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Depletion of praziquantel in muscle tissue and skin of cultured rockfish (Sebastes schlegeli) under the commercial culture conditions
Aquaculture 219 (2003) 1 – 7 www.elsevier.com/locate/aqua-online
Depletion of praziquantel in muscle tissue and skin of cultured rockfish (Sebastes schlegeli) under the commercial culture conditions Chun Soo Kim, Jae Bum Cho, Kyoung Jin Ahn, Jae Il Lee, Ki Hong Kim * Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Pusan 608-737, South Korea Received 30 March 2002
Abstract Depletion of praziquantel in muscle tissue and skin of cultured rockfish (Sebastes schlegeli) under the commercial net-pen farm conditions was analyzed. The levels of praziquantel were determined after the administration of three doses of either praziquantel 200 or 400 mg/kg of body weight (bw) given at 24-h intervals. Fish were collected daily following last drug administration and analyzed for praziquantel in muscle tissue and skin by reversed-phase high-performance liquid chromatography (RP-HPLC) using diazepam as an internal standard. In the group of fish fed 200 mg of praziquantel/kg bw, praziquantel was detected in muscle tissue until 1 day and in skin until 3 days post treatment. Following treatment with 400 mg of praziquantel/kg bw, praziquantel was found in muscle tissue and skin until 5 and 6 days post treatment, respectively. In both muscle tissue and skin, the praziquantel concentrations were consistently declined with the lapse of time. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Praziquantel; Depletion; Sebastes schlegeli; Muscle tissue; Skin
1. Introduction Praziquantel chemotherapy has been employed to control various internal helminth infections in mammals, and has recently been used to control monogenean diseases in fish
*
Corresponding author. Tel.: +82-51-620-6145; fax: +82-51-628-7430. E-mail address: [email protected] (K.H. Kim).
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 3 6 3 - 0
2
C.S. Kim et al. / Aquaculture 219 (2003) 1–7
by bath treatment (Schmahl and Mehlhorn, 1985; Moser et al., 1986; Buchmann, 1987; Schmahl and Taraschewski, 1987; Schmahl et al., 1989; Buchmann et al., 1990; Szekely and Molnar, 1990; Thoney, 1990; Santamarina et al., 1991). Recently, Kim et al. (1998) and Kim and Cho (2000) reported that oral administration of praziquantel was effective in treating Microcotyle sebastis, an blood-sucking gill polyopisthocotylean and a major parasitic disease agent of net-pen farmed rockfish, Sebastes schlegeli, in Korea (Kim and Cho, 2000). Administration of 200 mg praziquantel/kg of body weight (bw) for three times at 24-h intervals was a recommended dose for treatment of M. sebastis (Kim et al., 2001a,b). The use of praziquantel in food fish, however, may lead to residues in fish tissues, and the public health authorities require safe drug withdrawal periods. Muscle and skin are the edible products of fish and should be used for residue analysis in the depletion studies. Although Kim et al. (2001b) reported the depletion of praziquantel in plasma and muscle tissue of rockfish after oral and bath treatments under the laboratory conditions, no information is available on the depletion of praziquantel in the skin and no experiment is conducted under the commercial rockfish-culture conditions. The aim of the present study, therefore, was to determine depletion of praziquantel in muscle tissue and skin of cultured rockfish after oral treatment under the practical net-pen culture conditions. Determination of praziquantel was carried out by reversed-phase (RP)HPLC with ultraviolet detection at 217 nm, and diazepam was used as an internal standard.
