A sensitive microbioassay for the detection of antibacterial agents in the aquatic environment

Abstract A sensitive microbioassay for the detection of antibacterials used in the therapy of farmed fishes is described. Three indicator organisms ( Bacillus subtilis, Bacillus cereus and Escherichia coli ) were evaluated for their sensitivity to seven antibacterial agents (Enrofloxacin, Sarafloxacin, Florfenicol, Cosumix, oxolinic acid, amoxycillin and oxytetracycline). The limits of detection using the microbioassay were compared with those for a conventional radial diffusion assay. The microbioassay was two to four times more sensitive for all antibacterials tested except for amoxycillin, where the end point was the same. The sensitivity of the microbioassay to the presence of oxytetracycline in sea water was evaluated using Bacillus cereus as an indicator organism. The results indicate that the microbioassay can be adapted for use with sea water samples if the samples are filtered before use.     

Pharmacokinetics of oxolinic acid and oxytetracycline in kuruma shrimp, Penaeus japonicus

The pharmacokinetics of oxolinic acid and oxytetracycline were examined in kuruma shrimp (Penaeus japonicus) after intra-sinus (10 and 25 mg/kg, respectively) and oral (50 mg/kg) administration. The shrimp were kept in tanks with recirculated artificial seawater at a salinity of 22–23 ppt. The water temperature was maintained at 25±0.6 °C. The hemolymph concentrations of both drugs after intra-sinus dosing were best described by a two-compartment open model. The distribution and elimination half-lives (t_1/2 and t_1/2β) were found to be 0.59 and 33.2 h for oxolinic acid and 0.45 and 24.7 h for oxytetracycline, respectively. The apparent volume of distribution at a steady state (V_ss) and total body clearance (CL_b) were estimated to be 1309 ml/kg and 28.8 ml/kg/h for oxolinic acid and 748 ml/kg and 22.7 ml/kg/h, respectively. The hemolymph concentration–time curves after oral administration did not fit by the nonlinear least squares method using one- and two-compartment model with first-order absorption in either of the drugs. The peak hemolymph concentration (C_max), the time to peak hemolymph concentration (t_max) and the elimination half-life were found to be 17.8 μg/ml, 7 h and 34.3 h for oxolinic acid and 24.3 μg/ml, 10 h and 33.6 h for oxytetracycline, respectively. The bioavailability (F) after oral administration was 32.9% for oxolinic acid and 43.2% for oxytetracycline. The hemolymph protein binding in vivo was determined to be 36.7±8.5% for oxolinic acid and 22.9±4.8% for oxytetracycline.     

The in vitro susceptibility of Aeromonas salmonicida and other fish-pathogenic bacteria to 29 antimicrobial agents

Abstract. The minimum inhibitory concentrations (MICs) of 29 antimicrobial agents were determined for some representative Aeromonas salmonidda, Yersinia ruckeri and Vibrio isolates. Eleven of them, including three widely used in clinical practice, were further tested against 28–36 recent field isolates of A. salmonidda. The fluoroquinolones were inhibitory at very low concentrations with 50% of A. salmonidda isolates having a MIC of enro-floxacin ≤0·02μg ml-1. The distribution of MICs to the fluoroquinolones and to oxolinic acid were similar indicating possible cross resistance. Amoxycillin, florfenicol, minocycline, rifampicin and the potentiated sulphonamides had a single peak MIC distribution with values of ≤1·25μg ml-1. Minimum inhibitory concentrations of the fluoroquinolones were four or more times greater at 10°C compared with 22°C; this was not so with amoxycillin, florfenicol or the sulphachlorpyridazine-trimethoprim (S-TP) combination. A. salmonicida was rapidly killed at 22°C by the fluoroquinolones and S-TP whereas oxolinic acid and amoxycillin killed within 24h. Florfenicol was bacteriostatic. The therapeutic potential of some of these antibiotics is considered.     

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.     

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04‐A2 and VET03‐A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non‐wild type (NWT) using normalized resistance interpretation (NRI)‐determined cut‐off values (CO_WT). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC CO_WT ≤ 0.25 mg/L) and 42% were WT for oxytetracycline (MIC CO_WT ≤ 0.25 mg/L). In contrast, all the isolates tested were WT (MIC CO_WT ≤ 2 mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion‐based CO_WT values were ≥51 mm for 10 μg amoxicillin, ≥44 mm for 30 μg florfenicol, ≥30 mm for 2 μg oxolinic acid and ≥51 mm for 30 μg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).     

