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.     

Delivery of Bioencapsulated Oxytetracycline to the Marine Shrimp Penaeus monodon

Abstract Post larvae of shrimp Penaeus monodon were fed Artemia sp. enriched with oxytetracycline (OTC). The amounts of this antibiotic in the Artemia sp., the shrimp, and the water of the experimental system were measured by radial diffusion bioassay. The results indicated that 11% of the OTC was taken up by the Artemia sp. After 8 d of being fed OTC-enriched Artemia sp., the shrimp were found to contain 3.12 μg OTC/shrimp, which is twelve times greater than the average minimum inhibitory concentration (MIC) cited in the literature for sensitive strains of Vibrio spp. The recommended therapeutic dose for treatment of bacterial infections is four times the MIC.     

Bioencapsulation of Therapeutic Quantities of the Antibacterial Romet-30 in Nauplii of the Brine Shrimp Artemia and in the Nematode Panagrellus redivivus

Nauplii of the brine shrimp, Artemia , and cultures of the soil nematode, Panagrellus redivivus , were fed a suspension of the water-insoluble antibacterial Romet-30 (sulfadimethoxine plus ormetoprim) to determine if they could be used as a mechanism for delivery of the drug to larval penaeid shrimp.
Romet-30 was added to groups of brine shrimp nauplii and nematodes in 1 L beakers to achieve a concentration of 3 mg of drug per ml of seawater. After a 4 h uptake period, predetermined numbers of organisms were assayed for antibiotic content using the micro-plate diffusion method and a Romet-30 sensitive strain of Vibrio alginolyticus as the indicator. The resulting zones of inhibition were compared to that of a standard Kirby-Bauer sensitivity disc containing 25 μg of the drug that was placed on each plate as standard control. Negative controls consisted of equal numbers of nauplii and nematodes that were not exposed to the antibiotic. Artemia nauplii and nematodes accumulated approximately 0.1 μg of the drug per nauplius and approximately 0.25 μg per nematode. They exhibited no signs of toxicity from exposure to the drug and contained the antibiotic in sufficient quantity to provide a theoretical therapeutic dose of the drug when fed to penaeid larvae.
In subsequent tests, normal and diseased larval Penaeus stylirostris readily accepted Romet-30 enriched Artemia . Diseased larvae fed the drug in this manner exhibited a statistically greater overall survival and a greater survival to the post-larval stage by Student's t -test at the 5% significance level.     

Histamine and Histidine in New Zealand Marine Fish and Shellfish Species,Particularly Kahawai (Arripis trutta)

Forty-seven retail samples of fish (28 species) were tested for levels of free histidine, histamine and aerobic plate counts. Five samples had elevated levels (≥ 20 mg/100 g) of histamine and all had < 100 mg/100 g. Three species had free histidine levels of more than 1000 mg/l00 g and these have been implicated in scombroid poisoning in New Zealand. One species with high histidine levels, kahawai (Arripis trutta), was chosen to determine the conditions under which potentially hazardous levels of histamine might develop. Two trials were carried out in which kahawai were stored under 17 regimes at temperatures between 0 and 35°C. For 10 of these treatments the fish were transferred from elevated storage temperatures to refrigerated storage during the trials. Levels of histidine and histamine, aerobic plate counts at 20°C and 35°C and sensory quality were monitored. The levels of free histidine in kahawai varied with season, and in Trial 2 there was a net loss of histidine + histamine during storage. Bleeding kahawai did not significantly affect the levels of histidine. Histamine levels varied greatly in fish held under identical conditions. Fish held at ambient temperatures developed the highest levels of histamine. Elevated histamine levels (> 20 mg/100 g) were first recorded in fish stored for 0.9, 0.9, 1, 2, 2.7, and 8 days at 35, 30, 25, 20, 15 and 10°C respectively. Storage at 5°C after storage at higher temperatures did not result in elevated levels. Of the 59 samples with elevated histamine levels, 9 had acceptable sensory characteristics while all had aerobic plate counts exceeding 10⁶ colony-forming units/g. Aerobic plate counts at 20°C are recommended over those at 35°C. It is concluded that fresh kahawai will only present a hazard from scombroid poisoning under conditions of extreme temperature abuse and that the presence of high numbers of bacteria is a good indicator of the hazard while sensory quality is not.     

