Avermectin Use in Aquaculture

Current Pharmaceutical Biotechnology, 2012, 13, 1095-1102

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Avermectin Use in Aquaculture Tor E. Horsberg* Norwegian School of Veterinary Science, PO Box 8146 Dep., NO-0033 Oslo, Norway Abstract: The main indication for use of avermectins in aquaculture-produced fish is infestations with ectoparasitic copepods. The compounds ivermectin and emamectin benzoate are predominantly used as in-feed formulations on salmonid fish against copepods in the family Caligidae: Lepeophtheirus salmonis, Caligus elongatus and C. rogercresseyi. These agents are well-documented as very effective on all developmental stages of the parasites. The duration of effect can be up to 10 weeks. The safety margin for ivermectin is narrow, but better for emamectin benzoate. Environmental impact from these chemicals on bottom-dwelling and sediment-dwelling organisms occurs, but these are restricted to the immediate area around the production site. Avermectins are incompletely absorbed from the intestine of the fish and slowly excreted. They penetrate the blood-brain barrier of the fish, ivermectin more than emamectin benzoate. Resistance has developed against these agents in L. salmonis in almost all major salmon producing areas. The situation must be viewed as serious and can render these agents completely ineffective for salmon lice control.

Keywords: Emamectin, ivermectin, fish, salmon, trout, sea lice. PARASITES IN SALMONID AQUACULTURE The world aquaculture production of fish contributed in 2006 to 47 % of the world food fish supply [1]. The major species were carps and other cyprinids (59 %), mainly produced in China. Macrocyclic lactones as avermectins are though mainly used in salmonid production, which contributes to approximately 7 % of the world fish production in aquaculture. The main indication for use of avermectins in aquaculture-produced fish is infestations with ectoparasitic copepods. Nematodes rarely represent a clinical problem in intensive salmonid production, but when they occur, the preferred treatment is with benzimidazoles. Several crustaceans are parasitizing fish. In salmonid aquaculture, these are mainly copepods belonging to the order Caligoida, although other groups also contain fish parasites. For the marine salmonid aquaculture, the most important species are sea lice, a term used for copepods of the family Caligidae. Costello [2] estimated that sea lice infestations represented a cost of 300 million per year for the fish farming industry. Lepeophtheirus salmonis is the most important parasite for salmonids in the north Atlantic and north Pacific, and may cause skin ulcerations, mainly in the neck region as seen in Fig. (1), resulting in problems with osmoregulation and secondary bacterial infections. Besides causing direct harm to the fish, sea lice are suspected to be passive vectors for other pathogens as the infectious salmon anaemia virus [3]. Several Caligus species are also capable of infecting salmonids as well as a number of marine fish species. C. elongatus is found in the north Atlantic, while C. clemensi is a north Pacific species affecting farmed salmonids [4]. On the Southern hemisphere, C. rogercresseyi is the ectoparasite *Address correspondence to this author at the Norwegian School of Veterinary Science, PO Box 8146 Dep., NO-0033 Oslo, Norway; Tel: (+47) 22 96 49 83; E-mail: [email protected] 1/12 $58.00+.00

Fig. (1). Salmon with skin ulcerations in the neck region caused by Lepeophtheirus salmonis.

causing the most severe problems in marine salmonid aquaculture [5], although a number of other parasitic copepods have also been found on farmed salmonids [6]. These parasites do not survive in fresh water, thus the infestations are evident only after transfer to sea. The parasites have a direct lifecycle. Free-living nauplii larvae are released from the hatching eggs in the paired eggstrings attached to the adult female. The eggs can also hatch from detached eggstrings. The nauplii develop into the infective copepodide that jumps on to a passing fish, attaches itself firmly to the skin with a protein filament and stays attached through the chalimus stages. The filament is then lost and the parasite can move freely over the body of the fish. Caligus species develop directly from chalimus to adults, while Lepeophtheirus species go through pre-adult stages. The parasites feed from mucus and skin. Adult female L.

