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Anti-parasitic drugs used to control sea lice infestations in the salmonid aquaculture industry are a growing environmental concern due to their potential impacts on non-target crustacean species. This study examined the lethal effects of teflubenzuron, a common in-feed pharmaceutical drug used on Norwegian salmon farms, on a non-target species, rockpool shrimp Palaemon elegans, following an extended exposure period. The standard daily dose for treating salmon is 10 µg teflubenzuron g-1 fish. Adult shrimp were fed 1 of 6 low doses of teflubenzuron (0, 0.0025, 0.005, 0.05, 0.094, 0.188 and 1.88 µg g-1) twice a week for a period of 66 d. Cumulative mortality reached 15, 27, 82 and 100% amongst shrimp exposed to the highest treatment groups (0.05, 0.094, 0.188 and 1.88 µg g-1, respectively). Cumulative mortality amongst shrimp exposed to the 2 lowest teflubenzuron doses and control feed was low (5%). Dose response curves based on measured concentrations within the shrimp were used to calculate a series of lethal threshold concentrations (LCx). The LC5, LC50 and LC90 concentrations of teflubenzuron causing low, median and high levels of mortality in rockpool shrimp were estimated to be 1.2, 18.4 and 150.6 ng g-1, respectively. These concentrations are similar to those reported in wild crustacean species, including shrimp species in the vicinity of Norwegian fish farms, both during and after teflubenzuron medication, suggesting that exposure to low doses of this compound can pose a significant risk to wild shrimp populations.

Sugar kelp Saccharina latissima is one of the dominating kelp species on sheltered sublittoral sites along the Norwegian coastline, providing important habitats and ecosystem services. In finfish aquaculture in Norway, infections caused by salmon lice Lepeophtheirus salmonis are one of the biggest challenges the industry is currently facing, and presently the most-used therapeutant is hydrogen peroxide (H2O2). Commonly, this treatment involves bathing the fish in a solution of approximately 1700 mg H2O2 l-1 seawater before releasing the solution into the surrounding waters. The present study was conducted to increase the knowledge on how these H2O2 emissions from farm operations could impact juvenile S. latissima. This was achieved by determining the effects of a 1 h exposure to realistic H2O2 levels on S. latissima mortality and photosynthesis. Effects on photosynthesis were determined by incubating plants at 3 time intervals post-exposure. Toxicity potentials including lethal concentration for 50% of the population (LC50) and effective concentration (EC50) for photosynthetic capacity (PMAX) and efficiency (α) were determined based on these data. Juvenile S. latissima was highly sensitive, having an LC50 of 80.7 mg H2O2 l-1, which is less than 5% of the dose commonly used at farms and emitted to the environment. A concentration of 85 mg l-1 caused an immediate 90% reduction in both PMAX and α. The EC50 was found to be 27.8 and 35.4 mg l-1 for PMAX and α, respectively. This may indicate that natural S. latissima populations in the vicinity of fish farms can be negatively affected by H2O2 bath treatments.

The amount of hydrogen peroxide (H2O2) used in the treatment of salmon lice in Norwegian salmon farming increased from 308 tons in 2009 to 43246 tons in 2015. For 2016 and 2017, however, the consumption was reduced to 26597 and 9277 tons, respectively. The use of this compound may have negative impacts on benthic fauna underneath the fish farms and, in particular, on polychaetes, which can be found in large numbers at the bottom under fish farms where they play a key role in the turnover of organic waste from the farm. The tolerance of Capitella sp. and Ophryotrocha spp. to a 1 h exposure to H2O2 (0, 100, 200, 400, 800, 1200 and 1800 mg l-1) was evaluated. The recommended dose for treatment of the salmon is 1800 mg l-1. Following exposures, the polychaetes were reintroduced into clean sea water. Both polychaete species experienced high cumulative mortality during a 72 h post-exposure period. The mortality showed to be dose dependent, with the highest dose giving the highest mortality. The 50% lethal concentration (LC50) of Capitella sp. was significantly higher than the LC50 of Ophryotrocha spp. at the same exposure time (p < 0.05). The 50% lethal time of Capitella sp. was significantly longer than that of Ophryotrocha spp. at the same concentration (p < 0.05). The results show that 1 h exposures to H2O2 at all the tested concentrations had irreversible negative effects on both polychaete species.