斑点鳟陆海接力养殖初步研究

为有效突破中国北方网箱养殖品种少的缺陷,并合理利用闲置网箱,通过陆海接力养殖模式,开展斑点鳟(♀Oncorhynchus mykiss×♂Oncorhynchus mykiss)的陆海接力养殖试验,并与工厂化养殖进行了对比,达到提高斑点鳟的养殖存活率,增加经济效益的目的。结果显示:从2013年6月1日至2014年6月19日,3种规格斑点鳟的初始体质量分别为(225.1±36.2)g、(102.8±23.5)g、(55.3±12.3)g,经384 d的工厂化养殖,养成平均质量分别达到(2143.4±253.1)g、(1 763.8±210.3)g、(946.3±120.4)g,存活率分别为91.2%、90.6%、89.3%;经384 d的陆海接力养殖,养成平均质量分别达到(2 408.3±321.2)g、(2 065.5±256.3)g、(1 142.6±156.3)g,存活率分别为87.5%、88.1%、85.3%,其中大规格苗种的平均日增体质量达到8.5 g/d。由此可见,最适宜进行陆海接力养殖的斑点鳟规格为体质量100 g以上的中等规格斑点鳟,且"陆海接力"养殖的斑点鳟具有生长快、经济效益高等特点,是一种值得推广的新型斑点鳟养殖模式。     

Bioencapsulation of erythromycin using adult brine shrimp, Artemia franciscana (Latreille)

Live adult brine shrimp, Artemia franciscana (Latreille), were enriched with erythromycin to determine if Artemia could accumulate therapeutic levels for subsequent feeding to young fish. Three trials were conducted to determine the erythromycin incorporation and survival rates of enriched Artemia when fed either liposomes containing erythromycin or various erythromycin suspensions. Erythromycin concentration in Artemia fed a liposome suspension was low (∼ 5 μg mL⁻¹) relative to Artemia fed the direct suspension (> 100 μg mL⁻¹) over the same time period. When enriched with suspensions up to 1 g erythromycin L⁻¹ sea water for 14 h, Artemia survival was not significantly affected ( P > 0.05) relative to controls. Using a suspension of 1 g L⁻¹, tissue erythromycin concentrations of 109 ± 16 μg erythromycin mL⁻¹  Artemia homogenate (mean ± SEM) were achieved after 12 h. Concentrations above 170 μg mL⁻¹ were obtained using suspensions of 2–5 g L⁻¹, but Artemia survival significantly ( P < 0.05) decreased.     

Therapeutic aminoglycoside antibiotic levels in brown shark, Car char hinus plumb eus (Nardo)

Abstract. The kinetics of gentamicin and tobramycin in juvenile brown sharks, Carcharhinus plumbeus (Nardo), were studied using a commercially available radioimmune assay (Clinical Assays, Cambridge, Massachusetts). Preliminary recovery studies in vitro demonstrated 95% recovery of antibiotic spiking with no detection of interfacing substances. There were no differences between serum and plasma data using this assay. Both drugs demonstrated distinct two-compartment characteristics. The mean half-life of the alpha component was 5.4±1.0 h for gentamicin and 3.2±0.5 h for tobramycin. The half-lives of the beta components were markedly longer, 54±4.9 h for gentamicin and 48±2 h for tobramycin. The experimental designs could not exclude third compartments. A therapeutic dosage schedule for gentamicin of 2 mg/kg intramuscularly followed by 1 mg/kg at 8 and 72 h was tested. This schedule avoided plasma levels greater than 12μg/ml and troughs greater than 2 μg/ml but more frequent administration may be required to treat infections with bacteria which are less sensitive to aminoglycoside antibiotics. A therapeutic tobramycin schedule of 2.5 mg/kg intramuscularly followed by 1.0 mg/kg, 4 h later and daily thereafter, achieved plasma levels within the human therapeutic range (5.8μg/ml) on the second day of therapy. Levels considered toxic to humans were not reached for 5 days.     

Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity

Vitellogenin (VTG) from spotted wolffish, Anarhichas minor, a candidate species for cold-water marine aquaculture, was purified by MgCl₂/EDTA precipitation followed by a two-step chromatographic procedure. VTG had an apparent molecular mass of 470 kDa, as determined by gel filtration, and an amino acid composition similar to those of other teleosts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified VTG revealed a major band with a relative molecular weight of 166 kDa and some minor bands. Spotted wolffish VTG (sw-VTG) is relatively robust to in vitro degradation, as shown when samples of purified VTG and plasma from mature females subjected to various storage conditions or multiple freeze/thaw cycles were analyzed by Western blot. We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) using an antibody against Atlantic wolffish (Anarhichas lupus) VTG and purified sw-VTG. The ELISA had a detection limit of 6.7 ng/ml and a working range of 16.2–787.5 ng/ml, with intra- and inter-assay coefficients of variation ranging from 1.5 to 7.3 % and 7.1 to 14.3 %, respectively. The assay could distinguish males from immature females and discriminate maturing females at different stage of oocyte development. These results suggest that the sw-VTG ELISA would be useful in spotted wolffish aquaculture to determine sex and monitor female maturation.     

Bioencapsulation of five forms of erythromycin by adult Artemia salina (L.)

Uptake of five chemical forms of erythromycin by adult Artemia salina (L.) (erythromycin phosphate – EP, erythromycin stearate – ES, erythromycin estolate – EE, erythromycin hydrate – EH and crystalline erythromycin – CE) was investigated in two trials. In each trial, final erythromycin concentration in Artemia tissue and survival after a 12‐h bioencapsulation period were determined. In the first trial, Artemia tissue concentration after a 12‐h bioencapsulation period was significantly (P < 0.05) affected by erythromycin form with ES (68.5 ± 3.3 μg mL^?1, mean ± SEM) ≈ EH (61.2 ± 3.4 μg mL^?1) > CE (37.1 ± 10.7 μg mL^?1) > EP (16.4 ± 7.7 μg mL^?1) > control. In trial 2, Artemia tissue concentration was also significantly (P < 0.05) affected by erythromycin form with EE (111.4 ± 9.6 μg mL^?1) > CE (89.1 ± 1.7 μg mL^?1) > ES (78.9 ± 1.6 μg mL^?1) > EP (33.4 ± 5.2 μg mL^?1) > control. Survival was significantly affected by erythromycin form in trial 1 with EP=control (100 ± 0.0%) > ES (74.4 ± 2.0%) > CE (32.2 ± 0.3%) > EH (8.8 ± 4.4%). In trial 2, survival was also significantly affected by erythromycin form with EP=control (100 ± 0.0%) > ES (67.1 ±3.7%) > CE (52.5 ± 7.7%) > EE (5.0 ± 2.5%). Based on both uptake and survival, EP and ES appear to be appropriate compounds for bioencapsulation of erythromycin using live adult Artemia.     

Ultrasound Enhanced Immersion Protocols for Masculinization of Nile Tilapia, Oreochromis niloticus

Androgen immersion protocols have been unsuccessful in consistently producing all-male tilapia at a high enough ratio for them to be commercially viable. This study explored the use of ultrasound to improve on the results of previous immersion studies. Variables tested include two hormones (trenbolone acetate-TBA and 17α-methyldihydrotestoterone-MDHT) at two concentrations (100 and 250 μg/L) and with or without ultrasound (cavitation level). All hormone treatments with ultrasound and non-ultrasound resulted in significantly higher masculinization than the appropriate controls ( P < 0.05). Among pairs of treatments of the same hormone at the same dose, all ultrasound treatments resulted in significantly higher number of males compared with non-ultrasound treatments with the exception of MDHT 250 μg/L ( P 0.05). Comparing across all ultrasound treatments, TBA 250 μg/L with ultrasound had higher masculinization than all the other ultrasound treatments ( P 0.05). Comparing across all non-ultrasound treatments, TBA 250 μg/L had higher ( P 0.05) masculinization than MDHT 100 μg/L and TBA 100 μg/L with non-ultrasound. Two of the three replicates of TBA 250 μg/L ultrasound treatment resulted in 100% males and the highest mean percentage (98%) of males. This study thus demonstrated the potential of a short-term immersion protocol using ultrasound to more predictably produce all-male, commercially viable tilapia seed.     

