A better understanding of recirculating aquaculture system (RAS) biosecurity is crucial for the sustainable and ethical production of Atlantic salmon in these systems. We present a study that describes and evaluates the performance of a RAS facility for fish infection research with Atlantic salmon as the main animal model. It is an important reference for the design of future experiments in RAS facilities, and also for developing new knowledge to improve the RAS biosecurity in the Atlantic salmon aquaculture industry.
Atlantic salmon (Salmo salar) is one of the world’s major farmed finfish species with an annual production of around 2.4 million tons (FAO, 2020). Its aquaculture production cycle comprises two distinct phases: a land-based smolt production followed by a grow-out phase in sea cages until market size.
Recently, the land-based production has been shifting from using traditional flow-through systems toward recirculating aquaculture systems (RASs). The increased adoption of RASs to produce smolts and post-smolts is partially due to the benefits of a controlled production environment, including reduction of negative environmental impacts, a flexible location, and high biosecurity.
“In theory, biosecurity is tighter in RASs than in other production systems such as flow-through systems, although pathogen breaches are still occurring and causing mass mortality events and high economic losses.”
Adversity of pathogens ranging from bacteria, viruses, fungi, and parasites may infect fish cultured in RASs. In salmonid RAS-based farming, infectious pancreatic necrosis virus (IPNV), the bacterial gill disease agents Flavobacterium spp. and Ca. Branchiomonas cysticola, or enteric red mouth disease agent Yersinia ruckeri are among the pathogens that pose a serious problem.
The development of disinfection strategies to control pathogen outbreaks in the culture water and water treatment units in RASs without negatively affecting fish health and welfare or the nitrifying bacteria community in the biofilters is challenging.
We present a study that describe and evaluate a novel RAS facility for fish pathogens research with a focus on Atlantic salmon as an animal model.
Materials and Methods
The experimental trials were carried out at the fish health laboratory of the Tromsø Aquaculture research station (Havbruksstasjonen i Tromsø AS, Kårvik, Norway) (Figure 1).
The nine individual RAS units are located in one of the infection rooms of the research facility (Figure 2).
Each individual RAS contains a cylinder conical experimental tank (V = 0.5 m3 ) with a dual outlet drain, an emergency oxygen stone, and a sensor for oxygen and temperature (Oxyguard R, Farum, Denmark). The water flow scheme across the different RAS units is shown in Figure 3.
Briefly, the water flows out of the fish tank via a bottom center outlet and a sidewall outlet into a microscreen drum filter with a 40-µm screen to remove suspended solids. The backwash discharged from the microscreen is piped to a septic tank and to an underground pipe circuit (500 m long) where water is heated and disinfected (89ºC for 15 min.). The microscreen drum filtrate flows into the moving bed bioreactor (MBBR, V = 0.2 m3, 50% filled with bio-media).
During the experiments, the RAS units were operating without the ozone generator and UV-C units as
these units were added to the RAS posteriorly to the experiment’s conclusion. The bio-media was pre-acclimatized for 3–6 weeks using sodium bicarbonate (NaHCO3 ) and ammonium chloride (NH4 Cl) solutions (Permakem AS, Lørenskog, Norway).
Atlantic salmon eyed eggs (AquaGen Atlantic QTL-innOva PRIME, AquaGen AS, Trondheim, Norway) were hatched and fish were raised in a flow-through system at ± 7.5ºC under continuous light photoperiod until ∼10–26 g of body weight.
Fish were fed continuously (∼23 h/day) to satiation with commercial diets (1 and 2 mm pellet size, Nutra Olympic, Skretting, Norway) delivered through an automatic belt feeder. In experiment 5, fish were fed an experimental diet formulated for Atlantic salmon parr-smolt (2- and 3-mm pellet size, Nofima AS, Bergen, Norway).
Biological data, water quality, and system management parameters from five independent experimental trials conducted between September 2020 and July 2021 were used to evaluate the RAS units:
Experiment 1—A Pathogen Challenge Model: Make-Up. Water Vector.
A total of 495 juvenile Atlantic salmon (± 12 g) were randomly distributed among the nine RAS units. The fish were subjected to one of three treatments in triplicate.
Treatments consisted of a control group, where fish were not exposed to Y. ruckeri; a single entry group, where fish were exposed one time to a 24-h culture of Y. ruckeri administered via the make-up water and, a multi-entry group, where fish were exposed to a 24-h culture of Y. ruckeri administered via the makeup water on 3 consecutive days.
The experimental period lasted 15 days; the final fish weight was16 g, the biomass was 453 g, and the overall specific growth rate was 1.92%/day.
Experiment 2—A Pathogen Challenge Model: Infected. Fish Vector.
A total of 450 juvenile Atlantic salmon (± 12 g), either previously infected with Y. ruckeri or uninfected, were distributed among the nine RAS units using the following infection matrix:
Control = 0 infected fish and 50 uninfected fish per RAS unit; low pathogen load = 5 infected fish and 45 uninfected fish per RAS unit and a high pathogen load = 20 infected fish and 30 uninfected fish per RAS unit.
All three treatments were run in triplicates. The experimental period lasted for 14 days; the final fish
weight was16 g, the biomass was 493 g, and the overall specific growth rate was 2.05%/day.
Experiment 3—Chemical Disinfection: Peracetic. Acid Concentrations.
A total of 360 juvenile Atlantic salmon (± 15 g) were randomly distributed among the nine RAS units and acclimatized for 1 week. The fish were subjected to three different treatments, with three replicated RAS per treatment.
Treatments were a control (0.0 mg/L), low PAA concentration (0.1 mg/L), and a high PAA concentration (1 mg/L). The experimental trial lasted for 29 days; the final fish weight was 35 g, the biomass was 877 g, and the overall specific growth was of 2.92%/day.