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salmonis Fig. (2) are most pathogenic and also feed from fish blood [4].

strated [12]. The efficacy against all developmental stages of C. rogercresseyi, the predominant ectoparasite in the Chilean marine aquaculture, has also been demonstrated [13]. Ivermectin is effective against Salmincola californiensis in chinook salmon [14] and rainbow trout [15], as well as Lernathropus kroyeri in sea bass [16]. Ivermectin has never been licensed for fish, but has been used off label in Ireland, Canada and Chile up until year 2000. The formulations have been a 1 % injectable solution [9, 11], a 1 % oral drench [12] or an oral 0.6 % premix for pigs. Doramectin

Fig. (2). Adult female Lepeophtheirus salmonis with eggstrings.

No studies have to date been published on the efficacy of doramectin in salmonid fish. However, a study with experimental Lernaea cyprinacea infections in carps demonstrated that all parasites, both juvenile and adult stages, were killed within 20 days after oral administration of 1 mg·kg-1 for 10 days. The same result was obtained after a single intramuscular injection of 0.2 mg·kg-1 and was twice as rapid as the natural disappearance of the parasite on the carps. No adverse reactions were observed in the treated groups, whereas 20 % of the experimental infected control fish died from the infection [8]. Emamectin Benzoate

As these parasites are ectoparasites feeding from mucus, skin and blood, they can be controlled both by bath treatments where the agent (pyrethroids or organophosphates) penetrate the parasite directly, or by in-feed treatments (chitin synthesis inhibitors or avermectins) where the agent is distributed to the skin after being absorbed from the intestinal tract. Other routes of administration, as injections, are not practicable with the current technology for the large group sizes farmed fish represent. AVERMECTIN EFFICACY AGAINST FISH TOPARASITES

EC-

Only a few avermectins have been introduced as antiparasitic agents for fish. Emamectin benzoate, an avermectin also used against lepidopterous insects and other pests in crop protection, is most widely utilized. This avermectin is not used as a parasiticide in terrestrial animals. Off-label use of ivermectin has also to a limited extent been utilized, while for other macrocyclic lactones, only a few reports on nonsalmonid fish species exist [7,8]. Ivermectin In 1987, the first report of ivermectin used against salmon lice was published [9]. A single oral dose of 0.2 mg·kg-1 was demonstrated to be effective against juvenile and adult parasites, but the margin of safety was low. Mortality rates of 2 - 5 % were observed, and by doubling the dose, the mortality increased to up to 24 %. Later studies confirmed the efficacy of ivermectin on L. salmonis at a dose of 0.05 mg·kg-1 twice weekly with an elimination of parasites of around 90 % [10]. At this dosage, ivermectin did not cause mortalities. The same dosage also gave good results against C. elongatus [11]. These studies focused on the efficacy against adult parasites, but good efficacy against juvenile parasite stages of L. salmonis could also be demon-

The benzoate salt of emamectin, an avermectin closely resembling eprinomectin, was developed as an antiparasiticide for fish by Schering-Plough Animal Health. Several of the first studies were conducted with emamectin benzoate formulated as a propylene glycol solution mixed with fish oil prior to coating the feed pellets. Later, a premix containing 0.2 % emamectin benzoate (SLICETM) was developed. In a series of tank trials with Atlantic salmon artificially infested with L. salmonis, an optimal oral dosage of 0.05 mg·kg-1 daily for 7 consecutive days was developed [17]. This dosage was found highly effective against both juvenile and adult stages of the parasite recorded two weeks after termination of treatment. No adverse effects on the treated fish were observed, neither at this dose, nor at a dosage rate of 0.1 mg·kg-1 daily for 7 days. However, doubling the dose did not improve the efficacy. In another study by the same group, it was demonstrated that the 7 days treatment prevented development of L. salmonis juveniles on the fish for about 9 weeks after termination of the treatment [18,19]. However, in another study, the duration of protective effect was found to be three weeks shorter [20]. The latter study was conducted after problems with emamectin resistance had occurred in Norway. The shorter protection may therefore have been associated with reduced emamectin sensitivity in the parasites. As controlled tank trials can give different and frequently better results than field studies, several trials in sea water net pens containing salmon naturally infected with L. salmonis and C. elongatus were conducted. At temperatures above 13 °C, an efficacy of 91 - 99 % on L. salmonis (all developmental stages) was observed at 14 days after the end of the treatment. The efficacy recorded in the tank trials were thus confirmed, even though the test pens were surrounded by pens with heavily infested fish. The efficacy against C. elongatus (all developmental stages) was recorded to be 82 - 84