Fertilization and Development of Eggs of the Sea Urchin Lytechinus variegatus Maintained on an Extruded Feed

Sea urchin eggs are used extensively as models for studies in developmental and molecular biology. Developing aquaculture techniques and facilities for sea urchins would facilitate their use for this purpose and for production of young sea urchins for stock enhancement. A basic requirement for the aqua-culture of sea urchins is the availability of a feed that predictably produces eggs of a consistent, high quality. We tested an extruded feed with Lyrechinus variegatus for this purpose. Lytechinus variegatus (mean horizontal diameter 55 mm, 75 g wet weight) were collected in April 1998. Nine individuals were maintained in each of four aquaria with closed, recirculating filtered sea water. They were fed approximately 1 g extruded feedindividual per week. All feed was consumed. The mean weight of the sea urchins changed little with this feeding regime. The sea urchins spawned spontaneously on a monthly basis during water changes. Four females in February and one in March were spawned by injection with potassium chloride. Fertilization was 99–100% successful in each case. Egg diameters from these spawnings and a spontaneous spawning in April ranged from 102 × 6 to 128 × 1 μm. Gastrulation occurred in <20 h and metamorphosis after 22 to 37 d. Newly metamorphosed juveniles ranged in size from 435 × 38 to 473 × 56 μm. Egg size, larval size, the chronology of embryonic and larval development, and size of newly metamorphosed juveniles are similar to those obtained from field individuals reported in the literature. These results indicate that a predictable production of high quality eggs by sea urchins under controlled conditions in the laboratory with a prepared feed is feasible.     

Multiresidue determination of antibiotics in aquaculture fish samples by HPLC–MS/MS

An analytical method based on HPLC with MS/MS detection was developed and optimized in order to determine the most useful antibiotics (sulphonamides and tetracyclines) used in aquaculture. A simple extraction procedure, without any clean‐up step, was evaluated in order to obtain maximum analyte recovery from fish samples (Sparus aurata). A mixture of methanol:water 70:30 (v/v)+1 mL EDTA 0.1 M was selected as optimum extractant solution. Because no matrix effects were observed, a standard calibration curve prepared in mobile phase was used for quantification purposes. Antibiotic‐free fish samples were spiked at different concentration levels and analysed by the optimized HPLC method. The average recoveries (n=6) obtained were satisfactory, ranging from 88% to 110% at 100 μg kg^?1. The proposed methodology provided limits of detection for the tested antibiotics in the 1.2–16 μg kg^?1 range, lower than 100 μg kg^?1, the maximum residue level established by the European Union. Finally, commercial fish samples from different origins were analysed in order to confirm the usefulness of the developed methodology.     

Metamorphosis to Postlarvae of Penaeus chinensis Osbeck by Removal of Heavy Metals from Rearing Systems with Polymeric Absorbent

The effect of removing heavy metal ions (copper, zinc, lead, and cadmium) with a polymeric heavy metal absorbent (PHMA) on metamorphosis of Penueus chinensis Osbeck was investigated. There was a significant ( P < 0.05) reduction in metamorphosis of protozoea to mysis at concentrations more than 31.0 μg/L copper, 38.3 μg/L zinc, 32.5 μg/L lead, and 30.3 μg/L cadmium, in the absence of PHMA. At concentrations more than 61.0 μg /L copper, 68.3 μg/L zinc, 62.5 μg/L lead, and 60.3 μg/L cadmium there was a significant ( P < 0.01) interaction between the absence and the presence of PHMA for metamorphosis of mysis to postlarvae. At concentrations of 12.3 μg/L copper, 45.8 μg/L zinc, 106.2 μg/L lead, and 200.2 μg/L cadmium, the metamorphosis of protozoea to mysis was blocked, but it was 51.4% in the presence of PHMA. At concentrations of 252.3 μg/L copper, 335.8 μ/L zinc, 806.2 μg/L lead and 300.2 μg/L cadmium, the metamorphosis of mysis to postlarvae was stopped, but it was 51.0% in the presence of PHMA. The toxicity of copper, zinc, lead, and cadmium to P. chinensis was reduced with PHMA.     

Assessment of Aquatic Aeromonas spp. Isolates' Susceptibility to Cinnamaldehyde,Vanillin, and Crude Kigelia africana Fruit Extracts

