The Effects of Ozone on Atlantic Salmon Post-Smolt in Brackish Water-Establishing Welfare Indicators and Thresholds.

Ozone is a strong oxidant, and its use in aquaculture has been shown to improve water quality and fish health. At present, it is predominantly used in freshwater systems due to the high risk of toxic residual oxidant exposure in brackish water and seawater. Here, we report the effects of ozone on Atlantic salmon (Salmo salar) post-smolts (~100 g), in a brackish water (12 ppt) flow-through system. Salmon were exposed to oxidation reduction potential concentrations of 250 mV (control), 280 mV (low), 350 mV (medium), 425 mV (high) and 500 mV (very high). The physiological impacts of ozone were characterized by blood biochemical profiling, histopathologic examination and gene expression analysis in skin and gills. Fish exposed to 425 mV and higher showed ≥33% cumulative mortality in less than 10 days. No significant mortalities were recorded in the remaining groups. The skin surface quality and the thickness of the dermal and epidermal layers were not significantly affected by the treatments. On the other hand, gill histopathology showed the adverse effects of increasing ozone doses and the changes were more pronounced in the group exposed to 350 mV and higher. Cases of gill damages such as necrosis, lamellar fusion and hypertrophy were prevalent in the high and very high groups. Expression profiling of key biomarkers for mucosal health supported the histology results, showing that gills were significantly more affected by higher ozone doses compared to the skin. Increasing ozone doses triggered anti-oxidative stress and inflammatory responses in the gills, where transcript levels of glutathione reductase, copper/zinc superoxide dismutase, interleukin 1ß and interleukin were significantly elevated. Heat shock protein 70 was significantly upregulated in the skin of fish exposed to 350 mV and higher. Bcl-2 associated x protein was the only gene marker that was significantly upregulated by increasing ozone doses in both mucosal tissues. In conclusion, the study revealed that short-term exposure to ozone at concentrations higher than 350 mV in salmon in brackish water resulted in significant health and welfare consequences, including mortality and gill damages. The results of the study will be valuable in developing water treatment protocols for salmon farming.

Influence of Fishmeal-Free Diets on Microbial Communities in Atlantic Salmon (Salmo salar) Recirculation Aquaculture Systems.

UNLABELLED: Reliance on fishmeal as a primary protein source is among the chief economic and environmental concerns in aquaculture today. Fishmeal-based feeds often require harvest from wild fish stocks, placing pressure on natural ecosystems and causing price instability. Alternative diet formulations without the use of fishmeal provide a potential solution to this challenge. Although the impact of alternative diets on fish performance, intestinal inflammation, palatability, and gut microbiota has been a topic of recent interest, less is known about how alternative feeds impact the aquaculture environment as a whole. The recent focus on recirculating aquaculture systems (RAS) and the closed-containment approach to raising food fish highlights the need to maintain stable environmental and microbiological conditions within a farm environment. Microbial stability in RAS biofilters is particularly important, given its role in nutrient processing and water quality in these closed systems. If and how the impacts of alternative feeds on microbial communities in fish translate into changes to the biofilters are not known. We tested the influence of a fishmeal-free diet on the microbial communities in RAS water, biofilters, and salmon microbiomes using high-throughput 16S rRNA gene V6 hypervariable region amplicon sequencing. We grew Atlantic salmon (Salmo salar) to market size in six replicate RAS tanks, three with traditional fishmeal diets and three with alternative-protein, fishmeal-free diets. We sampled intestines and gills from market-ready adult fish, water, and biofilter medium in each corresponding RAS unit. Our results provide data on how fish diet influences the RAS environment and corroborate previous findings that diet has a clear influence on the microbiome structure of the salmon intestine, particularly within the order Lactobacillales (lactic acid bacteria). We conclude that the strong stability of taxa likely involved in water quality processing regardless of diet (e.g., Nitrospira) may further alleviate concerns regarding the use of alternative feeds in RAS operations. IMPORTANCE: The growth of the aquaculture industry has outpaced terrestrial livestock production and wild-capture fisheries for over 2 decades, currently producing nearly 50% of all seafood consumed globally. As wild-capture fisheries continue to decline, aquaculture's role in food production will grow, and it will produce an estimated 62% of all seafood consumed in 2020. A significant environmental concern of the industry is the reliance on fishmeal as a primary feed ingredient, as its production still requires harvest from wild fisheries. Our study adds to the growing body of literature on the feasibility of alternative, fishmeal-free diets. Specifically, we asked how fishmeal-free diets influence microbial communities in recirculating salmon farms. Unlike previous studies, we extended our investigation beyond the microbiome of the fish itself and asked how alterative diets influence microbial communities in water and critical biofilter habitats. We found no evidence for adverse effects of alternative diets on any microbial habitat within the farm.