Lufenuron is a benzoylurea insecticide currently in use to combat sea lice infestation in salmon aquaculture in Chile. With pending approval in Norway, the aim of this work was to study the uptake and toxicity of lufenuron in liver tissue of Atlantic salmon. Juvenile salmon weighing 40 g were given a standard 7-day oral dose, and bioaccumulation and transcriptional responses in the liver were examined 1 day after the end-of-treatment (day 8) and after 1 week of elimination (day 14). Bioaccumulation levels of lufenuron were 29 ± 3 mg/kg at day 8 and 14 ± 1 mg/kg at day 14, indicating relatively rapid clearance. However, residues of lufenuron were still present in the liver after 513 days of depuration. The exposure gave a transient inhibition of transcription in the liver at day 8 (2437 significant DEGs, p-adj < .05), followed by a weaker compensatory response at day 14 (169 significant DEGs). Pathways associated with RNA metabolism such as the sumoylation pathway were most strongly affected at day 8, while the apelin pathway was most profoundly affected at day 14. In conclusion, this study shows that lufenuron easily bioaccumulates and that a standard 7-day oral dose induces a transient inhibition of transcription in liver of salmon., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Chemotherapeutants used to control infestations by sea lice can be released into the marine environment surrounding aquaculture farms. Among these therapeutic agents, emamectin benzoate is extensively utilized even though its impact on non-target taxa has not been thoroughly examined. In this context, we explored the effects of emamectin benzoate on a common Norwegian habitat-forming species: the phosphorescent sea-pen Pennatula phosphorea. Specifically, we examined P. phosphorea metabolic and responses before, during and after exposure to emamectin benzoate. Results indicate that an 8-day emamectin benzoate exposure (0.8 mg/L) did not induce P. phosphorea mortality or significant behavioural or metabolic modifications. However, we highlighted the presence and persistence of emamectin benzoate in exposed P. phosphorea tissue. These results indicate that emamectin benzoate is unlikely to adversely impact P. phosphorea populations in the environment. However, persistence of emamectin benzoate in tissue constitutes a potential for bioaccumulation with repeated treatments and should be examined in further studies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bastien Taormina reports financial support was provided by Norwegian Ministry of Trade, Industry and Fisheries., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Biological burrowing behavior is an important driver shaping ecosystems that is being threatened by CO 2 -induced ocean acidification; however, the effects of ocean acidification on burrowing behavior and its neurological mechanism remain unclear. This study showed that elevated p CO 2 significantly affected the burrowing behaviors of the Manila clam Ruditapes philippinarum , such as increased foot contraction, burrowing time, and intrabottom movement and decreased burrowing depth. Delving deeper into the mechanism, exposure to elevated p CO 2 significantly decreased extracellular pH and increased [HCO 3 - ]. Moreover, an indicator GABA A receptor, a neuroinhibitor for movement, was found to be closely associated with behavioral changes. In situ hybridization confirmed that the GABA A receptor was widely distributed in ganglia and foot muscles, and elevated p CO 2 significantly increased the mRNA level and GABA concentration. However, the increase in GABA A receptor and its ligand did not suppress the foot movement, but rather sent "excitatory" signals for foot contraction. The destabilization of acid-base homeostasis was demonstrated to induce an increase in the reversal potential for GABA A receptor and an alteration in GABA A receptor function under elevated p CO 2 . This study revealed that elevated p CO 2 affects the burrowing behavior of Manila clams by altering GABA A receptor function from inhibitory to excitatory.

The proliferation of sea lice (Lepeophtheirus salmonis) represents a major challenge for the salmonid aquaculture industry in Norway. Hydrogen peroxide (H 2 O 2 ) is a chemotherapeutant frequently used on Norwegian farms, however, its toxicity to non-target benthic species and habitats remains poorly understood. Maerl beds are constructed by the accumulation of non-geniculate coralline algae and provide important ecological functions. Due to the rapid expansion of aquaculture in Norway and the continued use of H 2 O 2 as an anti-sea lice treatment, it is crucial to understand the impact of H 2 O 2 on the physiology of maerl-forming species. The effects of a 1 h exposure to H 2 O 2 on the photophysiology and bleaching of the coralline alga Lithothamnion soriferum were examined here through a controlled time-course experiment. PAM fluorimetry measurements showed that H 2 O 2 concentrations ≥ 200 mg l -1 negatively affected photosystem II (PSII) in thalli immediately after exposure, which was observed through a significant decline in maximum photochemical efficiency (F v /F m ) and relative electron transport rate (rETR). The negative effects on PSII induced by oxidative stress, however, appear to be reversible, and full recovery of photosynthetic characteristics was observed 48 h to 28 days after exposure to 200 mg H 2 O 2 l -1 and 2000 mg H 2 O 2 l -1 , respectively. At 28 days after exposure, there was evidence of two- to four-times more bleaching in thalli treated with concentrations ≥ 200 mg H 2 O 2 l -1 compared to those in the control. This indicates that despite the recovery of PSII, persistent damages can occur on the structural integrity of thalli, which may considerably increase the vulnerability of coralline algae to further exposure to H 2 O 2 and other chemical effluents from salmonid farms., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Lumpfish is a novel farmed species used as cleaner fish for the removal of lice from farmed salmon. As often with new, farmed species, there are challenges with bacterial infections. The frequency of prescription of antibiotic agents to lumpfish is increasing, despite the lack of knowledge about appropriate doses, duration of treatment and application protocols for the various antibacterial agents. In the current study, we have tested the effect of medicated feed with florfenicol (FFC), oxolinic acid (OA) and flumequine (FLU) on lumpfish experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica. We found that all three antibacterial agents efficiently treated lumpfish with vibriosis using 10 and 20 mg kg -1  day -1 of FFC, 25 mg kg -1  day -1 of OA and 25 mg kg -1  day -1 FLU, whereas only FFC (20 mg kg -1  day -1 ) had good effect on lumpfish with pasteurellosis. None of the antibacterial agents were efficient to treat lumpfish with atypical furunculosis. FFC 20 mg kg -1  day -1 showed promising results in the beginning of the experiment, but the mortality increased rapidly 14 days post-medication. Efficient treatment is important for the welfare of lumpfish and for reducing the risk of development of antibiotic-resistant bacteria. To our knowledge, this is the first study to establish protocols for antibacterial treatment of lumpfish., (© 2021 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Bath treatment pharmaceuticals used to control sea lice infestations in the salmonid industry, such as hydrogen peroxide (H 2 O 2 ), are released directly into the environment where non-target organisms are at risk of exposure. The aim of this study was to determine the threshold concentrations for mortality of the Northern krill, Meganyctiphanes norvegica, a major component of the north Atlantic marine ecosystem. To assess the lethal effects of H 2 O 2 , we carried out a series of 1 h acute toxicity tests and assessed mortality through a 48 h post-exposure period. One-hour exposure to 170 mg/L, corresponding to 10% of the recommended H 2 O 2 treatment, caused 100% mortality and a subsequent acute median-lethal concentration LC50 value of 32.5 mg/L. Increased mortality was observed with time in all exposed groups, resulting in successively lower LC 50 values during the post-exposure period. The suggested H 2 O 2 concentrations have the potential of causing negative effects to the Northern krill.