Aeromonas spp. are primary and opportunistic fish pathogens that are responsible for severe economic losses in aquaculture, and are exacerbated by their increasing incidence of antimicrobial resistance. Phytochemicals are being explored as alternatives to conventional antimicrobial agents since they have destressing, growth‐promoting, immune‐stimulating, and antimicrobial properties. The susceptibility of 93 aquatic Aeromonas spp. and six Plesiomonas shigelloides isolates (from cultured catfish, koi carp, tilapia, and seawater) to three phytochemicals, viz.: cinnamaldehyde (10–1250 µg/mL), vanillin (5–250 µg/mL), and crude Kigelia africana fruit extracts (4–10 mg/mL ethyl acetate, dichloromethane, methanol, and hexane) were assessed using the disk diffusion assay and compared to ampicillin and tetracycline, using activity indices. No zones of inhibition were obtained with 10 µg/mL of cinnamaldehyde, or with 5–250 µg/mL of vanillin. However, varying degrees of inhibition were observed with higher concentrations of cinnamaldehyde, as well as with the K. africana methanol extract. Cinnamaldehyde (≥500 µg/mL) and the K. africana methanol extract displayed better antimicrobial activity against study isolates in comparison to vanillin and ampicillin. They appear to be promising and sustainable phytochemicals that might be useful as alternatives to the antimicrobial agents currently in use in aquaculture.     

Morphometric changes in a strain of the lineage 'Nevada', belonging to the Brachionus plicatilis (Rotifera) complex

The rotifer Brachionus plicatilis is an important component of aquaculture as a larval feed. Its taxonomic status has been recently re-defined as a species complex, consisting of at least 14 new species/lineages. This study deals with the lineage Brachionus 'Nevada', which has been shown to occur in European hatcheries. A strain of B . 'Nevada' was mass cultured using two commonly applied feeding regimes and analysed in terms of its morphometry. A new formula was proposed for the calculation of volume, which can be used as an index of adequacy of rotifers as feed for fish larvae. The results were related to life cycle parameters. The pre-reproductive and reproductive phases were divided into distinct size groups. Differences were also found between the two diets in morphometry and demography. Rotifers of a larger size (yeast-based diet) showed a lower growth rate and a longer reproductive period, lifespan and mean generation time compared with smaller-sized rotifers (Culture Selco^®-based diet). In terms of lorica length, the present study's strain of B . 'Nevada' (238.5 μm) was intermediate between values reported for Brachionus ibericus (193.5 μm) and B. plicatilis sensu stricto (299 μm).     

Possibility for decreasing of mercury content in bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> by fish culture

ABSTRACT:   The bluefin tuna tested were reared for 22 months from eggs before the beginning of the experiment, and sampling was performed every 3 months over the following year. The experimental results showed that the mercury concentration in the muscle ranged from 0.32 to 0.85 μg/g, which is lower than that found in wild bluefin tuna of a similar size. Increase in the mercury concentration corresponding to the increase in body weight was not shown, and it was quite different with wild bluefin tuna. Furthermore, no significant relationship was found between the lipid concentration and the mercury concentration in muscle. Among the internal organs of cultured bluefin tuna, the heart (0.32–0.66 μg/g), liver (0.43–0.99 μg/g) and spleen (0.59–1.0 μg/g) contained higher concentrations of mercury. It was estimated that the full-cycle cultured bluefin tuna had been fed small fish containing lower concentrations of mercury, and that the mercury concentration of tuna would be almost equal throughout the year because the effect of mercury accumulation would be weakened by body growth. Therefore, it was concluded that selecting diet fish species might decrease mercury contents in cultured bluefin tuna.     

The Effects of Exogenous Bovine Growth Hormone and Placental Lactogen on Juvenile Striped Bass Morone saxatilis Feed and Growth Efficiency

Striped bass Morone saxatilis under normal pond-reared conditions usually requires 14–18 mo to grow out to market weight. This includes a winter of comparatively slower growth which could possibly be overcome through hormonally induced acceleration of growth. Thus, the objective of this experiment was to test the hypothesis that bovine growth hormone and bovine placental lactogen increase growth in striped bass in a dose dependent manner. A secondary objective was to determine the effect of these hormones on body composition. Variables tested were weight gain, length gain, condition factor, feed conversion, and body composition. Fish injected biweekly with 10 μg/g body weight, bovine placental lactogen ( P < 0.09) or bovine growth hormone ( P < 0.11) increased body weight. Fish injected with 1 or 10 μg/g body weight bovine placental lactogen ( P < 0.10) or bovine growth hormone ( P < 0.10) increased body length during the 12-wk study period. Proximate analysis indicated that bovine growth hormone increased protein content (10 μg/g; P < 0.10) and decreased fat content (1 and 10 μg/g; P < 0.10). No significant differences occurred in feed conversions. We conclude that bovine growth hormone and bovine placental lactogen are effective in increasing body growth in striped bass.     