Optimizing Hydraulic Retention Times in Denitrifying Woodchip Bioreactors Treating Recirculating Aquaculture System Wastewater.

The performance of wood-based denitrifying bioreactors to treat high-nitrate wastewaters from aquaculture systems has not previously been demonstrated. Four pilot-scale woodchip bioreactors (approximately 1:10 scale) were constructed and operated for 268 d to determine the optimal range of design hydraulic retention times (HRTs) for nitrate removal. The bioreactors were operated under HRTs ranging from 6.6 to 55 h with influent nitrate concentrations generally between 20 and 80 mg NO-N L. These combinations resulted in N removal rates >39 g N m d, which is greater than previously reported. These high removal rates were due in large part to the relatively high chemical oxygen demand and warm temperature (∼19°C) of the wastewater. An optimized design HRT may not be the same based on metrics of N removal rate versus N removal efficiency; longer HRTs demonstrated higher removal efficiencies, and shorter HRTs had higher removal rates. When nitrate influent concentrations were approximately 75 mg NO-N L ( = 6 sample events), the shortest HRT (12 h) had the lowest removal efficiency (45%) but a significantly greater removal rate than the two longest HRTs (42 and 55 h), which were N limited. Sulfate reduction was also observed under highly reduced conditions and was exacerbated under prolonged N-limited environments. Balancing the removal rate and removal efficiency for this water chemistry with a design HRT of approximately 24 h would result in a 65% removal efficiency and removal rates of at least 18 g N m d.

Aquacultured rainbow trout (Oncorhynchus mykiss) possess a large core intestinal microbiota that is resistant to variation in diet and rearing density.

As global aquaculture fish production continues to expand, an improved understanding of how environmental factors interact in fish health and production is needed. Significant advances have been made toward economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and toward defining the effect of rearing density on fish health and production. Little research, however, has examined the effects of fishmeal- and grain-based diets in combination with alterations in rearing density. Moreover, it is unknown whether interactions between rearing density and diet impact the composition of the fish intestinal microbiota, which might in turn impact fish health and production. We fed aquacultured adult rainbow trout (Oncorhynchus mykiss) fishmeal- or grain-based diets, reared them under high- or low-density conditions for 10 months in a single aquaculture facility, and evaluated individual fish growth, production, fin indices, and intestinal microbiota composition using 16S rRNA gene sequencing. We found that the intestinal microbiotas were dominated by a shared core microbiota consisting of 52 bacterial lineages observed across all individuals, diets, and rearing densities. Variations in diet and rearing density resulted in only minor changes in intestinal microbiota composition despite significant effects of these variables on fish growth, performance, fillet quality, and welfare. Significant interactions between diet and rearing density were observed only in evaluations of fin indices and the relative abundance of the bacterial genus Staphylococcus. These results demonstrate that aquacultured rainbow trout can achieve remarkable consistency in intestinal microbiota composition and suggest the possibility of developing novel aquaculture strategies without overtly altering intestinal microbiota composition.

Denitrifying woodchip bioreactor and phosphorus filter pairing to minimize pollution swapping.

