36 DECEMBER 2022 • WORLD AQUACULTURE • WWW.WA S .ORG a similar study with goldfish Carassius auratus. There were four treatment groups, each consisting of two 20-L fish tanks and five fish in each: A) Negative control, no exposure to Ich, no treatment with Biokos; B) Infection control, exposure to Ich, no treatment with Biokos; C) Treatment, “low dose”, exposure to Ich, treatment with Biokos at lowest known efficacious concentration; and D) Treatment, “high dose”, exposure to Ich, treatment with Biokos at double the concentration. The fish in treatment groups B, C and D were pre-infected with Ich by co-habitation for five days with Ich-infected goldfish (Fig. 2). Biokos was added every second day, starting at day 0 and 50 percent of the water was exchanged each day, starting at day 1. Fish mortality, behavior and the number of trophonts on skin, fins and gill surfaces was recorded throughout the 20-d study. All Ich-infected fish not treated with Biokos died before day 14 (Fig. 3). All fish treated with Biokos survived the full length of the study, and much longer. No trophonts were visible on fins, gills, or skin of Biokos-treated fish after day 15. Implicit in these data is that the lower dose of Biokos was sufficient for efficacious treatment. No abnormal behavior was observed in any of the fish in treatment or control groups. This is the second study showing that low concentrations of Biokos can completely counter/cure Ich infection in fish. The study results add another species of fish that is completely tolerant to efficacious concentrations of Biokos, the others being zebrafish and rainbow trout. Effect on Biofilter Functionality Biofilters in fish tanks and some fish farms with recirculation systems (RAS) have the crucial function of turning ammonia from the fish into less toxic compounds. Nitrosomonas bacteria turns ammonia into nitrite and Nitrobacter forms less harmful nitrate from nitrite. In a fish tank, nitrate can be used for growth by any plants present and/or removed by regular water exchange. In RAS aquaculture systems nitrate may be reduced to molecular nitrogen by denitrification bacteria or removed in other ways. Obviously, any remedy for parasites must not be toxic to these bacteria. To study this, we combined surviving fish from the goldfish challenge study described above and assessed total ammonia nitrogen (TAN), nitrite and nitrate for 15 days, using water quality test strips. The rationale for the study was that, if Biokos had harmed the bacteria that facilitates biofiltration, we would see this in this follow-up study as peaks of TAN or nitrite or both with continued feeding. There were six treatments in this study: A) No fish, no biofilter; B) No fish, biofilter, no Biokos; C) Fish (n=6), “low” Biokos, biofilter; D) No fish, “low” Biokos”, biofilter; E) Fish (n=6), “high” Biokos, biofilter; and F) No fish, “high” Biokos, biofilter. Feed was added every second day, and no water exchange was performed during the study. All goldfish from treatments C and E survived to the end of the study, indicating that water quality had been good. Biofilters remained fully functional, as indicated by measured concentrations of TAN, nitrite and nitrate (Fig. 4). The TAN assay has a sensitivity of 0.25 mg/L FIGURE 2. Goldfish scales with embedded Ich trophonts. FIGURE 3. Cumulative mortality of goldfish in challenge study, as percent survival per day since treatment start. Biokos is a new natural agent that can fill the need for an effective solution to Ich in aquaculture as well as in ornamental fish. It is a cyclic lipopeptide that kills the Ich parasites by affecting their cellular membrane integrity. Here we have shown that Biokos at very low concentrations can cure goldfish from Ich infection, confirming similar earlier results with rainbow trout. We have also shown that Biokos does not affect the health and functionality of biofilter bacteria needed for conversion of ammonia to nitrate. This means that it can be applied to RAS systems (and ornamental fish tanks) without problems for fish or biofilter health. Finally, we have shown that microorganisms in mature aquaculture and fish tank water will inactivate and degrade Biokos within hours or days into harmless components.
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