Rosie Coyne

Concentration and persistence of oxytetracycline in sediments under a marine salmon farm

Abstract The concentration of oxytetracycline in the sediment under two adjacent cage blocks in a marine salmon farm was determined following the therapeutic use of the drug. The sediment cores were grey, indicating some build-up of organic material. Infaunal polychaetes were present as were mobile fauna including crabs, starfish and flat fish. There were no significant accumulations of undigested feed pellets under the cages. At one block where 186 kg oxytetracycline was used over 10 days the oxytetracycline concentrations were determined under a single cage that received 8.65 kg oxytetracycline during the treatment. Peak concentrations in the top 2 cm of the sediment were 9.9±2.9 μg·g −1 . This declined at an exponential rate ( r 2 =0.99) with a half-life of 16 days. At the second block the oxytetracycline concentration was measured with a sampling programme designed to determine the horizontal and vertical distribution of oxytetracycline under the whole cage block during and after a treatment where 175 kg of oxytetracycline were used over 12 days. Peak concentrations, in the top 2 cm of the sediments under the cage block, were 10.9±6.5 μg·g −1 and this declined at an exponential rate ( r 2 =0.99) with a half-life of 13 days. Nineteen days after the end of the therapy oxytetracycline was detected at depths of up to 8 cm in the sediment, but the concentration of the antibacterial agent had decreased at all levels in the sediment 14 days later. At the end of the treatment oxytetracycline was detected in an area of the sediment less than twice the area of the cages themselves. Data on current flow and sedimentation rate were used to generate a predictive model of the area of sediment that would be subject to the deposition of both pelleted fish feed and fish faeces. Oxytetracycline was confined to the area of sediment predicted to be subject to feed deposition that was directly under and slightly to the west of the cage block. Oxytetracycline was not detected in the area predicted to be subject to faecal deposition only.

Fish feed as a source of oxytetracycline-resistant bacteria in the sediments under fish farms

Abstract Concentrations of oxytetracycline and the frequency of oxytetracycline resistance in the environmental microflora were monitored following the therapeutic use of this agent at a marine fish farm. 529 kg of oxytetracycline were administered over a 24 day period at an average of 1.4 kg per cage per day. Three days after the end of the therapy 4.6 ± 3.7 μg/g oxytetracycline were detected in the sediments and the frequency of resistance in the sediment microflora was 9.0 ± 5.3%. A rise in the frequency of resistance in this flora to 26 ± 8.7% occurred 24 days after the therapy. This rise was not associated with any increase in the concentrations of oxytetracycline in the sediment. At this time the frequency of resistance in the flora isolated from mussels suspended above the sediments (36 ± 8.5%) was significantly ( P = 0.005) higher than that present in the sediment flora. The feed used on the farm 24 days after the end of therapy was shown to contain 4.6 × 10 4 oxytetracycline-resistant cfu/g. The distribution of phenotypic groups in the oxytetracycline-resistant flora in this feed and in the sediments during the peak in resistance were compared with those from other marine environments. These data demonstrated that resistant flora in feed can, under certain circumstances, significantly contribute to the resistant flora detected in sediments under fish cages.

Spatial distribution of oxytetracycline and elevated frequencies of oxytetracycline resistance in sediments beneath a marine salmon farm following oxytetracycline therapy

Abstract The concentrations of oxytetracycline and the frequencies of oxytetracycline resistant microorganisms were determined in 11 samples taken from the sediments in the vicinity of a block of fish cages at a marine salmon farm. The cage block contained 10 tonnes of Atlantic salmon smolts and a total of 20 kg of oxytetracycline were administered during the 12 day treatment. Samples cores were collected by divers 5 days after the end of the period of therapy and the top 2 cm of each core was analysed. HPLC analysis was able to quantify the oxytetracycline concentrations in three of the six samples taken directly under the cage block. The mean concentration under the cage was between 0.65 and 1.2 μg g −1 ( n = 6) depending on the values attributed to samples where the concentrations were below the level of quantitation (1.2 μg g −1 ). In the five samples taken from locations not directly under the cage block oxytetracycline was only detected in the sample taken adjacent to, and down current from, the cage block. This sample was collected 10 m to the west of the cage block and contained 4.2 μg g −1 oxytetracycline. These data indicate that oxytetracycline was confined to an area of the sediment which was smaller in extent than the area of the cage block itself. The frequencies of resistance to oxytetracycline in the microflora cultured from the samples were determined by differential plating on 2216V media, containing 25 and 100 μg ml −1 oxytetracycline. Analysis of eighty three samples from sites free of overt human influence demonstrated that the background levels of resistance at these two selection concentrations were 1.3 ± 1.3% and 0.4 ± 0.6%, respectively. Elevated frequencies of resistance were detected in samples from a wider area than the cage block. The median frequency of resistance in the samples ( n = 6) taken from directly under the cage block was 1.4% at 100 μg ml −1 and 5.3% at 25 μg ml −1 . In the samples ( n = 5) taken from outside the cage block the frequencies were 5.3% at 100 μg ml −1 and 8.5% at 25 μg ml −1 . There was no correlation between the concentration of oxytetracycline in a sample and the frequency of resistance that was determined in the culturable microflora in that sample.