2. Materials and methods 2.1. Chemicals and reagents Praziquantel (2-cyclohexylcarbonyl-4-oxo-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1a]isoquinoline) and the internal standard, diazepam (7-chloro-1-methyl-5-phenyl-3H-1,4benzodiazepin-2[1H]-one) were kindly donated by Shinpoong Pharma (Seoul, Korea). Acetonitrile for the mobile phase and distilled water were of chromatographic grade (E. Merck, Germany). Standard solutions of praziquantel were made by dilution of stock solution (10 Ag of praziquantel/1 ml of mobile phase) with mobile phase. The internal standard solution was prepared by dissolving 10 Ag of diazepam into 1 ml of mobile phase. 2.2. Fish and experimental regime Three net-pens, each containing about 10,000 rockfish weighing about 200 g, in a local rockfish farm were used for the experiment. Before feeding praziquantel-supplemented diets, the consumed amount of a feed given at a.m. was enumerated for 3 days, and the quantity of praziquantel added to the food was calculated so that the amount of praziquantel ingested daily by fish was 200 or 400 mg/kg bodyweight (bw). Each group of fish in the three net-pens was designated as control (C), 200 mg of praziquantel/kg bw (200 P), and 400 mg of praziquantel/kg bw (400 P). The fish in group C were fed a moist-
C.S. Kim et al. / Aquaculture 219 (2003) 1–7
3
pelleted fish meal (control diet) throughout the experiment The fish in groups 200 P and 400 P were fed diets supplemented with praziquantel at a rate of 200 or 400 mg of praziquantel/kg bw, respectively, for three times at 24-h intervals. The medicated diet was made by adding praziquantel to the feed mix prior to pelleting. The water temperature was 19 –20 jC. At 1 –8 days after last treatment, 10 fish were taken randomly from each netpen daily, and were kept frozen at 70 jC until analyzed. 2.3. Chromatographic conditions The instruments used were a Hewlett-Packard (HP1100 Series, USA) high-performance liquid chromatograph equipped with QUAT pump (HP1100 Series G1311A), an automatic gradient controller (HP1100 Series G1324A), an injection valve fitted with 5 ml sampling loop, a variable-wavelength UV detector and a data module. Analysis was performed on a ODS2 C18 column (125 4 mm, Hewlett-Packard) with acetonitrile-water (45:55, v/v) as the mobile phase. The column was kept at room temperature (20 –24 jC) and the flow rate was kept constant at 1.0 ml/min. The detector wavelength was set 217 nm. Between each 200 Al injection, the column was washed for 30 min with 100% acetonitrile. The detection limit of the assay was 0.05 Ag/g in both skin and muscle tissues. 2.4. Sample preparation Both a 2-g portion of muscle tissue or skin was weighed into a 15-ml corning tube, and 8.6 ml of 100% acetonitrile was added. The sample was ground using a homogenizer (ART-Moderne Labortechnik, Mu¨lheim, Germany), and then 0.4 ml of internal standard solution was added. After allowing to stand for 10 min at 4 jC, the sample was centrifuged at 10,000 g for 10 min, and the supernatant was collected. The collected supernatant was evaporated to dryness with a speed vacuum (Heto-Holten, Copenhagen, Denmark). The dry residue was dissolved in 1 ml of mobile phase, and a portion of 200 Al was injected into the HPLC. 2.5. Calibration curve Skin homogenate was spiked with standard solutions of praziquantel and internal standard to yield concentrations of 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 Ag/ml of praziquantel and 4.0 Ag/ml of the latter. The sample preparation was conducted as the above procedure, and each level, was assayed in triplicate. The calibration curve of muscle tissue determined in an earlier study (Kim et al., 2001b) was used to determine the level of praziquantel in muscle tissue.
3. Results Chromatograms of skin homogenate for calibration are shown in Fig. 1. A linear relationship (R2 = 0.999) was found when the ratio of the peak height of praziquantel in
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C.S. Kim et al. / Aquaculture 219 (2003) 1–7
Fig. 1. Chromatograms of praziquantel (PQ) determination in rockfish skin for standardization. Skin samples (a) spiked with 10 A/ml of PQ; (b) spiked with 5 Ag/ml of PQ; (c) spiked with 2.5 Ag/ml of PQ; (d) spiked with 1 Ag/ ml of PQ; (e) spiked with 0.5 Ag/ml of PQ. IS, internal standard.
skin to that of the internal standard was plotted against the concentration of praziquantel in the range of 0.25 –10.0 Ag/ml. The levels of praziquantel found in skin and muscle tissue after oral administration of praziquantel are shown in Table 1, and those chromatograms are shown in Figs. 2 and 3. In Table 1 Praziquantel in rockfish muscle tissue and skin samples after three oral doses of praziquantel 200 (200 P) or 400 mg/kg bw (400 P) at 24-h intervals (10 fish per each sampling day) Sampling day (days after treatment)
1 2 3 4 5 6 7 8 Values are mean F standard deviation. nd, below limit of determination.