Avermectin Use in Aquaculture

% at 14 days after the end of the treatment. At lower temperatures (approximately 8 °C), the efficacy on L. salmonis was demonstrated to be somewhat poorer (90 %), but this was attributed to a slower onset of efficacy at low temperatures [21]. A similar field trial was conducted in eastern Canada and also revealed efficacy of up to 96 % on the total sea lice numbers [22]. In a more extensive field trial, about 1.2 million salmon from four different commercial fish farms and naturally infested with L. salmonis were treated orally with either the chitin synthesis inhibitor teflubenzuron (10 mg·kg-1 daily for 7 days) or emamectin benzoate (0.05 mg·kg-1 daily for 7 days). The efficacy was recorded for up to 51 days after treatment by parasite counts. It was demonstrated that the number of parasites declined gradually, and an efficacy on 89 % of chalimus stages and 100 % on preadult stages of L. salmonis was recorded at 51 days after termination of treatment with emamectin benzoate. The optimal effect of teflubenzuron was recorded at 36 days (96 % on chalimus stages), but thereafter new infections increased the parasite numbers significantly in groups receiving this treatment [23]. The long lasting efficacy of oral emamectin benzoate treatments was also confirmed by Sevatdal et al. [24]. In this study, post-chalimus stages of L. salmonis could not be observed on the salmon before 116 days after the end of the treatment. Oral emamectin benzoate treatments against C. rogercresseyi have been the only treatment option in the Chilean salmonid aquaculture between 2000 and 2007. Although no controlled clinical studies have been published, the efficacy following introduction in 2000 has been reported as very good [25]. Following the initial focus on efficacy of emamectin benzoate against sea lice, studies on other infestations with crustacean parasites have been conducted. The effect on Salmincola edwardsii infections in brook trout revealed that the parasite number was reduced by 40 - 60 % in a group treated with 0.05 mg·kg-1 daily for 7 days, while the parasite number in the control group increased by 20 % [26]. In sea bass naturally infected with Lernanthropus kroyeri, an oral treatment with 0.1 mg·kg-1 daily for 7 days had a 76 % efficacy on the total number of parasites [27]. The potential of avermectins in controlling crustacean ectoparasites in fish is thus substantial. Compared to bath treatments with organophosphates or pyrethroids, or oral treatments with chitin synthesis inhibitors, the advantage is a demonstrated efficacy against all developmental stages on the fish and the long residual protective effect. The method of administration, i.e. through medicated feed, is very simple compared to labour-intensive and sometimes risky bath treatments of caging holding large quantities of fish. Emamectin benzoate, the avermectin licensed in the main salmon producing countries (Chile, Canada, Norway, Scotland and Ireland) became the product of choice almost immediately after being introduced. There are, however, challenges related to toxicity of the compounds in the target animals, possible adverse environmental effects with this treatment when used in an aquatic environment, residues in edible tissues and resistance development.