Induced spawning of maturing Indian catfish, Clarias batrachus (L.), using low doses of steroid hormones and salmon gonadotropin

Abstract. Effects of low doses of salmon gonadotropin (SG-G100) and three steroids — 17α,20β-dihydroxyprogesterone (17α,20β-diOHprog), deoxycorticosterone (DOC) and progesterone — individually, or combinations of steroid with SG-G100, on ovulation and hatching in Clarias batrachus (L.) were investigated. None of the steroids at any of the three different dose levels (1μg, 1·5μg/g and 2μg/g BW) could induce ovulation when injected alone. SG-G100 at a dose level of 10 μg/g BW was not effective but at the dose level of 15 μg/g BW it could induce ovulation. All the three steroids at their lowest doses (1μg/g BW) when injected in combination with SG-G100 (10μg/g BW) were significantly effective in inducing ovulation. When hatching percentage was taken into account, 17α,20β-diOHprog in combination with SG-G100 was found to be the most effective combination in comparison with other treatments.     

Spawning season,spawning grounds,and egg production of red sea bream in Hiuchi-nada,Seto Inland Sea

The spawning season and grounds of red sea bream in Hiuchi-nada, the central part of the Seto Inland Sea, were described using a new method based on monoclonal antibodies for identifying Pagrus major eggs, and the daily egg production (standardized by the incubation time and survival rate) was estimated. At the peak of spawning (May), the ranges of sea temperature, salinity, and chlorophyll a where red sea bream eggs occurred were 14.8–17.4°C, 32.0–33.0, 0.5–4.4 μg/L, respectively. The main spawning grounds of the red sea bream were confirmed as being the areas near the Geiyo Islands, Misaki Peninsula, Saijyo, Niihama. The spatial spread of red sea bream eggs increased with the egg developmental stage. The ranges of daily egg production in 2005 and 2006 were 0.3–19.3 and 0.2–6.7 × 10⁹ eggs/day, respectively. In Hiuchi-nada, aquaculture farms are located close to the spawning grounds, and the potential spawning population from the aquaculture farms equaled or exceeded that of the estimated spawning population obtained by the egg production method. Red sea bream eggs in Hiuchi-nada might be produced by both wild and aquaculture-based spawning populations.     

Effect of different C/N ratios and hydraulic retention times on denitrification in saline,recirculating aquaculture system effluents

Data on operation and performance of cost-effective solutions for end-of-pipe removal of nitrate from land-based saltwater recirculating aquaculture systems (RAS) are scarce but increasingly requested by the aquaculture industry. This study investigated the performance of a (semi)commercial-scale fixed-bed denitrification unit using single sludge for treating effluent from a commercial, saltwater RAS used for production of Atlantic salmon (Salmo salar). A fixed-bed denitrification reactor was fed continuously with 3-days hydrolyzed sludge from the commercial RAS, and was operated at different hydraulic retention times (HRTs; 1.82, 3.64, 5.46, or 7.28 h) or influent C/N ratios (3, 5, 7, or 10). Twenty-four h pooled samples were collected from the inflowing RAS water and the hydrolyzed sludge as well as from the denitrification reactor outlet, and samples were analyzed for nutrients and organic matter content.Nitrate removal rates increased consistently with decreasing HRT (from 64.3 ± 5.2–162.7 ± 22.0 g NO₃-N/m³/d within the HRTs tested) at non-limiting C/N ratios, while nitrate removal efficiencies decreased (from 99.6 ± 0.3–58.2 ± 8.9 %). With increasing influent C/N ratios at constant HRT (3.64 h), nitrate removal rates increased until the removal efficiency was close to 100 % and nitrate concentration in the denitrification reactor became rate-limiting. A maximum nitrate removal rate of 162.7 ± 2.0 g NO₃-N/m³/d was achieved at a HRT of 1.82 h and an influent C/N of 6.6 ± 0.5, while the most efficient use of hydrolyzed sludge (0.19 ± 0.02 g NO₃-N removed/g sCOD supplied) was obtained with a HRT of 3.64 h and a C/N ratio of 2.9. Removal rates of organic matter significantly and consistently increased with decreasing HRT and increasing C/N ratio. In addition, reducing HRT and increasing C/N ratios significantly improved removal of total phosphorus (TP) and PO₄-P.In conclusion, optimal management of the operating parameters (HRT and C/N ratio) in a single-sludge denitrification process can significantly reduce the discharge of nitrogen, organic matter, and phosphorous from land-based saltwater RAS and thus contribute to increased sustainability.