Frequency and distribution of resistance to oxytetracycline in micro-organisms isolated from marine fish farm sediments following therapeutic use of oxytetracycline

Abstract the background level of resistance to oxytetracycline in sediments free of anthrogogenic influences was determined on 2216 V agar with 25 μg·g −1 oxytetracycline. The mean frequency of resistance in 153 samples taken in Galway Bay was 1.2 ±1.8%. The impact of oxytetracycline therapy on the frequency of resistance in the sediments under a marine fish farm was investigated on two occasions. In the first investigation, oxytetracycline was detected at a concentration of 9.9 ± 2.9 μg·g −1 in the sediments under a cage that received 865 g oxytetracycline per day for 10 days, but no significant rise in resistance frequency was detected. In the second investigation, oxytetracycline was detected at a concentration of 10.9 ± 6.5 μg·g −1 in the sediments under a cage block that received 175 kg oxytetracycline over 12 days. The frequency of resistance reached 16.0 ± 8.9% after the treatment. The frequency declined at an exponential rate ( r 2 =0.89) with a half-life of 26 days. At 73 days after the end of therapy the frequency, in under-cage samples, was not significantly higher than the background level. At the end of the therapy elevated frequencies of resistance were detected up to 75 m from the edge of the cage block and in samples where the levels of oxytetracycline were below the limit of detection (1.2 μg·g −1 ). Thirty-three days after the end of the therapy the frequency of resistance in all samples not directly under the cages was not significantly higher than in samples taken from sediments free of anthropogenic influence.

Transient presence of oxytetracycline in blue mussels (Mytilus edulis) following its therapeutic use at a marine Atlantic salmon farm

On 15 occasions, during and after a therapeutic treatment of fish in an Atlantic salmon farm, oxytetracycline concentrations were determined in samples of blue mussels collected at two sites in the vicinity of the farm. Oxytetracycline was not detected in any samples of mussels collected 20 m from the cage block at a depth of 1 m. Those mussels sampled from immediately under one cage (Cage 65) at the farm (10–11 m depth) contained a concentration of 10.2 μg oxytetracycline g−1 of soft tissue on the last day of treatment. After the end of oxytetracycline administration the concentration detected in these samples declined exponentially (r2 = 0.94) with a half-life of approximately 2 days. At the time of the treatment the farm was stocked with 144 tonnes of fish and 186 kg of oxytetracycline were administered during a ten day treatment. In muscle samples taken from fish (n = 5) in Cage 65, after 8 days of therapy the mean oxytetracycline concentration was 1.3 ± 0.9 μg g−1. Three days after the end of the therapy the top 2 cm of the sediments beneath Cage 65 contained 9.9 ± 2.9 μg g−1 (n = 5). These tissue and sediment concentrations lie within the ranges of values that have previously been reported for such treatments. It is argued that residues present in filter feeding bivalves that occur as a consequence of the therapeutic use of oxytetracycline in marine fish farms are unlikely to present a significant human health hazard.

The critical importance of sample site in the determination of the frequency of oxytetracycline resistance in the effluent microflora of a fresh water fish farm

Abstract The frequency of oxytetracycline resistance in the microflora was studied in the effluent of a fresh water Atlantic salmon farm which had not used any oxytetracycline for three years. Resistance was defined as the ability to grow on casein peptone starch agar containing 25 μg ml −1 oxytetracycline. The farm effluent passed through a rolling drum filter and the retentate flow then entered a sedimentation tank. The effluent was sampled from the filter retentate as it left the filter and from the settled solids in the sedimentation tank. 12 independent samples were taken on four sampling days from two sampling sites. The frequency of resistance was dramatically different in samples taken at the two different locations in the farm outflow. In samples from the retentate of the outflow filter the median frequency of resistance was 0.47% (range 0.25–1.2%). In contrast in samples taken from a sedimentation tank downstream from the filter the median frequency was 38% (range 10–48%). Studies in oxytetracycline-free laboratory mesocosms demonstrated that in the presence of anaerobically decomposing fish feed, river sediments and river water the frequency of oxytetracycline resistant strains increased rapidly during incubation at 18 °C. After 14 days incubation the frequency had risen from 1.0% to 25%. In a similar mesocosm from which feed was omitted the frequency of resistance remained below 1%. It is suggested that elevated frequencies of oxytetracycline resistance may be encountered in environments where fish feed accumulates. These results have important implications for the design of surveys of the impact of antimicrobial agent use in fresh water fish farms.

