Mass mortality in RAS – Solved?

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My intention is to try to explain why the industry has experienced a series of mass mortalities in recirculated aquaculture systems, or RAS, in recent years.

The mortalities are likely linked to the release of hydrogen sulphide, or H2S. What follows explains a simple, inexpensive and practical way to mitigate the release of hydrogen sulphide and mass mortality.

The Pisco Group has, with input from AquaCircle, the Danish Industry Cluster for Aquaculture development, elaborated this document. Further research and investigations in collaboration between the industry, universities and knowledge institutes needs to be carried out to verify the details of the theory and refine the recommendations. We feel that we need to bring this information to the table now, if there is a chance that it can save fish from H2S-induced mortality in RAS systems.

Keep nitrate up
During the last decade, it has become common to use seawater in all steps of salmon production. Salt is thus used in smolt production systems, where the aim is to control fungus, to posts-molt systems, where higher salinity is a prerequisite. Seawater contains more sulphate, or SO42-, than freshwater. Elevated sulphate concentration increase the likelihood of increased H2S generation in the systems.

Until recently, nitrate, or NO3-, has only been considered a byproduct that needed to be diluted out of the production water or removed in denitrification systems in order to not reach toxic levels. We have not paid attention to nitrate levels.

We believe that nitrate needs to be considered in connection with the mysterious mass mortalities that have been seen in RAS systems in recent years. In studying data from systems experiencing sudden mass mortality, we found that many of these have seen a sudden drop in nitrate 12 to 72 hours before fish-health incidents.

It is our belief that we have all been inattentive to the importance of nitrate in relation to hydrogen sulphide generation and control. The stoichemetric formula below describes a process that is central to recirculation systems.

5H2S + 8NO3- -> 5SO42- + 4N2 + 4H2O + 2H+

We cannot quantify the extent, but this reaction takes place in all RAS systems. Our hypothesis is that nitrate has been the “guardian angel” of RAS systems by keeping the hydrogen sulfide generation in check and avoiding toxic levels of the substance which can result in mass mortalities.

From the equation above it can be seen, that if nitrate is present it will scavenge the toxic hydrogen sulphide and prevent its release into the water, which could eventually lead to fish death. It is therefore beneficial to keep a certain amount of nitrate in the water at all times.

This practical recommendation is further elaborated later on in this text. The following considerations about biofilm and reactions are very simplified and serve to illustrate the principle.

Toxic H2S
Biofilm exists on almost all surfaces in a recirculated system (not just in the biofilter). Biofilm grows and becomes thicker with age. The biofilm can simply be viewed as two layers: an aerobic (oxygen present) top layer of approximately 200 μm and an anerobic (oxygen depleted) bottom layer.

The top layer plays an active role in RAS, as it converts the toxic ammonia, or NH3, and ammonium, or NH4+, to harmless nitrate through a bacterial process called nitrification. This process consumes oxygen.

The bottom layer contains bacteria that consume organic material and convert sulphate to toxic hydrogen sulphide. This layer contains no oxygen, and can in theory be very thick and contain large ammounts of hydrogen sulphide.

Again it must be emphazised, that many other processes take place simultaniously in a biofilm. The illustration below serves to further illustrate this possible pathway.

H2S releases
The following example explains a common production scenario where smolts or post-smolts fast for a number of days, before being collected by a wellboat and transported to sea cages.

Step 1
The system is in steady state. Ammonia and ammonium is converted to nitrate. The concentration of nitrate in the water is high. Hydrogen sulphide “eats” nitrate and coverts into harmless sulphate.

Step 2
Feeding has stopped. The excretion of ammonia from the fish is reduced, and the bacteria convert less ammonia into nitrate. The concentration of nitrate in the water decreases because of water exchange and denitrification. As the nitrification rate drops, nitrate is depleted in the system. The hydrogen sulphide is still consuming the nitrate in the bottom part of the biofilm.

Step 3
The toplayer of the biofilm is getting thinner, as the nitrifying bacteria are starved. The hydrogen sulphide is still “eating” the nitrate in the bottom part of the biofilm.

Step 4
The top layer is now completely depleted of nitrate and hydrogen sulphide is released into the water, with the risk of reaching toxic levels to the fish.

Reacting in time
It is essential to always have enough nitrate available in the water, so you should do the following:

Always have calcium nitrate Ca(NO3)2 or another nitrate source on stock to dose into the water if the nitrate level drops to a critical level.

  • Make sure to measure nitrate concentration at least once a day.
  • Keep the nitrate level above 40 mg per liter nitrate in order to be on the safe side.
  • Reduce water exchange rate with new make up water, if you experience a sudden drop in Nitrate concentration.
  • There are critical situations, where you need to pay special attention because of the risk of a sudden drop in the nitrate concentration. In these situations you should measure at least twice a day. These situations are when feeding has been stopped or reduced dramatically (remember to turn off the denitrification filter, in used, if you experience a sudden drop in nitrate concentration); when large amounts of water have been changed in the system; when restocking with fish, without having cleaned the system (remember to dose ammonia into the biofilter during the fallow periods in order to keep the nitrifying bacteria alive)

Dosing calcium nitrate
If the nitrate level drops below 40 mg/l, calcium nitrate should be dosed into the water. Calcium nitrate is a relatively inexpensive compound (about NOK10 to NOK 20 a kilogram). The compound can be purchased in granular or liquid form.

For each 1,000 cubic meters of water in the system, dose 5.85 kg of calcium nitrate to increase the concentration of nitrate by 1 mg/l. The following equation can be used to determine the amount of calcium nitrate to dose into your system:

kg Ca(NO3)2 needed=Volume (m3)∙mgl NO3− nitrate increase 0.00585

An example
In a system with a total volume of 280 m3, a drop down to 30 mg/L nitrate is observed. It is therefore recommended to increase the concentration by 20 mg/l to a final level of 50 mg/l nitrate. The necessary dosing of calcium nitrate in granular form is therefore:

kg Ca(NO3)2 needed=280 m3 20mgl 0.00585=32.8 kg