Muscle (Ag/g)
Skin (Ag/g)
200 P
400 P
200 P
400 P
0.24 F 0.27 nd nd nd nd nd nd nd
1.20 F 0.80 0.68 F 0.39 0.73 F 0.50 0.67 F 0.89 0.11 F 0.19 nd nd nd
0.72 F 0.10 0.21 F 0.05 0.09 F 0.15 nd nd nd nd nd
1.29 F 0.70 0.24 F 0.06 0.16 F 0.14 0.06 F 0.10 0.08 F 0.14 0.06 F 0.10 nd nd
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Fig. 2. Chromatograms of praziquantel (PQ) determination in rockfish skin after oral administration of three doses of either praziquantel 200 mg (200 P group) or 400 mg/kg b.w. (400 P group) given at 24-h intervals. After (a) 1 day of 200 P group; (b) 3 days of 200 P group; (c) 4 days of 200 P group; (d) 1 day of 400 P group; (e) 2 days of 400 P group; (f) 6 days of 400 P group. IS, internal standard.
the group of fish fed 200 mg of praziquantel/kg bw, praziquantel was detected in muscle tissue until 1 day and in skin until 3 days post treatment. Following treatment with 400 mg of praziquantel/kg bw, praziquantel was found in muscle tissue and skin until 5 and 6 days post treatment, respectively. The levels of praziquantel in muscle tissue and skin were sharply declined at 2 days post-treatment, and were consistently declined with the lapse of day in both praziquantel-administered groups.
4. Discussion The present results indicate that the withdrawal time of praziquantel in muscle and skin of rockfish when fed 200 mg of praziquantel/kg bw for three times at 24-h intervals was less than 4 days. The residue levels of praziquantel in muscle and skin of rockfish administered praziquantel 400 mg/kg bw, at a day’s interval for three times were eliminated within 7 days post treatment. According to the preliminary study of Rogstad et al. (1987), the highest residue level of praziquantel in muscle and serum of rainbow trout, which were given a single oral dose (10 mg/kg bw) of praziquantel, was obtained 7 h after treatment, and no residues were found 48 h after medication.
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Fig. 3. Chromatograms of praziquantel (PQ) determination in rockfish muscle tissue after oral administration of three doses of either praziquantel 200 mg (200 P group) or 400 mg/kg b.w. (400 P group) given at 24 h intervals. After (a) 1 day of 200 P group; (b) 2 days of 200 P group; (c) 1 day of 400 P group; (d) 3 days of 400 P group; (e) 5 days of 400 P group. IS, internal standard.
Using a bioassay with parasitic cercariae as test organisms for determination of praziquantel levels, Bjo¨rklund and Bylund (1987) reported that the peak values of praziquantel in different tissues (serum, muscle, liver, bile fluid, kidney) of rainbow trout were reached 4– 16 h after a single oral administration of praziquantel at a dose of 500 mg/kg bw, and by 32 h post administration, 67 – 96% of the maximum amounts had been eliminated from the tissues. Recently, Kim et al. (2001b) reported that the residue levels of praziquantel in plasma and muscle of rockfish, administered orally at a single dose of 400 mg/kg bw, were highest at the 9 h post treatment, and eliminated within 5 days post treatment. The sharp decrease of praziquantel levels in muscle and skin at 2 days post-administration in this study was similar with the results of those reports in spite of repeated doses. In the present study, withdrawal time of praziquantel in skin was longer than that in muscle tissue. The accumulation and persistence of several chemotherapeutics in the skin of fish as compared to the muscle tissue have been reported (Plakas et al., 1990; Steffenak et al., 1991; Iosifidou et al., 1997). The present results, also, suggest that the skin of rockfish is an important tissue for praziquantel residue, and should be analyzed in the determination of praziquantel withdrawal time.