The precision and robustness of published protocols for disc diffusion assays of antimicrobial agent susceptibility: an inter-laboratory study

The precision of the disc diffusion protocols previously published by Alderman and Smith (Aquaculture 196 (2002) 211) was analysed in a seven-laboratory trial using Escherichia coli ATCC 25922 as the test strain. Discs containing seven antimicrobial agents were employed and 2899 zone size measurements were generated. The total data generated in the trial was used to quantify the intra- and inter-laboratory precisions. The study design also facilitated the investigation of the influence of the source of media and the source of discs on zone sizes. A smaller two-laboratory trial was employed to investigate the influence of incubation time of zone size.

The intra-laboratory precision was relatively high with the mean of the coefficients of variation calculated for each laboratory and each agent being 4.7%. In contrast, the inter-laboratory precision was very much lower with the mean of the values for each agent being 11.1%. Significant influences on zone size were detected for all three parameters of the protocol. Media source effects were particularly notable with respect to oxytetracycline and oxolinic acid discs, disc source effects with respect to ampicillin and sulphamethoxazole/trimethoprim discs and incubation times with the ampicillin and amoxycillin discs. ANOVA analysis of the total data set confirmed that inter-laboratory variation was the major factor influencing the low precision of the protocol.

The overall precision of the protocols used here was found to be significantly lower than that implied by the control limits associated with the same bacterium in other validated disc diffusion protocols. The implications of these results, for the further development of the protocols under investigation, are discussed.     

Antibiotic resistance of Aeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland

Abstract. Antibiotic sensitivity patterns of 304 isolates of Aeromonas salmonicida from 229 outbreaks of furunculosis among salmon in Scotland between 1988 and 1990 were investigated. Fifty-five per cent were resistant to oxytetracycline and 37% resistant to oxolinic acid. Multiple resistance was common (52%) and 18 out of 19 antibiograms which were found in the first year recurred in the succeeding year. More than a quarter of the outbreaks were associated with two or more A. salmonicida variants distinguishable by their antibiotic sensitivity patterns. The implications of these findings in the control of furunculosis are considered.     

Changes in drug resistance of Vibrio anguillarum in cultured ayu, Plecoglossus altivelis Temminck and Schlegel, in Japan

Abstract. Two-hundred-and-fifty-nine strains of Vibrio anguillarum isolated from diseased cultured ayu, Plecoglossus altivelis in various districts of Japan from 1974 to 1977 were studied for their sensitivity to 12 different chemotherapeutic agents: chloramphenicol (CM), tetracycline (TC), streptomycin (SM), kanamycin (KM), aminobenzyl penicillin (ABP), colistin (CL), nalidixic acid (NA), oxolinic acid (OA), piromidic acid (PA), furazolidone (NF), sulpharaonomethoxine (SA) and trimethoprim (TMP). One-hundred-and-thirty-four of the strains were additionally tested for sensitivity to O/129(2,4-diamino-6,7-diisopropyl pteridine) and ormethoprim (OMP). All strains were susceptible to SM, KM and ABP and also highly sensitive to CL. A correlation of resistance was found among the chemically-related drugs NA, OA and PA, and TMP, OMP and O/129, Nine strains were sensitive to all the drugs tested. The remaining 250 strains were resistant to various combinations of six drugs (CM, TC, NA, NF, SA and TMP). In particular, strains resistant to NA and NF (20.1%), to CM, TC, SA, NA and NF (29.7%) and to all six drugs (28.2%) were frequently detected. NA- or NF-resistant strains have increased since 1974 and TMP-resistant strains have increased since 1976. Transferable R plasmids were detected in 165 out of 250 resistant strains. The most common type of R plasmid determined resistance to CM, TC and SA. These multiple drug-resistant strains with transferable R plasmids were isolated from many different districts.     

Evaluation of florfenicol in Atlantic salmon,Salmo salar L.: efficacy against furunculosis due to Aeromonas salmonicida and cold water vibriosis due to Vibrio salmonicida

Two replicated controlled trials were conducted to determine the efficacy of florfenicol against Aeromonas salmonicida and Vibrio salmonicida infections in Atlantic salmon, Salmo salar L., smolts kept in 25‰ salt water. Infection with A. salmonicida was treated with florfenicol, oxolinic acid, oxytetracycline, trimethoprim/sulphadiazine or flumequine, whereas the V. salmonicida infection was treated with florfenicol or oxolinic acid only. A. salmonicida infection was induced by the introduction of cohabitant fish previously inoculated intraperitoneally. Medication started simultaneously in all test tanks on the first day of specific mortality among test fish. V. salmonicida infection was induced by intraperitoneal inoculation of all test fish. Medication started 1 day after infection. Medicated feeds were produced by coating the antibacterials on standard feed pellets, and administered twice daily for 10 consecutive days. With the dose used in the present trials, florfenicol was highly effective in reducing specific mortalities due to both infections. It was slightly more effective than oxolinic acid and trimethoprim/sulphadiazine against A. salmonicida infection. There was no significant difference between florfenicol and oxolinic acid in reducing specific mortalities due to V. salmonicida.     

Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administration to Atlantic salmon in seawater

The present preclinical study was performed to investigate the pharmacokinetics of flumequine in Atlantic salmon (Salmo salar L.) in seawater after administration of different doses and dosage formulations. Flumequine was administered intravenously (dose 4.9 mg/kg fish) and orally from the drug delivery system Aqualets as Apoquin 5 g/kg (dose 25 mg/kg) and 10 g/kg (dose 50 mg/kg), respectively. Experiments were carried out with oxolinic acid administered in the same way for the purpose of comparing the two compounds. The seawater temperature was 5±0.2°C in all experiments.

The pharmacokinetic calculations showed that the distribution half-life for flumequine was and for oxolinic acid . The drugs were absorbed rapidly, and flumequine reached a plasma concentration of C_max = 2.26 μg/ml after a single oral dose of 25 mg/kg, whereas oxolinic acid reached C_max = 0.99 μg/ml. The apparent bioavailability of flumequine was found to be 40–45%, whereas the apparent bioavailability of oxolinic acid varied from 25% at a dose of 50 mg to 40% at a dose of 25 mg/kg body weight of fish. The distribution profile of flumequine in the various compartment of fish appeared to be different from that of oxolinic acid. After a single oral dose (25 mg/kg) the areas under the concentration-time curves showed that flumequine was 2.3 times more concentrated in plasma and 2.6 times more concentrated in liver compared to oxolinic acid. In muscle the difference was less pronounced, flumequine being 1.4 times more concentrated than oxolinic acid.     

Chemotherapy of Cytophaga/Flexibacter-like bacteria (CFLB) infections in fish: studies validating clinical efficacies of selected antimicrobials

Abstract. In vitro and in vivo efficacies of commonly used chemotherapeutants were determined for Cytophaga johnsonae, Cytophaga psychrophila, Flexibacter colutnnaris and Flexibacter maritimus. Treatmeat of barramundi, Lutes calcarifer (Bloch), with oxolinic acid (OA) as a bath (50 ppm) or by mouth (10 mg kg⁻¹ body weight) resulted in serum levels above the minimum inhibitory concentration (MIC) for F. columnaris and produced significant clinical efficacy ( P < 0.05). Amoxycillin (AM) was found to produce adequate serum levels against F. maritimus when used as a bath (200 ppm) or given orally (80 mg kg⁻¹ body weight) to Atlantic salmon, Salmo salar L. and rainbow trout, Oncorhynchus mykiss (Walbaum), respectively. At these dose rates, this antibiotic was also clinically efficacious against F. maritimus ( P <0.05). Trimethoprim produced more than adequate serum levels for the control of F. maritimus when given as a bath (50 ppm) or orally (10 mg kg⁻¹ body weight). Trimethoprim was significantly more protective than AM when tested in vivo ( P < 0.05). For C. johnsonae and C. psychrophila , the MIC values for OA and oxytetracycline were low, whereas that for TMP was high. Also, based on MIC values, C. psychrophila strains were more sensitive to AM and norfloxacin than C. johnsonae.     

Digestibility of chloramphenicol,oxolinic acid and oxytetracycline in rainbow trout and influence of these antibiotics on lipid digestibility

Chloramphenicol (CP), oxolinic acid (OA) and oxytetracycline (OT) were fed to rainbow trout (Salmo gairdneri Richardson) at two different concentrations (0.1 and 0.5%) in a dry diet. At either dose, the apparent digestibility of CP was close to 99%, whereas it was in the 7–9% range for OT. For OA, these percentages were 38.1 and 14.3% for the 0.1 and 0.5% doses respectively. The analyses of lipids in the different groups demonstrated that antibiotics have no significant effect on the total lipid content and total fatty acids of the trout. However, all the antibiotics significantly enhanced the digestibility of some unsaturated fatty acids when given at the 0.5% dose.     

A single-dose pharmacokinetic study of oxolinic acid and vetoquinol,an oxolinic acid ester,in cod,Gadus morhua L., held in sea water at 8 degrees C and in vitro antibacterial activity of oxolinic acid against Vibrio anguillarum strains isolated from diseased cod

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.     