Bath treatment chemotherapeutants, used to control sea lice infestations in the salmonid aquaculture industry, are released directly into the marine environment around fish farms and pose a serious risk to non-target species, particularly crustaceans. Hydrogen peroxide (H 2 O 2 ) is the most frequently used bath treatment chemotherapeutant on Norwegian fish farms, however, limited information is available on its toxicity to European lobsters (Homarus gammarus), a commercially important species at risk of exposure due to its distribution overlapping with salmon farm locations. The aim of this study was to investigate the lethal effects of H 2 O 2 on pelagic (stage I-IV) larvae/post-larvae and its sub-lethal effects on the benthic stage V H. gammarus. To assess the lethal effects of H 2 O 2 , we carried out a series of 1 h toxicity tests and assessed mortality after a 24 h post-exposure period. Exposure to H 2 O 2 was toxic to all pelagic larval stages tested, with estimated median lethal concentrations (LC 50 ) of 177, 404, 665 and 737 mg/L for stage I, II, III and IV, respectively. These concentrations represent approximately 10, 23, 40 and 43%, of the recommended H 2 O 2 concentrations used for delousing salmon on Norwegian fish farms, respectively. To assess the sub-lethal effects of H 2 O 2 on H. gammarus, stage V juveniles were exposed to H 2 O 2 at concentrations of 85, 170 and 510 mg/L for 1 h and shelter-seeking behaviour and mobility endpoints were assessed. Numerous behavioural parameters including distance travelled to shelter, time to locate shelter and the number of shelter inspections, were negatively affected in lobsters exposed to H 2 O 2 when assessed immediately after the exposure period. However, no differences between control and exposed lobsters were detected after a 24 h post-exposure period. Our results demonstrate that short term exposures to H 2 O 2 are lethal to pelagic H. gammarus life stages and can negatively affect the shelter seeking behaviour of benthic life stages, though these behavioural changes may be short-lived., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The chitin synthesis inhibitor teflubenzuron (TFB) is a feed antiparasitic agents used to impede molting of the salmon lice, an ecto-parasite that severely affects the salmon industry. Low absorption of oral administered TFB may cause elevated concentrations in the feces discharged from the salmon into the benthic environment. The polychaete Capitella sp. are often dominant in such habitats and consume organic waste deposited on the sediment. In the present study, Capitella sp. were exposed to doses of TFB in salmon feed of 1, 2 and 4 g TFB kg -1 (0 g TFB kg -1 in control group) over an experimental period of 32 days. Cumulative mortality was 12%-15% in both treatment groups with 1 and 2 g TFB kg -1 and reached 27% in the group with 4 g TFB kg -1 . Only the highest dose (4 g TFB kg -1 ) negatively affected feed intake, growth and respiration of the polychaetes while food conversion efficiency was not affected. At the end of the experiment, the concentrations of TFB in the Capitella sp. were high, in the range of 9.24-10.32 μg g -1 for the three treatment groups. It was suggested that a maximum level of absorption rate was reached, also for the lowest dose. High concentrations of TFB in the Capitella sp. might pose a risk to crustaceans that forage for polychaetes in the vicinity of fish farms. We conclude that the effects of TFB on Capitella sp. may therefore primarily be to the predators rather than the Capitella sp., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Anti-sea lice pesticides, used in the salmonid aquaculture industry, are a growing environmental concern due to their potential to adversely affect non-target crustaceans. Azamethiphos and deltamethrin are two bath treatment pesticides used on salmon farms in Norway, however, limited information is available on their impact on European lobster (Homarus gammarus) larvae in the Norwegian marine environment. Here, we firstly report the lethal (LC 50 ) and effective (EC 50 ) concentrations of azamethiphos and deltamethrin for stage I and stage II larvae, following 1-h exposures. Using a hydrodynamic model, we also modelled the dispersal of both compounds into the marine environment around selected Norwegian farms and mapped the potential impact zones (areas that experience LC 50 and EC 50 concentrations) around each farm. Our data shows that azamethiphos and deltamethrin are acutely toxic to both larval stages, with LC 50 and EC 50 values below the recommended treatment concentrations. We also show that the azamethiphos impact zones around farms were relatively small (mean area of 0.04-0.2 km 2 ), however deltamethrin impact zones covered much larger areas (mean area of 21.1-39.0 km 2 ). These findings suggest that deltamethrin poses a significant risk to European lobster in the Norwegian marine environment while the impact of azamethiphos may be less severe., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study examined the uptake, tissue distribution and elimination of the antibacterial agents oxolinic acid and flumequine in lumpfish ( Cyclopterus lumpus L.) by use of LC-MS/MS following a single oral administration of 25 mg/kg fish given in feed. Lumpfish are increasingly used as cleaner fish for removal of sea lice on commercially farmed salmon. The production of lumpfish is successful, but there are challenges with bacterial infections and the number of antibacterial treatments has increased in recent years. As the lumpfish is a novel species to farming, there is a need for pharmacokinetic data and establishment of protocols for efficient antibacterial treatment. The current study describes the pharmacokinetic properties of oxolinic acid and flumequine in lumpfish. Absorption of oxolinic acid was moderate and was characterized by a calculated peak plasma concentration (C max ) of 2.12 μg/ml after 10.3 h (T max ) and an elimination half-life (t 1/2 β) of 21 h. Area under curve (AUC) and AUC from 0 to 24 h (AUC 0-24h ) were calculated to be 60.9 and 34.0 h μg/ml, respectively. For flumequine, plasma C max was found to be 2.77 μg/ml after 7.7 h (T max ) with t 1/2 β of 22 h. The area under the curve (AUC) and AUC from 0 to 24 h (AUC 0-24 ) were calculated as 104.3 and 50.3 h μg/ml, respectively. Corresponding C max values in muscle, liver, and head-kidney for oxolinic acid were 4.01, 3.04, and, 4.68 μg/g, respectively and T max of 11.1, 9.2, and 10.0 h, respectively. For flumequine, C max values of 4.16, 4.01, and 7.48 μg/g were obtained in muscle, liver, and head kidney, respectively, with corresponding T max values of 10.2, 10.3, and 6.0 h. Antimicrobial susceptibility values as determined by minimum inhibitory concentration (MIC) analyses against 28 isolates of Aeromonas salmonicida isolated from diseased lumpfish ranged from 0.06 to 15 μg/ml for oxolinic acid and 0.024 to 6.25 μg/ml for flumequine. Bimodal distributions in susceptibility to both oxolinic acid and flumequine were observed. The combination of pharmacokinetic properties and MIC data make possible calculation of efficient treatment doses, which are needed to improve the welfare of lumpfish and minimize development of antibiotic resistant bacteria., (Copyright © 2019 Haugland, Kverme, Hannisdal, Kallekleiv, Colquhoun, Lunestad, Wergeland and Samuelsen.)