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TARGET ANIMAL TOXICITY In the efficacy trial of ivermectin by Palmer et al. [9], it was noted that the margin of safety was narrow. Even a single dose of 0.2 mg·kg-1, commonly used in other animals, resulted in some mortalities among the treated salmon, and by doubling the dose, mortalities increased dramatically. Some of the surviving fish also showed lethargy and darkening of their skin. In a toxicity study, Johnson et al. [28] tested the toxicity of different oral doses of ivermectin in Atlantic, chinhook, coho salmon and steelhead trout. Ivermectin was given every second day at dose levels of 0.05 or 0.1 mg·kg-1 for up to 50 days. The results from steelhead trout were inconclusive due to an outbreak of vibriosis. A cumulative mortality rate of 10 % was observed for Atlantic salmon given the lowest dose, while no mortalities were observed in chinook and coho salmon at this dosage level. At 0.1 mg·kg-1, the mortality rates were 14%, 10 % and 2 % for Atlantic, chinook and coho salmon, respectively. Atlantic and coho salmon were also given higher doses. 0.2 mg·kg-1 resulted in 80 % cumulative mortality in the Atlantic salmon, and 20 % in the coho salmon. Thus, coho salmon was most tolerant while Atlantic salmon was most sensitive to ivermectin. No specific histopathological lesions were found, but darkening of the skin and downrolling of the eyes was observed in the Atlantic salmon. Also, in a 96 h toxicity study with sea bream exposed to various concentrations of ivermectin in the water, no specific pathological changes were found [29]. The acute toxic effects at relatively low doses of ivermectin in Atlantic salmon were partly explained by Høy et al. [30] who studied the distribution of tritiated ivermectin in Atlantic salmon and found that the compound penetrated into the brain effectively. The blood-brain barrier did not seem to be effective enough to prevent ivermectin from accumulating in the brain, as the concentrations were higher than in muscular tissue. Also in sea bream, a substantial accumulation of ivermectin in brain tissues could be found [31]. As shown in Fig. (3), emamectin benzoate was not accumulated in the salmon brain to the same extent although this compound also crossed the blood-brain barrier [24]. Similar observations have been made in P-glycoprotein deficient mice administered ivermectin or eprinomectin, an avermectin structurally very similar to emamectin. The concentration of eprinomectin in the brain was significantly lower than that of ivermectin [32]. Thus, it is possible that the activity of Pglycoproteins in the blood-brain barrier of fish is substantially lower than in mammals. However, a behavioural study in killifish (Fundulus heteroclitus) exposed to ivermectin alone or a combination of ivermectin and cyclospoin A demonstrated that P-glycoproteins still may have a role in the fish blood-brain barrier [33]. A toxicity study of emamectin benzoate in Atlantic salmon and rainbow trout in sea water revealed a lower acute toxicity of this compound compared to ivermectin. Groups of salmon and trout were fed diets containing 0, 0.1, 0.25 and 0.5 mg·kg-1 daily for seven consecutive days, i.e. up to 10 times the recommended dose. Lethargy, anorexia, incoordination and darkening of the skin colour were observed in fish given the highest doses, but no mortalities occurred within the seven days observation period after treatment. No spe-


Tor E. Horsberg

Fig. (3). Autoradiograms of Atlantic salmon given 3H-labelled ivermectin or emamectin benzoate. Light areas indicate high concentrations of the compound. Note the high ivermectin concentration in the brain.

cific histopathological lesions were found [34]. A similar study was also conducted in freshwater to explore the possibility of using the treatment on smolts prior to sea transfer. The tolerance in smolts kept in fresh water was comparable to that in sea water [19]. On the gene regulation level, it has been demonstrated that a standard seven days treatment with emamectin benzoate only to a minor degree affected transcription of genes in the liver. Up-regulated genes were associated with oxidative stress followed by a mild secondary inflammatory response. Glutathione-S-transferase (GST) was also up-regulated, and the authors suggested this enzyme to be involved in detoxification and phase II metabolism of emamectin benzoate in salmon [35]. Pharmacokinetics and Residue Depletion The duration of emamectin benzoate efficacy after oral administration against sea lice infestations is long, up to nine weeks after termination of the treatment [18]. Comparable results are not available for ivermectin, but Johnson et al. [12] could demonstrate effect of an ivermectin treatment for the whole 28 days survey period after the end of the treatment. In a survey on C. rogercresseyi in Chile, it was demonstrated that ivermectin applied through feed was highly effective in the control of this parasite with a duration of effect similar to that of emamectin benzoate [13]. The long duration of the clinical effect is caused by a long residence time of the active compound in the target tissues. This, in turn, indicates that the elimination must be slow. A pharmacokinetic and disposition study on Atlantic salmon in sea water after a single oral dose of tritiated ivermectin demonstrated that maximum tissue concentrations occurred after four days. At this timepoint, 29 % of the administered dose could be found in various tissues. The depletion was slow, as 19 % of the administered dose still could be detected in the tissues 28 days after administration. Autoradiographic results demonstrated that the concentrations in blood and skin, the target tissues, were significant throughout the study period of 28 days. Thus, the elimination was slow, occurring mainly via the bile. The metabolism was not determined, but it was noted that at 15 days after administration, 58 % of the radioactivity in the bile consisted of ivermectin metabolites. Interestingly, the concentration in

