P. monodon (49%; Uno et al. 2006), whereas it is higher than that found in P. japonicus(23%; Uno 2004) and P. setiferus (14–21%; Reed et al. 2004).

Elimination half-life (t1/2b) and total body clearance (CLb) are important parameters

used to characterize drug disposition. The t1/2b of OTC in P. vannamei hemolymph was

shorter than that in P. japonicus (Uno 2004), P. setiferus (Reed et al. 2004) or P. monodon(Uno et al. 2006). The CLb in the shrimp was relatively large in comparison with

P. japonicus (Uno 2004) and P. monodon (Uno et al. 2006). Therefore, the shorter t1/2b in

P. vannamei is a consequence of a large CLb.

Fig. 5 Effect of thermal (boiling, baking and frying), acid (HCl) and alkaline (NaOH) processing onresidual OTC in muscle and shell tissues of vannamei shrimp (P. vannamei). Values are the mean andstandard deviations for four samples

Aquacult Int (2010) 18:1003–1015 1011

123

In the current study, following oral administration, the maximum hemolymph con-

centration (Cmax) of 50 mg/kg dose was 17.4 lg/ml, which was 5.1-fold higher than that of

10 mg/kg dose. The AUC of the 50 mg/kg was 4.5 times that of the 10 mg/kg dose fol-

lowing oral administration. The t1/2b and mean residence time (MRT) became slightly

shorter with increasing doses. However, there were little differences in the oral bioavail-

ability (F) between the groups treated with doses of 10 and 50 mg/kg, although no sta-

tistical treatment of the results was performed. Poapolathep et al. (2008b) showed that the

t1/2b and absorption half-life were no significant changes with increasing doses in giant

freshwater shrimp (Macrobrachium rosenbergii). Haug and Hals (2000) also reported that

the pharmacokinetics of OTC was not dose-dependent in Arctic charr (Salvelinus alpinus)

and the dosage had no significant effect on the bioavailability as well. Table 4 shows the

comparative F of OTC in shrimp species. The F of OTC dihydrate was generally much

higher than those of hydrochloride and Q-salt form (mono-alkyl trimethyl ammonium salt).

Therefore, the F of OTC in shrimp may be not dose-dependent but influenced greatly by

chemical form of OTC.

In Japan and the USA, legislation stipulates maximum residue limits (MRL) for OTC

used in the treatment of fish and shellfish. The current MRL for OTC is 0.2 lg/g in edible

tissue. In the present study, the residual OTC was rapidly eliminated from the edible tissue

(muscle) with elimination half-life values of 19.4 and 15.4 h at dose levels of 10 and

50 mg/kg, respectively. Both OTC levels fell below the MRL within 96-h post-dosing. On

the contrary, OTC was accumulated and retained strongly in the shell. In our previous

study, OTC accumulated in the shell tissue of P. japonicus (Uno 2002) and P. monodon(Uno et al. 2006) because Ca-tetracycline chelates were a considerable stabilizing factor.

A practical application of pharmacokinetic data is the design of clinical dosage regi-

mens in which blood levels of a drug can be maintained within the therapeutic range by

means of repeated dosages. The Vibrio bacteria isolated from diseased shrimp in Thailand

had a minimum inhibitory concentration (MIC) range of 0.39–100 lg/ml (Chanratchakool

Table 3 Comparative pharmacokinetic values for oxytetracycline following intra-sinus administration inshrimp species

Parameter P. vannameia P. vannameib P. monodonc P. japonicusd P. setiferuse

a (h-1) 3.249 3.158 3.5303 1.535 0.460

b (h-1) 0.0588 0.0422 0.0428 0.0281 0.036

t1/2a (h) 0.21 0.23 0.20 0.45 2.05

t1/2b (h) 11.8 16.4 16.2 24.7 22.3

AUC/dose 19.4 19.8 25.8 44.1 –

CLb (ml/kg/h) 51.6 – 38.7 22.7 78.0

Vc (ml/kg) 250 271 217 157 –

Vp (ml/kg) 589 869 652 591 –

Vss (ml/kg) 839 1,140 869 748 –

Vdb (ml/kg) 878 – 903 807 2,304

a This studyb Chiayvareesajja et al. (2006)c Uno et al. (2006)d Uno (2004)e Reed et al. (2004)

1012 Aquacult Int (2010) 18:1003–1015

123

et al. 1995), and the MIC required to inhibit 90% of the strains has been estimated to be

37.5 lg/ml (Ruangpan and Kitao 1992). Average MIC of OTC against the Vibrio and

Aeromonas species isolated from shrimp hatcheries and ponds in India was 35.59–

49.94 lg/ml (Vaseeharan et al. 2005). Hence, oral dosing of OTC at a dose of 50 mg/kg

(Css: 53 lg/ml) might be appropriate for use in P. vannamei farming.

More information about the effect of cooking on residues is required to give a more

accurate estimate of consumer exposure to these compounds, and of any breakdown

products, since most food of animal origin is cooked before consumption (Rose et al.