The benzoylurea chitin synthesis inhibitor teflubenzuron, widely used against sea lice in North Atlantic aquaculture, may pose an environmental threat to non-targeted crustaceans. In this experiment, laboratory acclimated pink shrimp (Pandalus montagui), a species found in fjords with Atlantic salmon farming, were exposed to dietary teflubenzuron for 46 days (control; low dose: 0.01 μg/g; high dose: 0.1 μg/g). The exposure doses represent 0.1% and 1% of a standard treatment dose for Atlantic salmon. Mortality and prevalence of deformities, pharmacokinetics, oxidative stress and transcriptomic and metabolomic profiling were used to assess the response to teflubenzuron exposure. Mortality in the high-dose group was 25% (five of 20 individuals). No control or low-dose group shrimps died. Phenotypic responses,i.e., leg deformities (0 control, 6 low, 8 high) and cloudy eyes (0 control, 3 low, 7 high), were observed in some surviving shrimps (control n = 15, low n = 17, high n = 15). Accumulated levels of teflubenzuron in shrimps from the high-dose group ranged from 4.7 to 369 ng/g wet weight. Transcriptomic profiling showed very few significantly altered genes in the exposed shrimps. Teflubenzuron-induced changes to the metabolome pointed to well-known effects of benzoylurea agents, with reduced levels of N-acetylglucosamine indicating an effect on chitin synthesis. The metabolomic profiling showed that teflubenzuron exposure was associated with reduced energy metabolism. Some metabolites pointed to increased necrosis and/or bacterial overgrowth in the teflubenzuron-exposed shrimps. In conclusion, this study shows that teflubenzuron causes phenotypic effects in P. montagui exposed to 0.1% of the treatment dose given to Atlantic salmon., (© 2018 John Wiley & Sons, Ltd.)

Infestations with salmon lice, a parasitic copepod, is a major problem in the salmon farming industry. Teflubenzuron is an in-feed pharmaceutical applied to control lice outbreaks; the standard medication is 10 mg per kg fish per day for seven days. Surveys reveal that teflubenzuron accumulates and persists in the sediment around fish farms and causes deformities and mortality in juvenile European lobster (Homarus gammarus), a species commonly found in the vicinity of salmon farms in Norway. To date, there is no information on sub-lethal effects of teflubenzuron on, for example, behavior. We conducted an experiment to assess possible difference in the shelter seeking behavior of teflubenzuron-exposed (N = 19) vs. not exposed (N = 19) H. gammarus juveniles. The teflubenzuron-exposed juveniles had been given very low concentrations, 1.7 µg per pellet twice per week for 113 days prior to this experiment. The concentration of teflubenzuron was estimated to be less than 1 ng/g lobster when they were tested in the behavior experiment. Animals were placed in a lane with a shelter at one end. Once a lobster had found and entered the shelter, they were repeatedly displaced back to the opposite end of the lane, for a total of 3 repeated runs per animal. Three of the exposed juveniles failed to settle in the shelter, and the remaining teflubenzuron-exposed animals took significantly more time to explore the environment and to find and recognize shelter. Furthermore, exposed lobsters also exhibited slower walking speed compared to the controls. These results demonstrate that teflubenzuron significantly reduces exploratory behavior, learning and activity of juvenile H. gammarus. Thus, exposure to teflubenzuron could increase predation mortality of juvenile lobsters in the wild., (Copyright © 2018. Published by Elsevier Inc.)