brain exceeded that of muscle and blood throughout the experimental period [30]. In a metabolism and residue depletion study after a single oral dose of tritiated ivermectin in fresh water kept rainbow trout, it was observed that the percentage of metabolites in fillet (skin and muscle) increased steadily over the 42 days study period, from 19 % at day 3 to 65 % at day 42. The half-life of the parent compound in muscle was calculated to 13 days, and the half life of parent compound plus metabolites was 22 days [36]. The pharmacokinetics of ivermectin have also been studied in sea bream [37]. Surprisingly, this study demonstrated quite a different kinetic profile of the compound compared to the salmonid species. The authors claimed the absorption and elimination to be fast. The half life given in the paper was 21 hours, however by recalculating the elimination half-life from the beta value given, the t was 54 hours or 2.2 days. The discrepancies are though intriguing, but may be a result of a lower fat depot in the sea bream, higher water temperatures and differences in the route of administration. It has been demonstrated that emamectin benzoate is stored in the intestinal mucosa and is released slowly to the bloodstream after oral administration [24]. The same pattern can be deduced from the autoradiographic pictures of ivermectin in salmon presented by Høy et al. [30]. By intraperitoneal injection, the gastrointestinal tract is bypassed. The pharmacokinetic properties of emamectin benzoate in fish have been studied in more detail due to the fact that this compound has been developed and licensed as a product against sea lice. A metabolism and residue depletion study using tritiated emamectin benzoate in Atlantic salmon in sea water at 5 °C demonstrated a low degree of metabolism for this compound, the parent compound accounted for over 80 % of total residues in muscle and skin. The study also indicated a very slow depletion of the parent compound from muscle and skin for 15 days after termination of a seven day standard dosage. For the remaining period (30 - 90 days), an extremely long half-life of approximately 60 days for total radioactivity (i.e. the sum of parent compound and metabolites) could be calculated from information in tables [38]. This may have been an erroneous observation as the study was conducted in a recirculation system. Although the water was filtered through charcoal before recirculation, the possibility of recirculation of the active compound cannot be ruled

Avermectin Use in Aquaculture

out. Supporting this hypothesis, another study of the depletion of emamectin benzoate from rainbow trout fillet in sea water at 6 °C demonstrated a half-life of approximately 33 days. At 15 °C, the half-life in fillet was approximately 10 days [39]. This corresponds well with a field study in Atlantic salmon in sea water at 15 - 19 °C where a half-life of 9.2 days in muscle was determined [24]. The kinetic properties of emamectin benzoate have also been studied in Atlantic cod at 9 °C. The depletion half-life after oral administration was calculated to 7.5 days. Also in cod, the peak plasma concentration was observed 3 days after administration. As an intravenous dosage route was included in this study, the bioavailability could be calculated to 38 % [40]. This parameter has not been determined in other species, but the peak plasma concentration reported after oral administration in this study on cod (15 ng·ml-1) is equivalent with the maximum blood concentration in salmon reported by Sevatdal et al. [24] of 14 ng·ml-1 after a single, oral dose. The tissue distribution of emamectin benzoate has been studied in Atlantic salmon using autoradiography [24]. The study demonstrated a high quantity of radioactivity in mucous membranes (gastrointestinal tract, gills) throughout the observation period (56 days). Interestingly, activity was high in the epiphysis and hypophysis throughout the study. Whether or not this may influence on endocrine functions has not been studied. The highest activity was observed in the bile, indicating this to be an important route for excretion. Thus the avermectins seem to be incompletely absorbed from the intestinal tract of fish. The absorption seems to be delayed, with peak concentrations in the tissues several days after the feed bolus has passed through the intestine. This is most likely a result of depots in the intestinal mucosa from which the compounds are relatively slowly released. The elimination is also very slow, with depletion half-lives from muscle tissues exceeding 7 days after oral administration. The main elimination route seems to be via the bile and the gastrointestinal tract. In spite of the slow elimination, the withdrawal times set by the authorities are rather short, in Norway 175 degree-days (water temperature in °C · days). This because the maximum residue limit has been set to 300 g·kg-1 and such tissue concentrations are hardly reached during a normal treatment. In a study where a number of blood samples were collected the day after standard treatments with emamectin benzoate, the overall median concentration was 116 ng·ml-1, varying from 6 to 440 ng·ml-1 [41]. ENVIRONMENTAL SAFETY Since avermectins and other antiparasitic agents are used to kill parasites, the potential for unwanted side-effects on non-target organisms is substantial. The target organisms are mainly crustaceans, but the aquatic environment also harbours many non-target crustaceans and other sensitive organisms. Treatment of large groups of individuals in an open net-cage system will inevitably result in release of the chemical to the environment. Thus, evaluation of environmental safety is an important issue to consider when deciding to use antiparasitic agents against sea lice. New veterinary products in the EU must be evaluated by the Committee for Veterinary Medicinal Products (CVMP)