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Acknowledgements This study was supported by a grant from the Dae Sung Microbiological Laboratories and a grant from the Ministry of Maritime Affairs and Fisheries, Republic of Korea. References Bjo¨rklund, H., Bylund, G., 1987. Absorption, distribution and excretion of the anthelmintic praziquantel (Droncit) in rainbow trout (Salmo gairdneri R.). Parasitol. Res. 73, 240 – 244. Buchmann, K., 1987. The effects of praziquantel on the monogenean gill parasite Pseudodactylogyrus bini. Acta Vet. Scand. 28, 447 – 450. Buchmann, K., Szekely, C., Bjerregaard, J., 1990. Treatment of Pseudodactylogyrus infestations of Anguilla anguilla: II. Trials with bunamidine, praziquantel and levamizole. Bull. Eur. Assoc. Fish Pathol. 10, 18 – 20. Iosifidou, E.G., Haagsma, N., Tanck, M.W.T., Boon, J.H., Olling, M., 1997. Depletion study of fenbendazole in rainbow trout (Oncorhynchus mykiss) after oral and bath treatment. Aquaculture 154, 191 – 199. Kim, K.H., Cho, J.B., 2000. Treatment of Microcotyle sebastis (Monogenea: Polyopisthocotylea) infestation with praziquantel in an experimental cage simulating commercial rockfish Sebastes schlegeli culture conditions. Dis. Aquat. Org. 40, 229 – 231. Kim, K.H., Park, S.I., Jee, B.Y., 1998. Efficacy of oral administration of praziquantel and mebendazole against Microcotyle sebastis (Monogenea) infestation of cultured rockfish (Sebastes schlegeli). Fish Pathol. 33, 467 – 471. Kim, K.H., Lee, E.H., Kwon, S.R., Cho, J.B., 2001a. Treatment of Microcotyle sebastis infestation in cultured rockfish Sebastes schlegeli by oral administration of praziquantel in combination with cimetidine. Dis. Aquat. Org. 44, 133 – 136. Kim, K.H., Kim, C.S., Kim, J.W., 2001b. Depletion of praziquantel in plasma and muscle tissue of cultured rockfish Sebastes schlegeli after oral and bath treatment. Dis. Aquat. Org. 45, 203 – 207. Moser, M., Sakanari, J., Heckmann, R., 1986. The effects of praziquantel on various larval and adult parasites from freshwater and marine snails and fish. J. Parasitol. 72, 175 – 176. Plakas, S.M., Dickey, R.W., Barron, M.G., Guarino, A.M., 1990. Tissue distribution and renal excretion of ormetoprim after intramuscular and oral administration in the channel catfish (Ictalurus punctatus). Can. J. Fish. Aquat. Sci. 47, 766 – 771. Rogstad, A., Hormazabal, V., Yndestad, M., 1987. Extraction of praziquantel from fish tissue and its determination by high-performance liquid chromatography. J. Chromatogr. 391, 328 – 333. Santamarina, M.T., Tojo, J.L., Ubeira, F.M., Quintero, P., Sanmartin, M.L., 1991. Anthelmintic treatment against Gyrodactylus sp. infecting rainbow trout Oncorhynchus mykiss. Dis. Aquat. Org. 10, 39 – 43. Schmahl, G., Mehlhorn, H., 1985. Treatment of fish parasites: 1. Praziquantel effective against Monogenea (Dactylogyrus vastator, Dactylogyrus extensus, Diplozoon paradoxum). Z. Parasitenkd. 71, 727 – 737. Schmahl, G., Taraschewski, H., 1987. Treatment of fish parasites: 2. Effects of praziquantel, niclosamide, levamisole-HCl, and metrifonate on Monogenea (Gyrodactylus aculeati, Diplozoon paradoxum). Parasitol. Res. 73, 341 – 351. Schmahl, G., Taraschewski, H., Mehlhorn, H., 1989. Chemotherapy of fish parasites. Parasitol. Res. 75, 503 – 511. Steffenak, I., Hormazabal, V., Yndestad, M., 1991. Reservoir quinolone residues in fish. Food Addit. Contam. 8, 777 – 780. Szekely, C., Molnar, K., 1990. Treatment of Ancylodiscoides vistulensis monogenean infestations of the European catfish (Silurus glanis). Bull. Eur. Assoc. Fish Pathol. 10, 74 – 77. Thoney, D.A., 1990. The effects of trichlorfon, praziquantel and copper sulphate on various stages of the monogenean Benedeniella posterocolpa, a skin parasite of the cownose ray, Rhinoptera bonasus (Mitchill). J. Fish Dis. 13, 385 – 389.