Concerns have been raised over the environmental impacts of antiparasitic drugs used to delouse farmed salmon. Released into the marine environment, some of these drugs can have negative impact on non-targeted crustaceans in the vicinity of farming facilities. In this study, we examined the molecular effect of the insecticide teflubenzuron on a shrimp species inhabiting the littoral zone, the rockpool shrimp (Palaemon elegans). Rockpool shrimp was exposed for 98days to a dose representing 2% of a regular teflubenzuron medication applied to Atlantic salmon. Accumulation of teflubenzuron was studied in whole body samples, except abdominal segments 5 and 6, which were used for gene expression analysis. Insight into sublethal mode of action was sought by examining the transcriptional responses of 38 genes encoding proteins linked to molting and exoskeleton change, stress and detoxification. The accumulated levels of teflubenzuron in exposed animals varied between 1.7 and 33.0ng/g. Significant transcriptional effects of exposure were seen for markers linked to molting and exoskeleton change (chh, ctbs, gap65), stress and apoptosis (hsp40, hsp70, casp3), as well for detoxification (cyp6a18). In conclusion, this study shows that teflubenzuron can bioaccumulate in shrimps living in the littoral zone and at sublethal concentrations affects molecular mechanisms in non-hepatopancreatic tissue., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In recent years, use of lumpfish (Cyclopterus lumpus L.) as cleaner-fish to remove sea-lice have been chosen by many salmon farmers in Europe and Canada as an alternative to medical treatment, which has led to large scale production of lumpfish. At present, there is limited knowledge of how lumpfish respond upon anaesthesia, which anaesthetics and concentrations that are efficient and conditions for euthanasia. We have therefore tested and developed protocols for bath immersion for three commonly used anaesthetics metacaine (Finquel, buffered tricaine methanesulfonate, MS-222 and Tricaine Pharmaq), benzocaine (Benzoak vet) and isoeugenol (Aqui-S), determined concentration for normal and fast anaesthesia and evaluated safety margin for each condition. Also, a behavioral matrix has been developed. We have examined the effect of fish size (10-20 g, 200-400 g and 600-1300 g) and sea water temperature (6°C and 12°C). We found that 200 mg L-1 metacaine is an efficient dose for deep narcosis independently for fish size and temperature due to good safety margins with regards to both exposure times and doses. However, for many tasks lighter anaesthesia is sufficient, and then 100 mg L-1 metacaine can be used. Benzocaine is less efficient than metacaine, but can be used as anaesthetic of fish < 400 g. The optimal doses of benzocaine were 100-200 mg L-1 for small fish (10-20 g) and 200 mg L-1 for medium sized fish (200-400 g). For larger fish (> 600 g), benzocaine is not suitable. Isoeugenol cannot be recommended for full anesthesia of lumpfish. The conditions for lethal doses varied with chosen anaesthetic, fish size and temperature. For small fish (10-20 g), exposure to 1600 mgL-1 of metacaine in 10 minutes it lethal. Guided protocols for non-lethal anaesthesia will contribute to ensure safe treatment of lumpfish according to an ethical standard for good fish welfare.

The persistence and stability of the oral administered anti salmon-lice drugs teflubenzuron and diflubenzuron were tested when associated to organic material as faecal particles from Atlantic salmon and medicated food pellets. This laboratory study was performed in seawater under aerobic conditions, at 7°C in the dark and showed that both compounds were remarkably persistent and stable since no significant reduction in the concentrations of flubenzurons in sediment were seen after 24 weeks. Therefore neither chemical or microbial degradation nor outwashing seems to be important pathways for these drugs to disappear from sediment under fish farms. Thus, it is more likely that the decrease of flubenzurons from marine sediments described in field investigations is caused by either bioturbation, resuspension of organic particles or a combination of these.

Increasing use of pharmaceutical drugs to delouse farmed salmon raises environmental concerns. This study describes an experiment carried out to elucidate the molecular mechanisms of the antiparasitic drug teflubenzuron on a non-target species, the European lobster. Juvenile lobsters (10.3±0.9 mm carapace length) were fed two environmentally relevant doses of teflubenzuron, corresponding to 5 and 20% of a standard salmon medication (10 mg/kg day), termed low and high dose in this study. After 114 days of dietary exposure, whole-animal accumulation of teflubenzuron was determined. One claw from each animal was collected for transcriptional analysis. Overall, exposed animals showed low cumulative mortality. Six animals, two from the low dose treatment and four from the high dose, showed exoskeletal abnormalities (claw deformities or stiff walking legs). Residual levels of teflubenzuron in juvenile lobster were 2.7-fold higher in the high dose (282 ng/g) compared to the low dose treatment (103 ng/g). The transcriptional examination showed significant effects of teflubenzuron on 21 out of 39 studied genes. At the transcriptional level, environmentally relevant levels of the anti-salmon lice drug impacted genes linked to drug detoxification (cyp3a, cyp6a2, cyp302a, sult1b1, abcc4), cellular stress (hsp70, hsp90, chh), oxidative stress (cat, gpx3) and DNA damage (p53), as well as molting and exoskeleton regulation (chi3l1, ecr, jhl1, chs1, ctbs, gap65, jhel-ces1) in claw tissue (muscle and exoskeleton). In conclusion, teflubenzuron at sub-lethal levels can affect many molecular mechanisms in European lobster claws., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The salmon louse (Lepeoptheirus salmonis) is a challenge in the farming of Atlantic salmon (Salmo salar). To treat an infestation, different insecticides are used like the orally administered chitin synthetase inhibitor teflubenzuron. The concentrations and distribution of teflubenzuron were measured in water, organic particles, marine sediment and biota caught in the vicinity of a fish farm following a standard medication. Low concentrations were found in water samples whereas the organic waste from the farm, collected by sediment traps had concentrations higher than the medicated feed. Most of the organic waste was distributed to the bottom close to the farm but organic particles containing teflubenzuron were collected 1100 m from the farm. The sediment under the farm consisted of 5 to 10% organic material and therefore the concentration of teflubenzuron was much lower than in the organic waste. Teflubenzuron was persistent in the sediment with a stipulated halflife of 170 days. Sediment consuming polychaetes had high but decreasing concentrations of teflubenzuron throughout the experimental period, reflecting the decrease of teflubenzuron in the sediment. During medication most wild fauna contained teflubenzuron residues and where polychaetes and saith had highest concentrations. Eight months later only polychaetes and some crustaceans contained drug residues. What dosages that induce mortality in various crustaceans following short or long-term exposure is not known but the results indicate that the concentrations in defined individuals of king crab, shrimp, squat lobster and Norway lobster were high enough shortly after medication to induce mortality if moulting was imminent. Considering food safety, saith and the brown meat of crustaceans contained at first sampling concentrations of teflubenzuron higher than the MRL-value set for Atlantic salmon. The concentrations were, however, moderate and the amount of saith fillet or brown meat of crustaceans to be consumed in order to exceed ADI is relatively large., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study describes experiments carried out to examine effects of the antiparasitic drug teflubenzuron, used in delousing farmed salmon, on a non-target species, the European lobster (Homarus gammarus). Juvenile lobsters were fed two doses of teflubenzuron, 10 and 20mg/kg successively for 7 days corresponding to a standard medication of the fish (10mg/kg day) and twice the standard dose (20mg/kg day). Monitoring lasted 3 months to include at least one moulting period for all individuals. Cumulative mortality was higher in all replicates given medicated feed compared with the control group. Mean cumulative mortality for each dosing was 41 ± 13% for 10mg/kg and 38 ± 8% for 20mg/kg, i.e. no difference. Drug residue was analysed in all juveniles that died, in addition to 12 juveniles at day 8 and the first 12 surviving lobsters. A decline in concentration of teflubenzuron from over 8,000 ng/g (day 5) to 14 ng/g (day 70) was observed in the juveniles that died during the experiment. Twelve individuals that died contained 82 ng/g or less whereas the mean concentration in the first 12 lobsters that survived moulting was 152 ng/g. Following a single oral administration, the half-life of teflubenzuron in lobster was estimated to 3.4 days and the initial concentration (C0) to 515 ng/g at time t0. At the end of the study a considerable number of juvenile lobsters were observed with deformities in various organs; carapace, walking legs, cheliped, tail fan, abdomen and antenna. The occurrence of observed deformities varied from 0 to 15% in treated replicates and will most likely affect ability to locate and consume food (antenna, claw and walking legs), respiration (carapace) and ability to move/swim (walking legs, tail fan and abdomen). In total, the mortality and senescent damages were close to 50% in all replicates. Juveniles that survived medication without deformities however, moulted and increased in size at each moult equally well as the unmedicated controls., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