恶喹酸与诺氟沙星对异育银鲫体内嗜水气单胞菌的抑菌效果比较

在水温(25±2)℃下,给体质量150~200g的异育银鲫分别口灌3种剂量(20、30、40mg/kg)的诺氟沙星和恶喹酸,结合这两种药物对嗜水气单胞菌AH10的体外药效学研究和对异育银鲫单次口灌不同剂量的诺氟沙星、恶喹酸的体内药代动力学,研究诺氟沙星、恶喹酸对异育银鲫体内嗜水气单胞菌AH10的抑菌效果。试验结果表明,恶喹酸和诺氟沙星对嗜水气单胞菌AH10的最小抑菌质量浓度分别为1μg/mL和0.5μg/mL。口灌上述3种剂量恶喹酸、诺氟沙星后,异育银鲫血浆中恶喹酸的最大药物质量浓度分别为4.1μg/mL、6.0μg/mL和8.89μg/mL;诺氟沙星的最大药物质量浓度分别为11.5μg/mL、15.1μg/mL和18.9μg/mL;恶喹酸的最大药物质量浓度/最小抑菌质量浓度分别为4.1、6.0和8.89;诺氟沙星的最大药物质量浓度/最小抑菌质量浓度分别为23、30.2和37.8;恶喹酸0~24h内药—时曲线下面积/最小抑菌质量浓度分别为21.6214、33.1449、39.1846;诺氟沙星0~24h内药—时曲线下面积/最小抑菌质量浓度分别为274.75、451.55、578.35。综合0~24h内药—时曲线下面积/最小抑菌质量浓度、最大药物质量浓度/最小抑菌质量浓度这两个指标可知,诺氟沙星对异育银鲫体内的嗜水气单胞菌AH10抑制效果强于恶喹酸。     

Some biochemical properties and antibiotic sensitivities of Pasteurella piscidda isolated in Greece and comparison with strains from Japan, France and Italy

Abstract The biochemical properties and antibiotic sensitivity patterns of 13 Pasteurella piscicida isolates from Greece are described and compared with 10 Japanese and five European (Italian and French) isolates. Morphologically and biochemically, all isolates of P. piscidda tested were nearly identical and only minor differences could be detected with reference to the level of acid production from sugars (especially from glucose). Greek, Italian and French isolates showed similar antibiotic sensitivity patterns, being resistant to erythromycin, kanamycin and streptomycin, and sensitive to most of the other antibiotics tested. Resistance was also demonstrated for some isolates towards the potentiated sulphonamides. The Japanese isolates appeared to be resistant to a larger range of antibiotics including erythromycin, kanamycin, streptomycin, oxolinic acid, oxytetracycline and chloramphenicol. Multiple resistance of Japanese strains was also noted. These results are discussed in the light of future measures for the control of pasteurellosis.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Delivery of quinolone antibacterials to turbot, Scophthalmus maximus (L.), via bioencapsulation: quantification and efficacy trial

Abstract. The incorporation of the quinolone antibacterials oxolinic acid and sarafloxacin into Artemia fransciscana and subsequently into turbot, Scophthalmus maximus (L.), was quantified, and the therapeutic efficacy of the bioencapsulated drugs against challenge with Vibrio anguillarum 8587 was investigated. Five-week-old turbot were fed for 10 days with Artemia enriched with either oxolinic acid or sarafloxacin. Therapeutic concentrations were reached with both drugs (11.8 μg oxolinic acid per gram fresh weight: 1.8 μg sarafloxacin per gram fresh weight). Both treatments proved effective against challenge, with V. anguillarum -specific mortality rates of 6 and 14% in the oxolinic acid and sarafloxacin treated groups, respectively, compared with 75% in the infected control. However, incorporation of the drug by brine shrimp was extremely low (<1%), and therefore, bioencapsulation cannot be considered as an appropriate method to reduce impact on the environment.     

