Introduction Materials and methods

Ž . Aquaculture 186 2000 221–231 www.elsevier.nlrlocateraqua-online ž Treatment of trichodiniasis in eel Anguilla anguilla reared in recirculation systems in Denmark: alternatives to formaldehyde Hans C.K. Madsen a , Kurt Buchmann b , Stig Mellergaard a, a Danish Institute for Fisheries Research, Fish Diseases Laboratory, Stigboejlen 4, DK-1870 Frederiksberg C, Denmark b Royal Veterinary and Agricultural UniÕersity, Stigboejlen 4, DK-1870 Frederiksberg C, Denmark Received 21 April 1999; received in revised form 10 December 1999; accepted 14 December 1999 Abstract Ž . Treatment of trichodiniasis in recirculation eel farms using formalin 37 has been found insufficient. We have screened 30 different chemical substances for efficacy against natural Ž . infections with trichodinids Trichodina jadranica on skin and gills of eels. The following Ž . Ž . substances showed high parasiticidal effect: acriflavin 25 ppm , bithionol 0.1 ppm , chloramine Ž . w Ž . Ž . Ž . T 50 ppm , Detarox AP 45 ppm , malachite green 1 ppm , raw garlic 200 ppm , potassium Ž . w Ž . permanganate 20 ppm and Virkon PF vet. 20 ppm . Preliminary screening revealed that the anthelmintic, bithionol, and the decomposable disinfectants Detarox AP and Virkon PF w vet. were potential therapeutics and these were tested on infected eel stocks in recirculation production units. The following treatments showed a high parasiticidal effect: bithionol at 0.1 ppm in a w Ž . recirculation system including the biofilters; Detarox AP 45 ppm in recirculation systems with w Ž the biofilters temporarily isolated for 1 h and Virkon PF vet. 40 ppm; 25 ppm at start . supplemented with 15 ppm 1 h later in rearing tanks temporarily isolated from recirculation systems for 3 h. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Recirculation aquaculture; Trichodiniasis; Eel; Treatment

1. Introduction

Ž . In Denmark, European eels Anguilla anguilla have been produced in heated Ž . 23–25 8C recirculation systems with biofilters during the last two decades. Infestations Corresponding author. Ž . Ž . E-mail addresses: kurt.buchmannvetmi.kvl.dk K. Buchmann , smedfu.min.dk S. Mellergaard . 0044-8486r00r - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S 0 0 4 4 - 8 4 8 6 9 9 0 0 3 7 9 - 8 Ž . with the trichodinid Trichodina jadranica Raabe, 1958 Ciliophora: Trichodinidae on Ž gills and skin of cultured eels have caused substantial financial losses Lyholt and . Buchmann, 1995; Lyholt et al., 1998 . Heavily infected eels become lethargic, secrete Ž excessive mucus and lose appetite resulting in inhibition of growth Amlacher, 1972; Bauer et al., 1973; Schaperclaus, 1979; Mellergaard and Dalsgaard, 1987; Sanmartin et ¨ . al., 1991; Lom and Dykova, 1992; Buchmann and Bresciani, 1997 . ´ Ž Previously, trichodiniasis was effectively controlled with formalin 50–120 ppm, one . to two treatments per week , but it now appears insufficient to control the infection. Little information about treatment of protozoan parasites under production conditions in aquaculture has been published. Also, relatively few agents are available. In addition to the lack of information some of the known efficacious potent antiprotozoan substances Ž . e.g., malachite green and acriflavin have been categorised as potentially carcinogenic. In order to find a more efficacious trichodiniasis control agent and reduce the human health hazards of formalin exposure we screened 30 potential chemical agents for efficacy against T. jadranica and tested the most promising candidates in recirculation production units.