Depletion of praziquantel in muscle tissue and skin of cultured rockfish (Sebastes schlegeli) under the commercial culture conditions Chun Soo Kim, Jae Bum Cho, Kyoung Jin Ahn, Jae Il Lee, Ki Hong Kim * Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Pusan 608-737, South Korea Received 30 March 2002
Abstract Depletion of praziquantel in muscle tissue and skin of cultured rockfish (Sebastes schlegeli) under the commercial net-pen farm conditions was analyzed. The levels of praziquantel were determined after the administration of three doses of either praziquantel 200 or 400 mg/kg of body weight (bw) given at 24-h intervals. Fish were collected daily following last drug administration and analyzed for praziquantel in muscle tissue and skin by reversed-phase high-performance liquid chromatography (RP-HPLC) using diazepam as an internal standard. In the group of fish fed 200 mg of praziquantel/kg bw, praziquantel was detected in muscle tissue until 1 day and in skin until 3 days post treatment. Following treatment with 400 mg of praziquantel/kg bw, praziquantel was found in muscle tissue and skin until 5 and 6 days post treatment, respectively. In both muscle tissue and skin, the praziquantel concentrations were consistently declined with the lapse of time. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Praziquantel; Depletion; Sebastes schlegeli; Muscle tissue; Skin
1. Introduction Praziquantel chemotherapy has been employed to control various internal helminth infections in mammals, and has recently been used to control monogenean diseases in fish
*
Corresponding author. Tel.: +82-51-620-6145; fax: +82-51-628-7430. E-mail address: [email protected] (K.H. Kim).
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 3 6 3 - 0
2
C.S. Kim et al. / Aquaculture 219 (2003) 1–7
by bath treatment (Schmahl and Mehlhorn, 1985; Moser et al., 1986; Buchmann, 1987; Schmahl and Taraschewski, 1987; Schmahl et al., 1989; Buchmann et al., 1990; Szekely and Molnar, 1990; Thoney, 1990; Santamarina et al., 1991). Recently, Kim et al. (1998) and Kim and Cho (2000) reported that oral administration of praziquantel was effective in treating Microcotyle sebastis, an blood-sucking gill polyopisthocotylean and a major parasitic disease agent of net-pen farmed rockfish, Sebastes schlegeli, in Korea (Kim and Cho, 2000). Administration of 200 mg praziquantel/kg of body weight (bw) for three times at 24-h intervals was a recommended dose for treatment of M. sebastis (Kim et al., 2001a,b). The use of praziquantel in food fish, however, may lead to residues in fish tissues, and the public health authorities require safe drug withdrawal periods. Muscle and skin are the edible products of fish and should be used for residue analysis in the depletion studies. Although Kim et al. (2001b) reported the depletion of praziquantel in plasma and muscle tissue of rockfish after oral and bath treatments under the laboratory conditions, no information is available on the depletion of praziquantel in the skin and no experiment is conducted under the commercial rockfish-culture conditions. The aim of the present study, therefore, was to determine depletion of praziquantel in muscle tissue and skin of cultured rockfish after oral treatment under the practical net-pen culture conditions. Determination of praziquantel was carried out by reversed-phase (RP)HPLC with ultraviolet detection at 217 nm, and diazepam was used as an internal standard.