The duration of efficacy of emamectin benzoate in the control of Caligus curtus infestations in Atlantic cod, Gadus morhua L., was studied following an administration of 50 μg kg⁻¹ for seven consecutive days. No lice were found on medicated fish when challenged 1 week (challenge 1) or 5 weeks (challenge 2) following termination of medication, whereas the mean abundance of lice among the unmedicated fish was 17.9 and 19.3 lice per fish in challenge 1 and 2, respectively. Muscle concentrations of 19.5 ± 8.2 ng g⁻¹ and 3.4 ± 0.9 ng g⁻¹, respectively, and skin concentrations of 23.1 ± 10.8 and 4.2 ± 1.0 ng g⁻¹, respectively, were found 27 and 55 days following the termination of medication. Tissue concentrations and the duration of efficacy indicate a dosing regime for emamectin in cod, similar to the regime used for Atlantic salmon, Salmo salar L., (© 2011 Blackwell Publishing Ltd.)

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3-4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2-2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.

The pharmacokinetic profile of the antiparasitic agent emamectin benzoate was studied in plasma after intravenous (i.v.) injection and in plasma, muscle and skin following oral (p.o.) administration to cod, Gadus morhua, held in sea water at 9 degrees C and weighing 100-200 g. Following i.v. injection, the plasma drug concentration-time profile showed two distinct phases. The plasma distribution half-life (t(1/2)alpha) was estimated as 2.5 h, the elimination half-life (t(1/2)beta) as 216 h, the total body clearance (Cl(T)) as 0.0059 L kg(-1) h(-1) and mean residence time (MRT) as 385 h. The volume of distribution at steady state, V(d(ss)), was calculated to be 1.839 L kg(-1). Following p.o. administration the peak plasma concentration (C(max)) was 15 ng mL(-1), the time to peak plasma concentration (T(max)) was 89 h and t(1/2)beta was 180 h. The highest concentration in muscle (21 ng g(-1)) was measured after 7 days and t(1/2)beta was calculated to be 247 h. For skin, a peak concentration of 28 ng g(-1) at 3 days was observed and a t(1/2)beta of 235 h was determined. The bioavailability following p.o. administration was calculated to be 38%.

Background: Emamectin benzoate (EB) is a dominating pharmaceutical drug used for the treatment and control of infections by sea lice (Lepeophtheirus salmonis) on Atlantic salmon (Salmo salar L). Fish with an initial mean weight of 132 g were experimentally medicated by a standard seven-day EB treatment, and the concentrations of drug in liver, muscle and skin were examined. To investigate how EB affects Atlantic salmon transcription in liver, tissues were assessed by microarray and qPCR at 7, 14 and 35 days after the initiation of medication., Results: The pharmacokinetic examination revealed highest EB concentrations in all three tissues at day 14, seven days after the end of the medication period. Only modest effects were seen on the transcriptional levels in liver, with small fold-change alterations in transcription throughout the experimental period. Gene set enrichment analysis (GSEA) indicated that EB treatment induced oxidative stress at day 7 and inflammation at day 14. The qPCR examinations showed that medication by EB significantly increased the transcription of both HSP70 and glutathione-S-transferase (GST) in liver during a period of 35 days, compared to un-treated fish, possibly via activation of enzymes involved in phase II conjugation of metabolism in the liver., Conclusion: This study has shown that a standard seven-day EB treatment has only a modest effect on the transcription of genes in liver of Atlantic salmon. Based on GSEA, the medication seems to have produced a temporary oxidative stress response that might have affected protein stability and folding, followed by a secondary inflammatory response.