2. Materials and methods

2.1. Locations Two different eel farms with a reputation of having frequent outbreaks of trichodinia- sis were selected for the experiments 2.2. Infection leÕel. Ž . Ž . Small pigmented eels elvers 1–30 g were examined by scraping mucus with a scalpel from the total surface of skin and of the four gill arches from one side of the eel. Ž . Larger eels 70–250 g were scraped only once on the central upper dorsal part of the body at the border to the dorsal fin in one third of the length of the eel. On eels of this Ž . Ž size the majority of trichodinids T. jadranica were found on the skin unpublished . data . Sampled mucus was transferred to glass slides and examined for trichodinids Ž microscopically. The approximate number of trichodinids number on skin and gills . pooled on small eels and on larger eels were converted to one out of five different infection categories for each fish: 0 parasites s category 0; 1–10 parasites s category 1; 11–100 parasites s category 2; 100–1000 parasites s category; 1000–10 000 parasites Ž . s category 4. The infection level in a group n G 10 of examined fish was expressed as the mean of the infection category of each fish. Mean infection categories were primarily used as an indicator of shifts in the infection levels within a stock of fish. 2.3. Aquarium trials 2.3.1. Screening substances The chemicals selected for screening of efficacy against T. jadranica are listed in Table 1. Low pH and sodium chloride are included in ranges, which could be of interest for the farmers. Extreme nitrate concentrations were tested, too, because relative high Ž y . concentrations 600–1000 mg NO are suspected to be unfavourable to the parasite 3 Ž . according to the eel farmers. Infected pigmented eels mean weight, 4.5 g removed Ž from the production systems were introduced into plastic aquaria 20 l water originating from the rearing tanks, characteristics: 5–15 mg organic dry matterrl, pH range . 6.5–7.0, temperature 25 1 8C . During the exposures ammonia was removed from the Ž . water using ‘‘biofilters’’ Power filter, 3 l, EHEIM 2213, Germany . Groups of 10 infected eels were exposed to the different chemicals in aquaria without any water exchange and the mean infection category was determined 24 h after the initial exposure. A substance was considered effective if all or almost all parasites were killed at a concentration not toxic to eels. In order to establish the therapeutic dose, replicate experiments were conducted with the effective substances in concentrations in the vicinity of the lowest dose needed for a good parasiticidal effect. Substances lethal to eels were considered ineffective. 2.3.1.1. Effects on eel. Substances were considered toxic if they caused nonreversible Ž . effects on eels e.g., spasms, loss of equilibrium, death and as nontoxic if only short-term reversible effects on eels were observed; e.g., slightly elevated mucus secretion was noted as a nontoxic effect. Ž . 2.3.1.2. Effect of exposure time. Five eelsrgroup were exposed to bithionol 0.1 ppm or w Ž . Virkon PF vet. 20 ppm for 1, 2, 3 or 4 h and afterwards transferred to water without substance and examined for trichodinids during the fourth hour. Eels were exposed to w Ž . Detarox AP 45 ppm for 1 2, 1, 11 2 or 2 h and examined during the second hour. The shortest exposure times needed to remove all or almost all parasites were used as exposure time for the experimental treatments under farmed conditions. All concentra- Ž . tions reported are nominal concentrations. Bithionol was suspended in ethanol 1:20 ; control eels were exposed to an equal amount of solvent as used in the treated group. 