A single-dose pharmacokinetic study of oxolinic acid and vetoquinol, an oxolinic acid ester, in Atlantic halibut, Hippoglossus hippoglossus L., held in sea water at 9 °C

The pharmacokinetic properties of the antibacterial agent oxolinic acid were studied after intravenous, intraperitoneal and oral administration to 1.5–3.0 kg Atlantic halibut, Hippoglossus hippoglossus L., held in sea water at 9 °C. Following intravenous injection, the plasma drug concentration-time profile showed two distinct phases. The terminal elimination half-life was estimated to be 52 h, whereas total body clearance (Cl_T) was determined to be 0.044 L kg^–1 h^–1. The volume of distribution at steady state, V_d(ss), was calculated to be 3.0 L kg^–1, indicating good tissue penetration of oxolinic acid in Atlantic halibut. The peak plasma concentration (C_max) and the time to peak plasma concentration (T_max) were estimated to be 1.2 and 2.7 μg mL^–1, and 21.5 and 80 h, respectively, following oral administration of medicated feed or intraperitoneal injection. The corresponding bioavailabilities were calculated to be 15% and 92%, respectively. Oral administration of vetoquinol, the carbitol ester of oxolinic acid, increased the bioavailability of oxolinic acid to 64% and the total bioavailability (oxolinic acid + vetoquinol) to 82%, whereas C_max and T_max values of 6.7 μg mL^–1 and 14.5 h, respectively, for oxolinic acid, and 1.0 μg mL^–1 and 6.3 h, respectively, for vetoquinol were obtained. Based on a minimum inhibitory concentration (MIC) of 0.0625 μg mL^–1 for susceptible strains, a single intraperitoneal injection of 25 mg kg^–1 of oxolinic acid maintains plasma levels in excess of 0.25 μg mL^–1, corresponding to four times the MIC value, for ≈12 days. The corresponding values for a single oral dose of 25 mg kg^–1 of oxolinic acid and vetoquinol were 5 and 10 days, respectively. For resistant strains with a MIC of 1 μg mL^–1, a single oral dose of vetoquinol (25 mg kg^–1) maintained plasma levels in excess of 4 μg mL^–1 for 34 h.     

Bioencapsulation and pharmacokinetic study of oxolinic acid in rotifers (Brachionus plicatilis), Artemia franciscana nauplii and metanauplii

The uptake of oxolinic acid by the rotifer Brachionus plicatilis, Artemia franciscana nauplii and metanauplii was studied as a function of its concentration in the enrichment medium and the duration of the enrichment period. An emulsion containing 5, 10, 20 or 30% (w/w) oxolinic acid was administered and the enrichment period lasted 4, 8, 12 or 36 h. Highest incorporation of oxolinic acid was achieved using a 20% emulsion and a 12 h enrichment for rotifers (205.05 ± 17.1 μg g^?1 dry weight), a 24 h enrichment for nauplii (2528.8 ± 254.6 μg g^?1 dry weight), and an 8 h enrichment for metanauplii (1236.58 ± 22.9 μg g^?1 dry weight). Higher concentrations of oxolinic acid in the enrichment emulsion or longer enrichment times resulted in decreased survival. Two hours post enrichment the contents of the drug appeared significantly decreased. The concentration data of oxolinic acid were best fit to a two phase exponential elimination model, the first phase elimination half‐life (t_1/2α) being 1.86, 1.08 and 1.74 and the terminal phase elimination half‐life (t_1/2β) 26.83, 29.67 and 17.48 in rotifers, nauplii and metanauplii correspondingly. Enrichment with an emulsion containing 20% oxolinic acid is recommended employing a duration of 12, 24, or 8 h enrichment for rotifers, nauplii and metanauplii respectively, while enriched carriers should be used shortly after enrichment.     

Leaching of taurine from commercial type aquaculture feeds

Leaching of soluble compounds from pelleted feeds is an issue for the aquaculture industry through increased environmental impact and reduced ingestion essential components. This study was undertaken to examine the leaching rates of taurine, a non‐protein amino acid with critical physiological roles in teleosts. To this end we adapted a new liquid chromatography mass spectrometry (LC‐MS) method for quantifying taurine. Twelve different feeds (4 mm dia.) varying in protein source and taurine levels were examined. Fishmeal content ranged from 0.0% to 45.5% with taurine supplementation ranging from 0.0% to 5.0%. Taurine was extracted and quantified from individual pellets in triplicate at six time points (0, 1, 5, 10, 20 and 40 min). Leaching rates ranged from 0.026 ± 0.005 to 0.826 ± 0.121 mg min^?1 over 40 min at 27°C and were strongly correlated to initial taurine content of the feeds (for distilled water n = 12, P < 0.001, R² = 0.91 for artificial seawater, 25 ppt, n = 4, P = 0.020, R² = 0.96). Loss of taurine from feeds was 59.5 ± 16.5% after 40 min. This study shows that a significant amount of taurine is lost over time from uneaten feed and that taurine supplementation should exceed requirement levels for slow consumers or feed being delivered as multiple additions.