This study was performed to determine the efficacy of orally administered florfenicol in the treatment of experimentally induced vibriosis (Listonella anguillarum) in cod, Gadus morhua. The L. anguillarum strain HI-610 was used. This strain has a minimal inhibitory concentration value of 0.5 mg L(-1) against florfenicol. Fifteen groups of 40 fish each were challenged by bath with 1.7 x 10(5) CFU mL(-1) for 1 h. Three days following challenge, medication with florfenicol was introduced in 12 of the groups. The dosages used were 10 mg kg(-1) day(-1) for 10 consecutive days in marine or salmonid pellets, 10 mg kg(-1) day(-1) for five consecutive days in marine pellets or administered at days 1, 2, 4, 6 and 8 following initiation of treatment. Among challenged unmedicated fish mortality started at day 3 post-challenge reaching a final cumulative mortality of 77% at day 15. The experiment was terminated at day 26. In the medicated groups, the majority of deaths occurred from days 3-7 post-challenge reaching final cumulative mortalities of 31% and 52%, respectively, for the fish given marine and salmonid pellets for 10 consecutive days. The fish treated with medicated marine pellets for five consecutive days and at days 1, 2, 4, 6 and 8 (sequential feeding) following initiation of treatment had cumulative mortalities of 52% and 38%, respectively. Survival of medicated fish in all groups was significantly (P < 0.005) greater than survival of challenged unmedicated fish. Furthermore, a significant difference (P < 0.001) in survival was found between fish treated for 10 consecutive days using marine pellets and the groups using marine pellets for five consecutive days and salmonid pellets for 10 consecutive days. Twenty four hours following last medication, six fish had mean plasma concentrations of 3.3 +/- 1.7 and 3.5 +/- 2.8 microg mL(-1), respectively, in fish treated for 10 consecutive days using marine and salmonid pellets. Corresponding values for fish treated for five consecutive days and by sequential feeding were 2.2 +/- 2.3 and 1.7 +/- 0.7 microg mL(-1), respectively.

A one-step simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of oxytetracycline (OTC) in fish tissue. The method involves liquid extraction of muscle tissue, precipitation of proteins and reversed phase HPLC analysis with spectrophotometric detection. The limit of quantitation of OTC in spiked fish muscle was 0.04 microg/g and the method showed high linearity (r(2) = >0.999) in the working range of 0.04-2 microg/g. The precision (%R.S.D.) was between 1.9 and 7.5% for the concentration range 0.04-1.0 microg/g and there was no significant difference between the concentrations determined on three different test days for all four spiked concentrations. The percentage recovery over the spiked concentration range 0.04-1.0 microg/g was consistently within a narrow range of 33-35%. While the method had the advantage of high precision, sensitivity and linearity, the method's additional salient advantages included high sample through-put (60 individual preparations per day) and minimum amount of consumables, time and labour required to perform the analysis. The method was successfully applied to a pharmacokinetic study.

The efficacy of a single intraperitoneal injection of oxolinic acid to control an outbreak of atypical Aeromonas salmonicida infection in goldsinny wrasse (Ctenolabrus rupestris) and in the treatment of systemic vibriosis in corkwing wrasse (Symphodus melops) was examined. In addition a field study was performed to examine the effect of medication on the survival rate of goldsinny wrasse in Atlantic salmon cages. Four groups of wild caught goldsinny wrasse, each of 50 fish, were treated with an intraperitoneal injection of propylene glycol:saline (50:50) (control) or 50 mg/kg oxolinic acid at a concentration of 50 mg/mL. Three days after medication the fish in all groups were treated by an intraperitoneal injection of prednisolone acetate and an increase in seawater temperature from 9.0 to 11.5 degrees C. Cumulative mortalities were 18% in the two groups treated with oxolinic acid and 94 and 100% in the unmedicated control groups, giving a 'relative percentage survival' (RPS) value of 82%. A laboratory maintained population of originally wild caught corkwing wrasse experiencing high daily mortality was treated with oxolinic acid (50 mg/kg) or propylene glycol:saline (control). Cumulative mortalities were 84% (control) and 42% (oxolinic acid medicated group) giving an RPS value of 50%. In a field investigation using goldsinny wrasse approximately 30% were medicated with oxolinic acid (50 mg/kg) prior to stocking in cages with Atlantic salmon. In two of three cages the cumulative mortality was significantly lower (P = 0.025 and P < 0.001) in the medicated groups.

The pharmacokinetic properties of the antibacterial agent oxolinic acid and vetoquinol, the carbitol ester of oxolinic acid, were studied after intravenous (i.v.) and oral (p.o.) administration to 100-150 g cod, Gadus morhua L., held in sea water at 8 degrees C. Following i.v. injection, the plasma drug concentration-time profile showed two distinct phases. The distribution half-life (t1/2alpha) was estimated at 1.3 h, the elimination half-life (t1/2beta) as 84 h and the total body clearance (Cl(T)) as 0.047 L kg(-1) h(-1). The volume of distribution at steady state, Vd(ss) was calculated to be 5.5 L kg(-1), indicating good tissue penetration of oxolinic acid in cod. Following p.o. administration of oxolinic acid or vetoquinol, the peak plasma concentrations (C(max)) of oxolinic acid and the time to peak plasma concentrations (T(max) were estimated to be 1.2 and 2.5 microg mL(-1) and 24 and 12 h, respectively. The bioavailabilities of oxolinic acid following p.o. administration of oxolinic acid and vetoquinol were calculated to be 55 and 72%, respectively. The in vitro minimum inhibitory concentration (MIC) values of oxolinic acid against three strains of Vibrio anguillarum isolated from diseased cod were 0.016 microg mL(-1) (HI-610), 0.250 microg mL(-1) (HI-618) and 0.250 microg mL(-1) (HI-A21). Based on a MIC value of 0.016 microg mmL(-1) a single p.o. administration of 25 mg kg(-1) of oxolinic acid maintains plasma levels in excess of 0.064 microg mL(-1), corresponding to four times the MIC-value, for approximately 12 days. The analogous value for a single p.o. dose of 25 mg kg(-1) of oxolinic acid administered as vetoquinol was 13 days.