2. Materials and methods 2.1. Chemicals and reagents Praziquantel (2-cyclohexylcarbonyl-4-oxo-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1a]isoquinoline) and the internal standard, diazepam (7-chloro-1-methyl-5-phenyl-3H-1,4benzodiazepin-2[1H]-one) were kindly donated by Shinpoong Pharma (Seoul, Korea). Acetonitrile for the mobile phase and distilled water were of chromatographic grade (E. Merck, Germany). Standard solutions of praziquantel were made by dilution of stock solution (10 Ag of praziquantel/1 ml of mobile phase) with mobile phase. The internal standard solution was prepared by dissolving 10 Ag of diazepam into 1 ml of mobile phase. 2.2. Fish and experimental regime Three net-pens, each containing about 10,000 rockfish weighing about 200 g, in a local rockfish farm were used for the experiment. Before feeding praziquantel-supplemented diets, the consumed amount of a feed given at a.m. was enumerated for 3 days, and the quantity of praziquantel added to the food was calculated so that the amount of praziquantel ingested daily by fish was 200 or 400 mg/kg bodyweight (bw). Each group of fish in the three net-pens was designated as control (C), 200 mg of praziquantel/kg bw (200 P), and 400 mg of praziquantel/kg bw (400 P). The fish in group C were fed a moist-
C.S. Kim et al. / Aquaculture 219 (2003) 1–7
3
pelleted fish meal (control diet) throughout the experiment The fish in groups 200 P and 400 P were fed diets supplemented with praziquantel at a rate of 200 or 400 mg of praziquantel/kg bw, respectively, for three times at 24-h intervals. The medicated diet was made by adding praziquantel to the feed mix prior to pelleting. The water temperature was 19 –20 jC. At 1 –8 days after last treatment, 10 fish were taken randomly from each netpen daily, and were kept frozen at 70 jC until analyzed. 2.3. Chromatographic conditions The instruments used were a Hewlett-Packard (HP1100 Series, USA) high-performance liquid chromatograph equipped with QUAT pump (HP1100 Series G1311A), an automatic gradient controller (HP1100 Series G1324A), an injection valve fitted with 5 ml sampling loop, a variable-wavelength UV detector and a data module. Analysis was performed on a ODS2 C18 column (125 4 mm, Hewlett-Packard) with acetonitrile-water (45:55, v/v) as the mobile phase. The column was kept at room temperature (20 –24 jC) and the flow rate was kept constant at 1.0 ml/min. The detector wavelength was set 217 nm. Between each 200 Al injection, the column was washed for 30 min with 100% acetonitrile. The detection limit of the assay was 0.05 Ag/g in both skin and muscle tissues. 2.4. Sample preparation Both a 2-g portion of muscle tissue or skin was weighed into a 15-ml corning tube, and 8.6 ml of 100% acetonitrile was added. The sample was ground using a homogenizer (ART-Moderne Labortechnik, Mu¨lheim, Germany), and then 0.4 ml of internal standard solution was added. After allowing to stand for 10 min at 4 jC, the sample was centrifuged at 10,000 g for 10 min, and the supernatant was collected. The collected supernatant was evaporated to dryness with a speed vacuum (Heto-Holten, Copenhagen, Denmark). The dry residue was dissolved in 1 ml of mobile phase, and a portion of 200 Al was injected into the HPLC. 2.5. Calibration curve Skin homogenate was spiked with standard solutions of praziquantel and internal standard to yield concentrations of 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 Ag/ml of praziquantel and 4.0 Ag/ml of the latter. The sample preparation was conducted as the above procedure, and each level, was assayed in triplicate. The calibration curve of muscle tissue determined in an earlier study (Kim et al., 2001b) was used to determine the level of praziquantel in muscle tissue.
3. Results Chromatograms of skin homogenate for calibration are shown in Fig. 1. A linear relationship (R2 = 0.999) was found when the ratio of the peak height of praziquantel in
4
C.S. Kim et al. / Aquaculture 219 (2003) 1–7
Fig. 1. Chromatograms of praziquantel (PQ) determination in rockfish skin for standardization. Skin samples (a) spiked with 10 A/ml of PQ; (b) spiked with 5 Ag/ml of PQ; (c) spiked with 2.5 Ag/ml of PQ; (d) spiked with 1 Ag/ ml of PQ; (e) spiked with 0.5 Ag/ml of PQ. IS, internal standard.
skin to that of the internal standard was plotted against the concentration of praziquantel in the range of 0.25 –10.0 Ag/ml. The levels of praziquantel found in skin and muscle tissue after oral administration of praziquantel are shown in Table 1, and those chromatograms are shown in Figs. 2 and 3. In Table 1 Praziquantel in rockfish muscle tissue and skin samples after three oral doses of praziquantel 200 (200 P) or 400 mg/kg bw (400 P) at 24-h intervals (10 fish per each sampling day) Sampling day (days after treatment)
1 2 3 4 5 6 7 8 Values are mean F standard deviation. nd, below limit of determination.
