Abstract
Bioencapsulation is a fairly recent technique developed to deliver medications to fish that may be difficult to medicate by traditional means. Several papers have been published concerning the bioencapsulation of various antimicrobials by brine shrimp (Artemia sp.). These studies use zones of inhibition, efficacy trials and HPLC to determine the availability of drug in the brine shrimp.1,5,6 In this study, the authors determined the ability of live adult brine shrimp to bioencapsulate metronidazole in sufficient levels to treat aquarium fish with the accepted oral dosages for both anaerobic bacteria and protozoal parasites. To mimic clinical situations, pulverized over-the-counter metronidazole pills (TEVA pharmaceuticals, Sellersville, PA USA 18960), comprising of 65.25% pure metronidazole, were mixed in artificial seawater (ASW) with brine shrimp. High-performance liquid chromatography was used to determine the solubility of the pill (including inert products) in artificial seawater (pH = 8.16 ± 0.03, 27.1 ± 0.3°C, 31.4 ± 0.9% Sal) to be 17.000 ppt (g/L) yielding a concentration of pure metronidazole of 8597 ± 343 ppm. The amount of metronidazole encapsulated in adult brine shrimp was quantified over a range of concentrations and time periods. At a constant duration of 180 min (3 hr), the amount of metronidazole accumulated in Artemia increased as the quantity of pill added to the solution increased. A maximum pure metronidazole concentration in the brine shrimp of 1858 ± 55 ppm was reached with the addition of 50.000 g of pill per L of ASW. In timed experiments with a constant bath of 33.640 g of pill per L of ASW, metronidazole accumulated in the brine shrimp until the 720 min (12 hr) mark, reaching a maximum concentration of metronidazole in brine shrimp of 1708 ± 143 ppm. Additional time did not yield higher concentrations of metronidazole in the Artemia. The addition of an enrichment diet (DC DHA Selco®, INVE Aquaculture NV, Hoogveld, Dendermonde, Belgium) had a significantly negative effect on the uptake of metronidazole by the Artemia (p = 0.001). A pilot study, with 3 species of fish (yellow tang, Zebrasoma flavescens; french grunt, Haemulon flavolineatum; and clownfish, Amphiprion sp.) indicated the fish daily consumed 2.5 ± 0.5% body weight in nonmedicated Artemia. This study indicated that brine shrimp immersed in a metronidazole bath can bioaccumulate metronidazole at levels which are considered therapeutic doses in humans and other animals.2,3,4 Even though further work needs to be performed to determine if these doses produce therapeutic blood levels in fish, this study suggests previous empiric recommendations for soaking brine shrimp in metronidazole solutions may be of insufficient concentration or duration to achieve therapeutic dosages for treating fish.
References
1. Majack TJ, MB Rust, KC Massee, GW Kissil, RW Hardy, ME Peterson. 2000. Bioencapsulation of erythromycin using adult brine shrimp, Artemia franciscana (Latreille). Journal of Fish Diseases 23: 71-76.
2. Noga EJ. 2000. In: Fish Disease: Diagnosis and Treatment. Iowa State University Press, Iowa. Pp. 290.
3. Physician's Desk Reference. 2001. Pp. 3002-3004
4. Plumb DC. 1999. In: Veterinary Drug Handbook, 3rd ed. Iowa State University Press, Iowa. Pp. 572-575.
5. Robles R, P Sorgeloos, H Van Duffel, H Nelis. 1998. Progress in biomedication using live foods. Journal of Applied Ichthyology 14: 207-212.
6. Touraki M, P Rigas, P Pergandas, C Kastritsis. 1995. Determination of oxytetracycline in the live fish feed Artemia using high-performance liquid chromatography with ultraviolet detection. Journal of Chromatography 663: 167-171.