1996). Several studies have been reported on degradation of residual OTC after cooking in

fish tissue (Kitts et al. 1992; Du et al. 1997; Huang et al. 1997; Maruyama and Uno 1997).

In general, the residual OTC was relatively heat stable in fish tissue under ordinary cooking

procedures. In the present investigation, the cooking procedures could not completely

degrade residual OTC in shrimp muscle, although boiling and frying were more effective

than baking in reducing OTC residues in muscle. In addition, OTC residues in shell were

considerably thermostable. Similar findings have been reported for OTC residues in muscle

and shell from P. japonicus and P. monodon (Uno 2002; Uno et al. 2006). On the contrary,

OTC residues in shell were considerably reduced ([80%) by the acid treatment because the

Ca-tetracycline chelates were released from shell into hydrochloric acid solution. In

industrial processing, chitin is extracted from shrimp shell by acid treatment with hydro-

chloric acid to dissolve calcium carbonate followed by alkaline extraction with sodium

hydroxide to solubilize proteins. The results indicate if chitin is made from shrimp shell

containing detectable levels of OTC, chitin products might contain negligible or near zero

levels of OTC. However, further investigations are needed to elucidate these points.

Conclusions

The present study revealed the pharmacokinetic profile of OTC in P. vannamei as to be

characterized by rapid absorption, wide distribution to the tissues and rapid elimination.

The oral bioavailability of OTC in the shrimp was satisfactory (44–48%). There were little

differences in the oral bioavailability between the two groups treated with doses of 10 and

50 mg/kg. The thermal processing, such as cooking, could not completely degrade residual

OTC in shrimp muscle and shell. However, OTC residues in shell were considerably

reduced ([80%) by the acid treatment.

Table 4 Comparative bioavailability (F) of oxytetracycline in shrimp species

Species Chemical form Dose (mg/kg) F (%) Reference

P. japonicus Hydrochloride 50 43.2 Uno (2004)

P. monodon Hydrochloride 10 59.9 Sangrungruang et al. (2004)

Hydrochloride 50 35.6 Uno et al. (2006)

P. setiferus Dihydrate 100 92.2 Reed et al. (2006)

Q-salta 100 49.5 Reed et al. (2006)

P. vannamei Dihydrate 50 80.6 Faroongsarng et al. (2007)

Hydrochloride 10 48.2 This study

Hydrochloride 50 43.6 This study

a Mono-alkyl trimethyl ammonium salt

Aquacult Int (2010) 18:1003–1015 1013

123

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (C) from the JapanSociety for the Promotion of Science (no. 18500613). We are grateful to Mrs. Bangorn Srimukda, Directorof Chanthaburi Coastal Fisheries Research and Development Center (Chanthaburi, Thailand) for providingfacilities for this experiment. The staff at this center is acknowledged for technical advice and assistance.We also thank Mr. John Armstrong of Selkirk College (British Columbia, Canada) for reviewing the Englishof this manuscript.

References

Bermudez-Almada MC, Perez-Tello MG, Valenzuela-Quintanar AI, Vazquez-Moreno L (1999) Oxytetra-cycline residues in cultured white shrimp tissue by HPLC and a microbial receptor assay. J Food Sci64:638–640

Brillantes S, Tanasomwang V, Thongrod S, Dachanantawitaya N (2001) Oxytetracycline residues in giantfreshwater prawn (Macrobrachium rosenbergii). J Agric Food Chem 49:4995–4999

Chanratchakool P, Pearson M, Limsuwan C, Roberts RJ (1995) Oxytetracycline sensitivity of Vibrio speciesisolated from diseased black tiger shrimp, Penaeus monodon Fabricius. J Fish Dis 18:79–82

Chiayvareesajja S, Chandumpai A, Theapparat Y, Faroongsarng D (2006) The complete analysis of oxy-tetracycline pharmacokinetics in farmed Pacific white shrimp (Litopenaeus vannamei). J Vet Phar-macol Ther 29:409–414

Du WX, Marshall MR, Xu DH, Santerre CR, Wei CI (1997) Retention of oxytetracycline residues in cookedchannel catfish fillets. J Food Sci 62:119–122

FAO (2009) Fish stat plus. http://www.fao.org/fishery/statistics/software/fishstat. Cited 28 Jul 2009Faroongsarng D, Chandumpai A, Chiayvareesajja S, Theapparat Y (2007) Bioavailability and absorption

analysis of oxytetracycline orally administered to the standardized moulting farmed Pacific whiteshrimp (Litopenaeus vannamei). Aquaculture 269:89–97

Gomez-Jimenez S, Espinosa-Plascencia A, Valenzuela-Villa F, Bermudez-Almada MC (2008) Oxytetra-cycline (OTC) accumulation and elimination in hemolymph, muscle and hepatopancreas of whiteshrimp Litopenaeus vannamei following an OTC-feed therapeutic treatment. Aquaculture 274:24–29