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according to specific guidelines for environmental risk assessments (EudraLex - Volume 7 Scientific guidelines for medicinal products for veterinary use). The assessment is vital for decisions regarding approval and restrictions on the use for new products. Off-label use of products approved for other indications and/or other species, e.g. the 0.6 % premix for pigs, is though not evaluated by the CVMP. In such cases, the utilisation of the treatment must be justified according to the cascade principle of Article 11 in Directive 2001/82/EC, as amended by Directive 2004/28/EC. An evaluation of whether or not possible adverse environmental effects precludes the use of the product must be evaluated by the prescriber and in some cases also by environmental authorities. Application method (bath or oral), pharmacokinetics, water solubility, binding to particulate matter, water currents and stability in water and sediments are all factors determining how much, how long and where the active ingredient ends up. Avermectins generally have very low water solubility. Thus, they will not be found in high concentrations in water, and adverse effects on pelagic organisms by ivermectin have been evaluated as unlikely [42]. Most of the concerns are related to possible adverse effects on bottom-dwelling and sediment-dwelling organisms. In a study where the acute impact on benthic organisms of ivermectin was assessed, the greatest effect was found on the rag worm, Hediste diversicolor. The mud shrimp, Corophium volutator was also affected, but to a lesser degree [43]. A clear dose-response effect was seen, but effects were evident at all concentrations tested (0.8 - 80 mg ivermectin per m2). The doses were chosen from a model and not based on tests of actual concentrations. Davies et al. [44] found a degradation half-life of ivermectin in sediments of more than three months. They estimated the risk for harmful effects on polychaetes by spilled medicated feed and faeces containing ivermectin to be significant. The effects were evaluated to be most prominent directly below and around the fish cages. This was confirmed by Cannavan et al. [45], who after a sea lice treatment with ivermectin did not detect the compound in sediment samples taken further away from the cages than 31 meters. The highest level found directly under a cage was 0.067 mg/m2, less than 1/10 of the lowest concentration tested by Collier et al. [43]. Cannavan et al. [45] concluded that the detected concentrations of ivermectin in the sediments were unlikely to cause adverse effects on benthic organisms. The environmental fate and environmental toxic properties of emamectin benzoate were studied in a farm applying a single treatment with recommended doses. The monitoring lasted for one year. The sediment concentrations accumulated to 0.2 mg/m2 and the compound could be detected in low concentrations for up to a year after the treatment. However, the benthic fauna did not seem to be affected and the authors concluded that the treatment did not seem to have any toxic impacts on sediment-dwelling organisms [46]. In another study, the toxic effects of emamectin benzoate on planctonic marine copepods were studied. Toxic effects were not seen on concentrations obtained in the water after an emamectin treatment [47].


For bottom-dwelling crustaceans like crabs and lobsters, subtoxic effects may be of importance for growth and reproduction. In particular, a premature moulting of an ovigerous lobster female may lead to the eggs being shed together with the chitin shell. Waddy et al. [48] demonstrated that this could happen in American lobsters exposed to emamectin benzoate. The observation was puzzling, as avermectins generally inhibit moulting in insects. The authors speculated that there could be differences in the neuroendocrine regulation of moulting between insects and crustaceans. Thus, the potential for adverse environmental effects on bottom-dwelling and sediment-dwelling organisms exist. Avermectins are degraded by light, but little light reach sediments under a fish farm. The half-life of the avermectins in sediments is therefore long, most likely more than 6 months. Under field conditions, treatments with emamectin benzoate may have to be applied several times per year, and concentrations significantly higher than those reported by Telfer et al. [45] could build up. Under such practices, adverse effects on the bottom-dwelling fauna must be considered probable. However, the concentrations that accumulate will most likely only affect the immediate area under and around the fish farm. RESISTANCE The development of resistance towards an antiparasitic agent can render the agent ineffective for parasite control in a region. This was evident in several regions in Norway, where organophosphates in the early and mid 1990s totally lost their effect as sea lice control agents [49]. Later, evidences of treatment failures with pyrethroids have been reported from Norway, Scotland and Ireland [50,51]. Treatment failures have also been reported for emamectin benzoate. Initially, these incidents were isolated cases and could frequently be attributed to erroneous calculations of biomass, concurrent diseases and other factors. The appetite can vary considerably between individual fish, causing huge variations in the obtained tissue concentrations of the agent [41] and this can be misinterpreted as resistance. However, in 2006 several reports from Chile indicated a systematic failure of efficacy by emamectin benzoate towards C. rogercresseyi. Bioassay tests were established, and demonstrated significantly reduced sensitivity in the parasite [25]. In addition, there were reports of reduced efficacy of emamectin against L. salmonis in Ireland and Scotland. In a Scottish epidemiological survey of sea lice burdens linked to emamectin treatments between 2002 and 2006, a trend towards gradually reduced efficacy could be seen. Although sea lice infestations were reduced following treatments, not all treatments were effective [52]. From 2006, significant efficacy problems with emamectin benzoate in all salmon producing regions have developed, perhaps with the exception of the Pacific coast of Canada. These problems have been documented through efficacy monitoring (parasite counts before and after treatments) and bioassays (toxicological tests of the susceptibility of parasites towards increasing concentrations of the agent in question) [50]. However, a comprehensive overview has not yet made its way into the scientific literature. In a Norwegian survey in 2009, 129 salmon lice strains were tested for sensi-