A high performance liquid chromatographic (HPLC) method was developed to determine the concentration of diflubenzuron, a delousing agent used in fish farming, in marine mud and shell sand. The recovery of diflubenzuron from mud was 100.8+/-1.1% and 105.5+/-4.3% for shell sand. The limit of quantitation was found to be 0.1 microg g(-1). The stability of diflubenzuron was studied under laboratory conditions in marine sediments at different temperatures (4 and 14 degrees C). No degradation of diflubenzuron occurred in the organic rich mud sediment or in the shell sand sediment during the experimental period of 204 days. Increasing the temperature from 4 to 14 degrees C had no effect on the stability. Furthermore, diflubenzuron showed to be persistent in both mud and shell sand sediment since no detectable diffusion from the sediment to the water phase occurred during the experimental period of 204 days. Increasing the water current in the tanks had no effect on the persistence. Under field conditions, the concentrations of diflubenzuron found in the organic material from sediment traps placed 2 m from the bottom under the cage in a fish farm during medication were high and ranged from 71 to 259 microg g(-1). The concentrations of diflubenzuron in the sediment under the fish farm were, however, low, with a maximum concentration of 5.4 microg g(-1). The dispersion of diflubenzuron to the sediment was limited to less than 20 m from the edge of the cage in every direction. Fifteen months following the medication, only traces (< 0.1 microg g(-1)) of diflubenzuron were detected in the sediment under the fish farm. Possible explanations for this decrease are resuspension and redistribution of sediment and/or oxic degradation of the drug.

The pharmacokinetic properties of the antibacterial agents oxolinic acid and flumequine were studied in corkwing wrasse (Symphodus melops) after either intraperitoneal injection or bath treatment. Following intraperitoneal administration the peak plasma concentrations (Cmax) and the time to peak plasma concentrations (Tmax) were estimated to be 2.0 microg/mL and 12 h, respectively, for oxolinic acid and 2.6 microg/mL and 12 h, respectively, for flumequine. In muscle, Cmax and Tmax were estimated to 6.7 microg/g and 12 h, respectively, for oxolinic acid with corresponding values of 8.5 microg/g and 13 h, respectively, for flumequine. In liver, Cmax and Tmax were calculated to 7.0 microg/g and 12 h, respectively, for oxolinic and 12.2 microg/g and 11 h, respectively, for flumequine. Elimination half-lives (t1/2 beta) of 26, 24 and 29 h, respectively, for plasma, muscle and liver were calculated for flumequine. For oxolinic acid two distinct elimination phases were found and calculated to be 16 h (t1/2 beta) and 57 h (t1/2 gamma) in plasma, 15 and 59 h, respectively, in muscle and 20 and 72 h, respectively, in liver. Bath treatment using 150 mg/L of flumequine or 200 mg/L of oxolinic acid for 72 h resulted in flumequine concentrations of 1.0 microg/mL in plasma, 5.0 microg/g in muscle and 12.4 microg/g in liver. Corresponding values for oxolinic acid were 1.0 microg/g in plasma, 2.5 microg/g in muscle and 4.9 microg/g in liver.

1. Uptake, bioavailability, tissue disposition and elimination of sulphadimethoxine (SDM) and ormetoprim (OMP) were examined in Atlantic salmon (Salmo salar) following intravenous and oral administration of Romet at a dose of 5 mg OMP and 25 mg SDM kg-1 fish. 2. Plasma clearance was rapid for both drugs following a single i.v. dose, characterized by t1/2 alpha = 0.48 and 0.54h, t1/2 beta = 9.9 and 25.6h for SDM and OMP respectively with a volume of distribution (Vss) = 0.389 and 2.478 l kg-1. 3. Following oral administration, peak plasma concentrations of 1.13 and 9.99 micrograms ml-1 were achieved after 17.6 and 20.3h for OMP and SDM respectively. Bioavailabilities were 85% for OMP and 39% for SDM. 4. Oral administration revealed the highest concentration of OMP in kidney and liver whereas the highest concentrations of SDM were found in muscle and bile. 5. High concentrations of N4-acetylated SDM were found in the bile indicating significant metabolism of SDM.

A rapid clean-up and high-performance liquid chromatographic method for the simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of Atlantic salmon (Salmo salar) has been developed. Sample preparation is based on protein precipitation using trichloroacetic acid or methanol for plasma and muscle, respectively. The drugs are separated using a reversed-phase C18 analytical column and phosphate buffer-acetonitrile (80:20, v/v) containing 1-heptanesodiumsulphonate and triethylamine, as mobile phase. Detection was performed at 270 nm. The average recovery of ormethoprim was 97.2% in muscle and 95.7% in plasma, whereas the average recovery of sulphadimethoxine was 86.5% in muscle and 90.2% in plasma. The limit of detection at a signal-to-noise ratio of 3 was 50 ng/g and 30 ng/ml for ormethoprim in muscle and plasma respectively and 30 ng/g and 15 ng/ml in muscle and plasma respectively for sulphadimethoxine.

A sensitive high-performance liquid chromatographic procedure was developed for the determination of oxolinic acid in fish silage. Oxolinic acid was extracted with a mixture of McIlvaine buffer (pH 3.6) and methanol (55:45) and re-extracted into dichloromethane. After successive clean-up by liquid-liquid partitioning, oxolinic acid was determined by HPLC with fluorimetric detection (exitation at 325 nm, emission at 360 nm). The analytical column was 3-microns MOS-Hypersil (150 x 4.6 mm I.D.) and the mobile phase contained (A) 0.025 M oxalic acid (pH 3.2)-acetonitrile-methanol-tetrahydrofuran (80:2.5:15:2.5, v/v) and (B) oxalic acid (pH 3.2)-acetonitrile-methanol-tetrahydrofuran (50:20:25:5), v/v) with the following elution profile: 0-5 min, linear gradient from 50 to 100% B; 5-10 min, isocratic at 100% B; 10.1-15 min, isocratic at 50% A-50% B. The calibration graph was linear over the concentration range studied (0.025-0.2 microgram/g). The limit of detection was 0.01 microgram/g (signal-to-noise ratio = 4).