Fate of oxytetracycline in a fresh water fish farm : influence of effluent treatment systems

Abstract The fate of orally administered oxytetracycline in a fresh-water hatchery was investigated using high-performance liquid chromatography. A filter of nominal porosity 50 μm in the farm effluent was capable of at least a 500–650-fold concentration of the oxytetracycline into the filter retentate flow. No oxytetracycline was detected in the filtered farm effluent (limit of detection 0.02 μg·ml−1). Analysis of hourly samples of the retentate taken over a 24-hour period allowed an estimate of the daily amount of oxytetracycline retained by the filter. This estimate (1250 g) was of the same order as the amount of oxytetracycline used on the farm (904 g on the day of sampling and 1104 g on the preceding day). Thus the quasi-totality of the input oxytetracycline was removed from the farm effluent by the filter. A sedimentation trap on the filter retentate flow removed oxytetracycline from this flow at a variable and lower (12–92%) efficiency. It is argued that correct design of effluent treatment systems could significantly reduce the environmental impact of land-based fish farms.

Concentration of 22,23-dihydroavermectin B1a detected in the sediments at an Atlantic salmon farm using orally administered ivermectin to control sea-lice infestation

Abstract An HPLC analytical method based on a methanol extraction, C 8 clean-up, derivatization using trifluoroacetic acid anhydride and reverse-phase HPLC was developed for the detection of 22,23-dihydroavermectin B 1a in marine sediment. The limit of quantitation was determined as 0.93 ng 22,23-dihydroavermectin B 1a (H 2 B 1a ) per g of sediment. This method was used to determine the concentrations of H 2 B 1a in sediments taken from under a fish farm where ivermectin had been administered orally, at 0.05 mg/kg twice weekly, to control sea-lice infestation in 350,000 post-smolt Atlantic salmon. Prior to sampling, a total of 23 g of ivermectin had been administered during the current production cycle. The sediment was sampled using diver-collected cores at sample sites 25 m apart. The mean concentration of H 2 B 1a detected in the six sediment cores taken directly under the cage block was 5.0 ng/g in the top 3 cm, 3.1 ng/g in the 3–6 cm fraction and 0.7 ng/g in the 6–9 cm fraction. Concentrations were under the limit of quantitation at depths below 9 cm in all cores taken from directly under the cage block. H 2 B 1a was not detected in any samples taken further than 22.5 m from the cage block in the eastern, western and southern directions. To the north of the cage block, H 2 B 1a was detected in samples taken 31 m from the cage block and the limit of distribution in this direction was not established. Despite the limitations of this study, it is difficult to reconcile the concentrations of H 2 B 1a detected in this study with long term accumulation and persistence of ivermectin in sediments under fish farms.

Treatment of the early life stages of scallop (Pecten maximus) with antimicrobial agents; searching for an alternative to chloramphenicol

High mortality rates are often observed in rearing the early stages of the great scallop, Pecten maximus. The addition of antibacterial agents has been necessary to improve larval survival. However, as one antibacterial agent, chloramphenicol, is banned in Norway and Europe the aim of this study is to investigate alternative antibacterial agents. The therapeutic agents investigated in this study were florfenicol, oxytetracycline, oxolinic acid, neomycin and Pyceze. The mean minimum inhibitory concentration (MIC) values were determined for oxytetracycline, oxolinic acid and Pyceze against bacteria isolated from scallop larvae. Two types of treatment regime were investigated on an intermediate scale (20 L). One regime involved continuous exposure of scallop larvae to the therapeutic agent while the other involved a short exposure lasting two hours. All intermediate scale treatments were performed in parallel to large-scale production (800 L) treatment with chloramphenicol. Of the therapeutants investigated, oxolinic acid was the most effective, although only at high concentrations. The short exposure of two hours was ineffective for all therapeutics.

Failure of flumisol bath treatments during commercial transport of Atlantic salmon smolts to prevent the activation of stress inducible furunculosis

Abstract This paper reports the development of protocols for the bath administration of flumequine to Atlantic salmon (Salmo salar L.) smolts during their transfer from a fresh water hatchery to a marine farm in buckets suspended under a helicopter. The fish were treated at a density of 365 kg m−3 in water of pH 6.3 and hardness 13.2 mg 1−1 CaCO3. Serum flumequine concentrations showed a linear increase with respect to time of the bath and were dose dependent. The addition of benzocaine to the baths had little effect on uptake during the first 20 min of the bath. 720 000 smolts with covert stress inducible furunculosis were transported to 20 sea cages during 180 helicopter trips in buckets containing 100 μg ml−1 flumequine and 5.7 μg ml−1 benzocaine. Furunculosis was detected in fish in all sea cages shortly after transfer and the treatment was considered to have failed to control the activation of the covert infections. The pharmacokinetics of flumequine were determined in fish that were introduced to sea water at the termination of a bath treatment in fresh water. This data was compared to that obtained from fish that remained in fresh water following a similar treatment. The introduction of the fish into sea water resulted in a very rapid excretion of flumequine via the intestine. It is argued that this rapid elimination of the antimicrobial agent may have been an important factor in the failure of the bath treatment under commercial conditions.