Tor E. Horsberg

tivity against emamectin benzoate using the bioassay method by Westcott et al. [53]. Of these, 83 strains (64.3 %) were classified as strains with reduced sensitivity towards emamectin benzoate, as the concentration necessary to immobilize or kill 50 % of the population was more than 4 times higher (EC50 > 120 ppb) that the corresponding concentration for fully sensitive individuals (EC50 ~ 30 ppb). Out of the 83 strains, 37 (44.5 %) were classified as fully resistant as they tolerated more than 10 times the emamectin benzoate concentration that sensitive strains could tolerate (EC50 > 300 ppb) (Horsberg & Sevatdal, unpublished). Although the diagnostic methods need further validation, there is little doubt that resistance against emamectin benzoate is spreading rapidly. The mechanisms behind resistance development against avermectins in sea lice have not yet been determined. Several mechanisms have been described from other pests. The most prominent are enhanced detoxification of the therapeutic agent by the parasite, decreased accumulation of the agent in the parasite or mutations in genes coding for the target protein of the drug [54]. Tribble et al. [55] described that the expression level of two multidrug transporter Pglycoproteins in L. salmonis increased after exposure to emamectin benzoate. However, no direct link to resistance could be established. The direct mechanism(s) causing resistance in sea lice have thus not been elucidated. The situation concerning resistance development in sea lice, especially L. salmonis must be considered serious. In Norway, Scotland, Ireland and eastern Canada, the number of salmon in fish farms by far outweighs the number of wild salmons. Thus, the main source for reinfestation comes from the farms themselves where regular parasite treatments put a constant selection pressure on resistance development. In these countries, the influx of naive parasites from wild fish hosts is very limited. Thus, the problem will not disappear by itself. New chemicals may only be valuable for a limited time period. Preservation of fully sensitive parasite strains, management practices with co-ordinated production zones, synchronized treatments and synchronized fallowing of sites in larger areas seem to be the most promising way to handle the problem. CONFLICT OF INTEREST None declared. ACKNOWLEDGEMENT None declared. REFERENCES [1]

[2] [3] [4]

FAO Fisheries and Aquaculture Department, Rome. The state of world fisheries and aquaculture 2008. Bi-annual report, 196 pp. http://www.fao.org/docrep/011/i0250e/i0250e00.htm. (Accessed June 18, 2010). Costello, M.J. The global economic cost of sea lice to the salmonid farming industry. J. Fish Dis., 2009, 32(1), 115-118. Nylund, A.; Hovland, T.; Hodneland, K; Nilsen, F.; Lovik, P. Mechanisms for transmission of infectious salmon anemia (ISA). Dis. Aquat. Org., 1994, 19(2), 95-100. Johnson, S.C.; Treasurer, J.W.; Bravo, S.; Nagasawa, K.; Kabata, Z. A review of the impact of parasitic copepods on marine aquaculture. Zool. Stud., 2004, 43(2), 229-243.

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Received: June 18, 2010

Accepted: December 09, 2010

Tor E. Horsberg [50]

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