Pairing denitrifying woodchip bioreactors and phosphorus-sorbing filters provides a unique, engineered approach for dual nutrient removal from waters impaired with both nitrogen (N) and phosphorus (P). This column study aimed to test placement of two P-filter media (acid mine drainage treatment residuals and steel slag) relative to a denitrifying system to maximize N and P removal and minimize pollution swapping under varying flow conditions (i.e., woodchip column hydraulic retention times (HRTs) of 7.2, 18, and 51 h; P-filter HRTs of 7.6-59 min). Woodchip denitrification columns were placed either upstream or downstream of P-filters filled with either medium. The configuration with woodchip denitrifying systems placed upstream of the P-filters generally provided optimized dissolved P removal efficiencies and removal rates. The P-filters placed upstream of the woodchip columns exhibited better P removal than downstream-placed P-filters only under overly long (i.e., N-limited) retention times when highly reduced effluent exited the woodchip bioreactors. The paired configurations using mine drainage residuals provided significantly greater P removal than the steel slag P-filters (e.g., 25-133 versus 8.8-48 g P removed m-3 filter media d-1, respectively), but there were no significant differences in N removal between treatments (removal rates: 8.0-18 g N removed m-3 woodchips d-1; N removal efficiencies: 18-95% across all HRTs). The range of HRTs tested here resulted in various undesirable pollution swapping by-products from the denitrifying bioreactors: nitrite production when nitrate removal was not complete and sulfate reduction, chemical oxygen demand production and decreased pH during overly long retention times. The downstream P-filter placement provided a polishing step for removal of chemical oxygen demand and nitrite.

Denitrifying bioreactor clogging potential during wastewater treatment.

Chemoheterotrophic denitrification technologies using woodchips as a solid carbon source (i.e., woodchip bioreactors) have been widely trialed for treatment of diffuse-source agricultural nitrogen pollution. There is growing interest in the use of this simple, relatively low-cost biological wastewater treatment option in waters with relatively higher total suspended solids (TSS) and chemical oxygen demand (COD) such as aquaculture wastewater. This work: (1) evaluated hydraulic retention time (HRT) impacts on COD/TSS removal, and (2) assessed the potential for woodchip clogging under this wastewater chemistry. Four pilot-scale woodchip denitrification bioreactors operated for 267 d showed excellent TSS removal (>90%) which occurred primarily near the inlet, and that COD removal was maximized at lower HRTs (e.g., 56% removal efficiency and 25 g of COD removed per m3 of bioreactor per d at a 24 h HRT). However, influent wastewater took progressively longer to move into the woodchips likely due to a combination of (1) woodchip settling, (2) clogging due to removed wastewater solids and/or accumulated bacterial growth, and (3) the pulsed flow system pushing the chips away from the inlet. The bioreactor that received the highest loading rate experienced the most altered hydraulics. Statistically significant increases in woodchip P content over time in woodchip bags placed near the bioreactor outlets (0.03 vs 0.10%P2O5) and along the bioreactor floor (0.04 vs. 0.12%P2O5) confirmed wastewater solids were being removed and may pose a concern for subsequent nutrient mineralization and release. Nevertheless, the excellent nitrate-nitrogen and TSS removal along with notable COD removal indicated woodchip bioreactors are a viable water treatment technology for these types of wastewaters given they are used downstream of a filtration device.

Observations on side-swimming rainbow trout in water recirculation aquaculture systems.

During a controlled 6-month study using six replicated water recirculation aquaculture systems (WRASs), it was observed that Rainbow Trout Oncorhynchus mykiss in all WRASs exhibited a higher-than-normal prevalence of side swimming (i.e., controlled, forward swimming but with misaligned orientation such that the fish's sagittal axis is approximately parallel to the horizontal plane). To further our understanding of this abnormality, a substudy was conducted wherein side swimmers and normally swimming fish were selectively sampled from each WRAS and growth performance (length, weight), processing attributes (fillet yield, visceral index, ventrum [i.e., thickness of the ventral "belly flap"] index), blood gas and chemistry parameters, and swim bladder morphology and positioning were compared. Side swimmers were found to be significantly smaller in length and weight and had less fillet yield but higher ventrum indices. Whole-blood analyses demonstrated that, among other things, side swimmers had significantly lower whole-blood pH and higher Pco2. Side swimmers typically exhibited swim bladder malformations, although the positive predictive value of this subjective assessment was only 73%. Overall, this study found several anatomical and physiological differences between side-swimming and normally swimming Rainbow Trout. Given the reduced weight and fillet yield of market-age side swimmers, producers would benefit from additional research to reduce side-swimming prevalence in their fish stocks.