Muscle (Ag/g)
Skin (Ag/g)
200 P
400 P
200 P
400 P
0.24 F 0.27 nd nd nd nd nd nd nd
1.20 F 0.80 0.68 F 0.39 0.73 F 0.50 0.67 F 0.89 0.11 F 0.19 nd nd nd
0.72 F 0.10 0.21 F 0.05 0.09 F 0.15 nd nd nd nd nd
1.29 F 0.70 0.24 F 0.06 0.16 F 0.14 0.06 F 0.10 0.08 F 0.14 0.06 F 0.10 nd nd
C.S. Kim et al. / Aquaculture 219 (2003) 1–7
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Fig. 2. Chromatograms of praziquantel (PQ) determination in rockfish skin after oral administration of three doses of either praziquantel 200 mg (200 P group) or 400 mg/kg b.w. (400 P group) given at 24-h intervals. After (a) 1 day of 200 P group; (b) 3 days of 200 P group; (c) 4 days of 200 P group; (d) 1 day of 400 P group; (e) 2 days of 400 P group; (f) 6 days of 400 P group. IS, internal standard.
the group of fish fed 200 mg of praziquantel/kg bw, praziquantel was detected in muscle tissue until 1 day and in skin until 3 days post treatment. Following treatment with 400 mg of praziquantel/kg bw, praziquantel was found in muscle tissue and skin until 5 and 6 days post treatment, respectively. The levels of praziquantel in muscle tissue and skin were sharply declined at 2 days post-treatment, and were consistently declined with the lapse of day in both praziquantel-administered groups.
4. Discussion The present results indicate that the withdrawal time of praziquantel in muscle and skin of rockfish when fed 200 mg of praziquantel/kg bw for three times at 24-h intervals was less than 4 days. The residue levels of praziquantel in muscle and skin of rockfish administered praziquantel 400 mg/kg bw, at a day’s interval for three times were eliminated within 7 days post treatment. According to the preliminary study of Rogstad et al. (1987), the highest residue level of praziquantel in muscle and serum of rainbow trout, which were given a single oral dose (10 mg/kg bw) of praziquantel, was obtained 7 h after treatment, and no residues were found 48 h after medication.
6
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Fig. 3. Chromatograms of praziquantel (PQ) determination in rockfish muscle tissue after oral administration of three doses of either praziquantel 200 mg (200 P group) or 400 mg/kg b.w. (400 P group) given at 24 h intervals. After (a) 1 day of 200 P group; (b) 2 days of 200 P group; (c) 1 day of 400 P group; (d) 3 days of 400 P group; (e) 5 days of 400 P group. IS, internal standard.
Using a bioassay with parasitic cercariae as test organisms for determination of praziquantel levels, Bjo¨rklund and Bylund (1987) reported that the peak values of praziquantel in different tissues (serum, muscle, liver, bile fluid, kidney) of rainbow trout were reached 4– 16 h after a single oral administration of praziquantel at a dose of 500 mg/kg bw, and by 32 h post administration, 67 – 96% of the maximum amounts had been eliminated from the tissues. Recently, Kim et al. (2001b) reported that the residue levels of praziquantel in plasma and muscle of rockfish, administered orally at a single dose of 400 mg/kg bw, were highest at the 9 h post treatment, and eliminated within 5 days post treatment. The sharp decrease of praziquantel levels in muscle and skin at 2 days post-administration in this study was similar with the results of those reports in spite of repeated doses. In the present study, withdrawal time of praziquantel in skin was longer than that in muscle tissue. The accumulation and persistence of several chemotherapeutics in the skin of fish as compared to the muscle tissue have been reported (Plakas et al., 1990; Steffenak et al., 1991; Iosifidou et al., 1997). The present results, also, suggest that the skin of rockfish is an important tissue for praziquantel residue, and should be analyzed in the determination of praziquantel withdrawal time.
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