Features Water Quality Week
Ask The Experts: Risks and benefits of using ozone

Ozone (O₃) is regarded as a powerful oxidant due to its highly unstable nature. This oxidising potential provides potent O₃-related germicidal functions, making it an effective well-known strategy for water disinfection. When O₃ dissolves in the water, it produces a broad spectrum of biocides destroying bacteria, viruses and cysts; hence, water treatment facilities often use  O₃ for disinfection.

In aquaculture, its benfits to improve water quality have been well-documented. In recirculating aquaculture systems (RAS), using O₃ results in the reduction of bacterial load, nitrite, ammonia, suspended solids, and yellow substances. 

However, the use of O₃ is mainly limited to freshwater RAS, as disinfection by-products (total residual oxidants) can form, and are higly toxic to fish in high salinity conditions. In particular, the formation of toxic bromines, following contact of O₃ with bromide in seawater, is of great concern. Therefore, a cautious attempt at using O₃ in saline RAS must begin with establishing thresholds that provide a favourable environment but do not negatively affect the health, welfare and production of farmed fish. 

CtrlAQUA develops technological and biological innovations supporting the adoption of closed containment systems for Atlantic salmon aquaculture. We are exploring the use of O₃ in salmon production in brackish water closed systems. 

Earlier, it was demonstrated that salinity of 12 ppt improved growth, survival and welfare of salmon in RAS and was identified to be cost-efficient for prolonged land-based rearing of post-smolts before transfer to open sea cages. 

The first trial aimed to identify the O₃ threshold for salmon post-smolts. To isolate the effects of ozone alone, the experiment was performed in a brackish water flow-through system. O₃ was injected into a header tank, and the desired O₃ concentration in each tank was achieved by mixing ozonated and non-ozonated water. 

The O₃ concentration in each tank was expressed in millivolts and monitored regularly. We tested five different levels of ozone: (1) control = 230 mV, (2) low = 280 mV, (3) medium = 350 mV, (4) high = 425 mV, and (5) very high = 500 mV. 

This short-term exposure study lasted for 10 days. Significant mortality was found, around 36 per cent in the very high group on day six, which was above the humane endpoint set for the trial. This prompted termination of the treatment group. Mortality was also recorded in the high group, registering a cumulative mortality of around 33 per cent on day 10. The medium group’s cumulative mortality was no more than one per cent. 

One of the drastic impacts of O₃ application was how O₃, particularly at 425 mV and higher, compromised gill health. Histopathology revealed extensive damage to branchial features, including widespread hyperplasia, lamellar clubbing and fusion, necrosis. Additionally found, was fish with compromised gill health had reduced chloride ion levels in the blood, which was a finding indicative of respiratory acidosis and a higher haematocrit level, showing the fish experienced compromised oxygen transport. 

During the trial, we also documented that fish from the two high groups were schooled near the water inlet prior to observed mortality which somehow supported the hypothesis that O₃ resulted in respiratory compromise. Interestingly, the skin was not significantly affected by the treatments. Therefore, based on the results from this foundational study, we concluded that O₃ levels of 300 to 350 mV was the safest range for post-smolt in brackish water.

This foundational study was used to further investigate the biological consequences of continuous O₃ application on post-smolts in a brackish water RAS. In a 45-day trial, we had two treatment groups: (1) a control group that did not receive ozonation and (2) a group that was continuously dosed with O₃. The latter received an average O₃ dose of 330 mV during the trial. 

No significant differences in survival, external operational welfare indicators and average weight at termination between the O₃-treated and control groups were found. Plasma was analysed for a panel of stress status and internal organ health indicators. Cortisol, lactate, glucose, alkaline phosphatase, and lactate dehydrogenase levels, as well as total antioxidant capacity remained unaltered by the treatments. Nonetheless, the increased level of plasma creatinine in the O₃-treated group at termination, suggest interference with renal function, warranting further investigation. This trial supported results from the foundational study, demonstrating the skin was less responsive to O₃ than the gills. Histological and gene expression analyses showed little skin changes after O₃ treatment. 

Contrastingly, changes in the gills showed it was a suitable organ for assessing the impact of O₃ on salmon. Quantitative histopathology disclosed that the O₃ group exhibited better gill health status than the control group, particularly at the end of the trial. The number of healthy gill filaments was higher in the O₃ group, while the prevalence of non-specific pathologies, such as lifting, clubbing, hypertrophy, and hyperplasia were higher in the control group. 

The gene expression profile revealed the potential of O₃ to induce local oxidative stress responses in the gills, as several genes affected by O₃ treatment had a crucial role in maintaining redox balance and antioxidant defence. 

Exploring the impact of O₃ treatment on salmon response to secondary stressors provided insights into the consequences of using O₃. O₃ treatment didn’t alter the capability of post-smolts to respond to a secondary stressor as both the control and O₃-treated groups mounted the classical adaptive respons handling confinement stress. 

When optimising the RAS environment,  physiological consequences of these rearing manipulations in fish should be considered. While we develop technologiesto make salmon production viable and economically feasible in closed systems, emphasis must be put on the effects on their health, welfare, and performance. 

Dr. Carlo C. Lazado is a senior research scientist in fish health, and Dr. Kevin T. Stiller is a research scientist in production biology with Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research based in Norway.