Assessing the suitability of a partial water reuse system for rearing juvenile Chinook salmon for stocking in Washington State.

To assess the suitability of water reuse technology for raising Pacific salmon Oncorhynchus spp. for stocking purposes, fish health and welfare were compared between two groups of juvenile Chinook salmon O. tshawytscha from the same spawn: one group was reared in a pilot partial water reuse system (circular tanks), and the other group was reared in a flow-through raceway. This observational study was carried out over a 21-week period in Washington State. Reuse and raceway fish were sampled repeatedly for pathogen screening and histopathology; fin erosion and whole-blood characteristics were also evaluated. By the study's end, no listed pathogens were isolated from either cohort, and survival was 99.3% and 99.0% in the reuse and raceway groups, respectively. Condition factor was 1.28 in raceway fish and 1.14 in reuse fish; this difference may have been attributable to occasional differences in feeding rates between the cohorts. Fin indices (i.e., length of the longest dorsal or caudal fin ray, standardized by fork length) were lower in reuse fish than in raceway fish, but fin erosion was not grossly apparent in either cohort. The most consistent histological lesion was gill epithelial hypertrophy in reuse fish; however, blood analyses did not suggest any corresponding physiological imbalances. Overall, results suggest that water reuse technology can be employed in rearing juvenile anadromous salmonids for stocking purposes.

Evaluation of a membrane biological reactor for reclaiming water, alkalinity, salts, phosphorus, and protein contained in a high-strength aquacultural wastewater.

The capacity of a membrane biological reactor to provide nitrification, denitrification, and enhanced biological phosphorus removal of a high-strength aquaculture backwash flow (control condition), or the same flow amended with 100mg/L of NO(3)-N and 3mg/L of dissolved P (test condition), was assessed using only endogenous carbon. Permeate TSS and cBOD(5) concentrations were <1mg/L under control and test conditions, achieving 99.97-100% removal efficiencies, respectively. Permeate TN concentrations were 1.8+/-0.5mg/L and 2.1+/-1.4 mg/L, while permeate TP concentrations were 0.05+/-0.01 mg/L and 0.10+/-0.03 mg/L, respectively, under control and test conditions. Our findings suggest that permeate flow could be reclaimed to recycle alkalinity, salts, and heat for fish culture and that the waste activated sludge does not produce metals concentrations that would prevent its land application (reclaiming phosphorus) or prevent its use as a protein source in animal feeds.

The cost and effectiveness of solids thickening technologies for treating backwash and recovering nutrients from intensive aquaculture systems.

The cost and effectiveness of three solids thickening processes, i.e., gravity thickening settlers (GTS), inclined belt filters (IBF), geotextile bag filters (GBF), were individually evaluated with the biosolids backwash produced in intensive aquaculture systems equipped with microscreen drum filters and radial-flow settlers. The IBF produced the cleanest discharge and highest treatment efficiencies, likely reflecting the rapid efficiency with which solids are separated from wastewater. The GBF was the least effective process, i.e., GBF leachate contained the highest concentrations of TP, TN, and cBOD. However, GBF was most effective for sludge volume reduction. Capital cost estimates for an IBF were more than twice that of GTS and GBF of similar treatment capacity. The GTS had the lowest capital and annual operating cost estimates. The estimated annual operating cost of the GBF was orders of magnitude higher than the IBF and GTS, due to the high cost to replace bags.