2.3.2. Tolerance of eels to chemicals Ž . w Ž . The acute and chronic toxity of bithionol 0.05–1 ppm , Detarox AP 10–150 ppm w Ž . Ž . and Virkon PF vet. 10–100 ppm in pure tap water pH s 7.2, 24 1 8C was Ž . Ž . examined in pigmented mean weight, 6.6 g and glass eels mean weight, 0.34 g . Because Virkon PF w vet. may catalyse the conversion of chloride ions to gaseous Ž chlorine at relatively high salinities personal communication, Pharmacia and Upjohn, . w Copenhagen the toxicity of Virkon PF vet. to pigmented eels was determined in tap water modified to salinities of 5, 10 and 15 ppt. The various salinities were obtained by adding NaCl to tap water at 24 8C. Groups of seven eels were exposed to different concentrations of the substances for 5 and 96 h. The exposed groups were transferred to tap water after 5 h of exposure and monitored for an additional 91 h. 2.4. Full-scale trials in eel farms w w Ž . Ž Bithionol, Detarox AP and Virkon PF vet. as well as formalin 37 as a . reference therapy were tested on whole stocks of T. jadranica infected eels in recirculation production units. Important data on treatment designs are shown in Table 3. Feeding was suspended 3–4 h after the introduction of the substance to the water. The substances were added either to the total water volume in the recirculation unit Table 1 Parasiticidal effect of various chemicals on Trichodina jadranica in eel. Trials were conducted as 24 h water Ž . bath exposure 25 8C of infected eels. Infection levels are stated as infection categories posttreatment w x Ž . Ž . w x Ž . w x Ž . w Origin of substances product number other data : 1 Sigma A 8251 . 2 Sigma A 8126 . 3 Sigma T x Ž . w x Ž . Ž 9881 . 4 BDH, UK 27670 4E . 5 Perdomini, Verona, Italy 15–25 peracetic acid, 4–5 hydrogen . Ž . Ž . Ž . w x Ž . w x Ž . peroxide . 6 : Raw and squeezed . 7 Merck 1.01398.0025 . 8 Fluka 60459 . 9 Antec International, UK Ž Ž . decomposable inorganic salts, alcylbenzene-sulphonate 90 biogradability under OECD test conditions . Ž . w x Ž . w x Ž . Ž . w x 10 Sigma A 0542 . 11 Sigma S 8259 . 12 Jansen Pharmaceutica, Beerse, Holland. 13 Fluka 25745 . Ž . Ž . Ž . w x Ž . Ž . w x Ž . 14 Duphar, Wees, Holland diflubenzuron . 15 Sigma D 7044 . 16 Hoechst. 17 DAK 726604 . 18 w x Ž . Ž . w x Ž . w x Ž . Ž Riedel-de Haen, Germany 31440 sodium nitrate . 19 Sigma M 2004 . 20 Sigma O 0877 . 21 Ph. Eur., ¨ . Ž . Ž . Ž . index 1272 . 22 Lowered with hydrochloric acid, eels came from pH 7.2 . 23 Pedersen J.L., Nibe, Ž . w x Ž . Ž . Ž . Ž . Denmark. 24 RBI, MA, USA Q-106 . 25 Raised from 3000 to 13,000 ppm . 26 Ph. Eur., index 1552 . Ž . Ž . Ž . Ž . w x 27 pH s 7.2. 28 Orion. 29 Bayer. 30 Fluka 53260 . Substances with parasiticidal Treatment Control, mean Treated, mean Dead eels Ž . effect data on substance concentration infection category infection category in treated group Ž . Ž . Ž . ppm S.D. S.D. due to toxicity Ž . Ž . Ž . Acriflavin hydrochlorid 1 25 2.2 0.6 0.0 0.0 Ž . Ž . Ž . Acriflavin neutral 2 20 2.4 0.7 0.1 0.3 Ž . Ž . 25 2.2 0.6 0.0 0.0 Ž . Ž . Ž . Bithionol 3 0.1 2.8 0.5 0.5 0.5 Ž . Ž . Ž . 0.25 1 h 2.8 0.4 0.0 0.0 Ž . Ž . Ž . Chloramine T, 24 4 40 2.6 0.5 1.1 1.1 Ž . Ž . 50 2.2 0.4 0.5 0.6 60 w a Ž . Ž . Ž . Detarox AP 5 20 2.0 0.0 0.3 0.5 a Ž . Ž . 30 2.0 0.0 0.0 0.0 Ž . Ž . 