Haug T, Hals PA (2000) Pharmacokinetics of oxytetracycline in Arctic charr (Salvelinus alpinus L.) infreshwater at low temperature. Aquaculture 186:175–191

Holmstrom K, Graslund S, Wahlstrom A, Poungshompoo S, Bengt-Eric B, Kautsky N (2003) Antibiotic usein shrimp farming and implications for environmental impacts and human health. Int J Food Tech38:255–266

Huang TS, Du WX, Marshall MR, Wei CI (1997) Determination of oxytetracycline in raw and cookedchannel catfish by capillary electrophoresis. J Agric Food Chem 45:2602–2605

Kitts DD, Yu CWY, Aoyama RG, Burt HM, McErlane KM (1992) Oxytetracycline degradation in thermallyprocessed farmed salmon. J Agric Food Chem 40:1977–1981

Maruyama R, Uno K (1997) Oxytetracycline residues in tissues of cultured eel and ayu and the effect ofcooking procedures on the residues. J Food Hyg Soc Jpn 38:425–429

Mohney LL, Williams RR, Bell TA, Lightner DV (1997) Residues of oxytetracycline in cultured juvenileblue shrimp, Penaeus stylirostris (Crustacea: Decapod), fed medicated feed for 14 days. Aquaculture149:193–202

Nogueira-Lima AC, Gesteira TCV, Mafezoli J (2006) Oxytetracycline residues in cultivated marine shrimp(Litopenaeus vannamei Boone, 1931) (Crustacea, Decapoda) submitted to antibiotic treatment.Aquaculture 254:748–757

Park ED, Lightner DV, Milner N, Mayersohn M, Park DL, Gifford JM, Bell TA (1995) Exploratorybioavailability and pharmacokinetic studies of sulphadimethoxine and ormetoprim in the penaeidshrimp, Penaeus vannamei. Aquaculture 130:113–128

Percot A, Viton C, Domard A (2003) Optimization of chitin extraction from shrimp shells. Biomacro-molecules 4:12–18

Poapolathep A, Poapolathep S, Imsilp K, Wannapat N, Klangaew N, Kusutjarit N, Kumagai S (2008a)Distribution and residue depletion of oxytetracycline in giant freshwater shrimp (Macrobrachiumrosenbergii). J Food Prot 71:870–873

Poapolathep A, Poapolathep S, Jermnak U, Imsilp K, Wannapat N, Sugita-Konishi Y, Kumagai S (2008b)Muscle tissue kinetics of oxytetracycline following intramuscular and oral administration at twodosages to giant freshwater shrimp (Macrobrachium rosenbergii). J Vet Pharmacol Ther 31:517–522

Reed LA, Siewicki TC, Shah JC (2004) Pharmacokinetics of oxytetracycline in the white shrimp, Litope-naeus setiferus. Aquaculture 232:11–28

1014 Aquacult Int (2010) 18:1003–1015

123

Reed LA, Siewicki TC, Shah JC (2006) The biopharmaceutics and oral bioavailability of two forms ofoxytetracycline to the white shrimp, Litopenaeus setiferus. Aquaculture 258:42–54

Rose MD, Bygrave J, Farrington WHH, Shearer G (1996) The effect of cooking on veterinary drug residuesin food: 4. oxytetracycline. Food Addit Contam 13:275–286

Ruangpan L, Kitao T (1992) Minimal inhibitory concentration of 19 chemotherapeutants against Vibriobacteria of shrimp, Penaeus monodon. Dis Asian Aquac I:135–142

Sangrungruang K, Chotchuang A, Ueno R (2004) Comparative pharmacokinetics and bioavailability ofoxytetracycline in giant tiger prawn. Fish Sci 70:467–472

Uno K (2002) Oxytetracycline and oxolinic acid residues in kuruma prawn (Penaeus japonicus) and theeffect of cooking procedures on the residues. J Food Hyg Soc Jpn 43:62–67

Uno K (2004) Pharmacokinetics of oxolinic acid and oxytetracycline in kuruma shrimp, Penaeus japonicus.Aquaculture 230:1–11

Uno K, Aoki T, Kleechaya W, Tanasomwang V, Ruangpan L (2006) Pharmacokinetics of oxytetracycline inblack tiger shrimp, Penaeus monodon, and the effect of cooking on the residues. Aquaculture 254:24–31

Vaseeharan B, Ramasamy P, Murugan T, Chen JC (2005) In vitro susceptibility of antibiotics against Vibriospp. and Aeromonas spp. isolated from Penaeus monodon hatcheries and ponds. Int J AntimicrobAgents 26:285–291

Weifen W, Hong L, Changhu X, Jamil K (2004) Elimination of chloramphenicol, sulphamethoxazole andoxytetracycline in shrimp, Penaeus chinensis following medicated-feed treatment. Environ Int 30:367–373

Yamaoka K, Nakagawa T, Uno T (1978) Application of Akaike’s information criterion (AIC) in theevaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm 6:165–175

Aquacult Int (2010) 18:1003–1015 1015

123