Experiment 4—Chemical Disinfection: Pulse vs. Continuous. PAA Administration.
A total of 360 juvenile Atlantic salmon (± 26 g) were randomly distributed among the nine RAS units and acclimatized for 1 week. The fish were subjected to three different treatments, with three replicates per treatment.
Treatments were control (no PAA), pulse PAA (1 mg/L every 72 h), and continuous PAA (1 mg/L). The experimental trial lasted for 29 days; the final fish weight was 54 g, the biomass was 1,181 g, and the overall specific growth rate was 2.52%/day.
Experiment 5—Smoltification: Effect of Dietary Fat Levels on. ParrSmolt Transformation.
A total of 990 juvenile Atlantic salmon (± 19 g) were randomly distributed among the nine RAS units and acclimatized for 1.5 weeks. Fish were subjected to three different dietary treatments in triplicates: control diet, low-fat diet (−5% fat), and high-fat diet (+5% fat).
The parr were smoltified using a square wave photoperiodic regime consisting of 6 weeks “winter signal” (LD 12:12), followed by 9 weeks of LD 24:0. The experimental trial lasted for 57 days; the final fish weight was 94 g, the biomass was 4,721 g, and the overall specific growth rate was 2.81%/d.
Fish Parameter Variability
Table 1 lists fish body weight and fork length figures derived from the five experimental trials at the terminal sampling. The inter-class correlation coefficient (ICC) was 0.1 on average, ranging from 0.0 to 0.4. Overall, the variation within tanks (CVe) was larger than the variation between tanks (CVß): 36 vs. 11% for weight and 6 vs. 2% for length, respectively, CVe and CVß.
The largest difference was observed for the weight, where the CVe values ranged from 22 to 97%, whereas the CVß values ranged from 5 to 20%. The average realized power of the five experiments was 59% for weight and 47% for length.
The realized power for weight presented the highest variation among experiments, ranging from 21 to 95%. The statistical power vs. number of fish sampled for three treatment effect sizes (small = 0.2, medium = 0.5, and large =0.8) at three ICC (0.0, 0.1, and 0.2) is shown in Figures 4A–C.
Here, it is observable that:
(1) statistical power decreases with ICC increase,
(2) statistical power increases with treatment effect increase, and
(3) statistical power increases with the increase of the number of fish
sampled per tank.
Water Quality and System Management
Average water quality parameters from the five experimental trials are summarized in Table 2. The CVExp. values (among the five experimental trials) were on average 243.7% higher compared to CVTreat. values (within the five experimental trials).
The CVExp. values of the water quality parameters controlled by sensors such as dissolved oxygen, pH, and temperature were relatively low 1.7–3.6%, whereas parameters depending on biofilter maturation level and performance such as NH4-N/NH3-N and NO2-N presented a very high CVExp. 146.4–202.6% among the five experimental trials.
Disinfection Between Experiment Trial Evaluation
The real-time RT-PCR results from all 18 swabs were negative for the presence of Y. ruckeri.
In this study, data from five independent experimental trials were used to establish a variation baseline for fish performance, water quality, and system management metrics of a novel and unique research facility for pathogen research in RAS.
A statistical power analysis model was developed for different experimental scenarios and can be used as a tool to reduce and refine the number of animals for experimental use. Moreover, the design of the nine single RAS units was described in detail to facilitate the planning and design of future experiments to address biosecurity challenges in the Atlantic salmon RAS industry.
“The current RAS facility reuses water and this feature opens a new pathogen research area: how to eliminate pathogens in water. This study evaluated the efficacy of a chemical disinfection method that combined a low-high pH cycle to eliminate pathogens, in this specific case the bacterium Y. ruckeri.”
The evaluation and description of novel research facilities are important for effective resource utilization, high-quality scientific output, and to raise awareness about the research facility for national and transnational collaboration.
These authors described the water treatment components, water process flow, and water quality limits for the target species, information that can be used for other research and industry users to develop and refine their own production systems.
“Overall, the relatively low CVß reported in this study is a good indication that the order of magnitude of the tank/RAS variance is low.”
Consequently, the research facility shows great promise for the replicability of the experimental conditions, which is especially important for complex biological and technological environments such as RAS, with the added benefit that the RAS units are independent and do not violate statistical assumptions.
This study describes and evaluates a RAS facility specifically designed to conduct research on Atlantic salmon pathogen infection dynamics and respective disinfection strategies.
A number of four major objectives were taken into account when designing and building this facility:
(1) to establish challenge models with pathogens using in vivo fish,
(2) to offer flexibility on disinfection technologies including ozonation, UV irradiation, and water chemicals,
(3) to produce industry-relevant fish performance and water quality within optimal levels for Atlantic salmon,
(4) to have identical and independent replicated RAS units to support robust experimental designs and statistical data analysis.
The data from the five trials analyzed indicate that the facility successfully delivers the expected results, and it is expected to be useful for developing new knowledge to improve the RAS biosecurity in the Atlantic salmon aquaculture industry.
This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “EVALUATION OF A RECIRCULATING AQUACULTURE SYSTEM RESEARCH FACILITY DESIGNED TO ADDRESS CURRENT KNOWLEDGE NEEDS IN ATLANTIC SALMON PRODUCTION” developed by: VASCO C. MOTA – Nofima AS; ANJA STRIBERNY – Nofima AS; GERHARDUS C. VERSTEGE – Nofima AS, Havbruksstasjonen i Tromsø AS; GARETH F. DIFFORD – Nofima AS, Norwegian University of Life Sciences; CARLO C. LAZADO – Nofima AS.
The original article, including tables and figures, was published on APRIL, 2022, through FRONTIERS IN ANIMAL SCIENCE.
The full version can be accessed online through this link: doi: 10.3389/fanim.2022.876504