30 1.9 0.7 1.9 0.6 Ž . Ž . 40 2.0 0.8 1.0 0.9 Ž . Ž . 45 2.4 0.5 0.4 0.5 a Ž . Ž . Formalin, 37 50 2.1 0.4 1.0 0.8 a Ž . Ž . 75 2.3 0.5 0.5 0.5 Ž . Ž . 50 2.4 0.5 2.0 0.7 Ž . Ž . 75 3.0 0.0 2.1 0.6 Ž . Ž . 100 3.0 0.0 1.0 0.0 90 Ž . Ž . Ž . Garlic, Allium saÕitum 6 100 2.0 0.8 1.0 0.7 Ž . Ž . 200 2.0 0.8 0.0 0.0 Ž . Ž . Ž . Malachite green 7 1 2.0 0.0 0.0 0.0 3 100 Ž . Ž . Ž . Potassium permanganate 8 10 2.4 0.7 1.2 0.4 Ž . Ž . 15 3.0 0.0 0.6 0.7 Ž . Ž . 20 2.0 0.0 0.0 0.0 25 100 w Ž . Ž . Ž . Virkon PF vet. 9 15 2.0 0.0 0.8 0.4 Ž . Ž . 20 2.6 0.5 0.0 0.0 Ž . Ž . Ž . Amprolium 10 50 2.3 0.5 2.0 0.5 Ž . Ž . Ž . Bithionol sulfoxide 11 0.25 2.3 0.5 2.1 0.3 w Ž . Ž . Ž . Carnidazole 12 25 2.5 0.5 2.3 0.3 Ž . Ž . Ž . Chloroquine diphosphate 13 100 2.0 0.0 1.3 0.7 w Ž . Ž . Ž . Dimilin 14 50 2.5 0.5 1.7 0.6 Ž . Ž . Ž . Halazone 15 10 2.5 0.5 2.6 0.5 50 Ž . Ž . Ž . Hydrogen peroxide, 40 1000 4 h 2.7 0.5 2.3 0.5 60 Ž . Ž . Ž . Imidocarb dipropionate 16 30 2.6 0.5 1.3 0.5 Ž . Ž . Ž . Metronidazole 17 40 2.2 0.4 2.6 0.5 Ž . Table 1 continued Substances with parasiticidal Treatment Control, mean Treated, mean Dead eels Ž . effect data on substance concentration infection category infection category in treated group Ž . Ž . Ž . ppm S.D. S.D. due to toxicity Ž . Ž . Ž . Nitrate 18 3000 2.0 0.8 2.2 0.4 Ž . Ž . Ž . N-methyl-glucamine 19 50 2.5 0.5 1.4 0.5 Ž . Ž . Ž . Oxolinic acid 20 50 2.2 0.4 2.8 0.4 Ž . Ž . Ž . Oxytetracycline 21 80 2.5 0.5 2.1 0.6 Ž . Ž . Ž . pH 5 22 2.1 0.6 2.4 0.5 Ž . Ž . Ž . Potassium oleat, 35 23 4 2.0 0.8 2.0 0.0 60 Ž . Ž . Ž . Quinidine sulphate 24 30 2.6 0.5 2.6 0.5 Ž . Ž . Ž . Sodium chloride 25 4000 1.8 0.5 1.5 0.5 Ž . Ž . 10 000 2.8 0.4 2.6 0.5 Ž . Ž . Ž . Sulfadimidinum 26 75 2.5 0.5 2.5 0.5 Ž . Ž . Ž . Tap water 27 2.8 0.4 2.5 0.5 w Ž . Ž . Ž . Tiguvon 28 20 2.8 0.4 3.0 0.0 20 Ž . Ž . Ž . Toltrazuril 29 5 2.3 0.7 2.6 0.5 20 Ž . Ž . Ž . 4-Hexylresorcinol 30 1 2.3 0.6 1.3 0.6 70 a In tap water. Ž Ž .. including the biofilters bithionol and formalin , to the recirculation unit while Ž w . biofilters were isolated temporarily 1 h, Detarox AP and formalin or to one or more Ž w . rearing tanks temporarily isolated from recirculation 3 h, Virkon PF . In the recircula- tion unit, the substances were added to the water reservoir supplying the rearing tanks. If biofilters were included during the experiment, treated fish were examined 3–4 h after the initial exposure. If biofilters were isolated during the experiment the treatment was regarded finished when rearing tanks were reconnected to biofilters and the fish were examined the following hour. For the bath treatments in separated tanks, untreated eels in the same system were used as controls. Groups of eels caught before treatment and transferred to the aquarium with system water were used as controls for treatments involving the entire recirculation system. Number and size of eels in a control group Ž . equalled the number of eels from the treated group Table 3 . Reinfection of treated eels Ž was examined 3 and 7 days after some of the treatments no control groups on days 3 . Ž and 7 . Changes in feeding activity and mortality were evaluated subjectively based on . experience 3–4 weeks posttreatments. Effects of treatment on biofilters were examined Ž w y1 . by determining the ammonia concentration Merck , resolution: 0.5 mg l every second day for 1 week posttreatment. High increase of ammonia was assumed to indicate a negative effect of treatment.

3. Results