Ammonia exists in two species: un-ionized ammonia (NH3) and ionized ammonium (NH4+), the amount of each type depending on the pH and temperature of the water. At a lower pH, the excess hydroniums (H+) in the water tend to drive the balance toward ammonium. At a higher pH, the lack of hydroniums tends to produce un-ionized ammonia. Temperature has a lesser effect, with the amount of un-ionized ammonia increasing with increasing temperature at any given pH.
This is of more than academic interest to aquaculturists, since only the un-ionized form is toxic. So, the total ammonia (ionized and un-ionized combined) that is measured by water quality analysis must be evaluated in light of pH and temperature. The generally accepted rule of thumb is that un-ionized ammonia in excess of 0.02 mg/L is potentially toxic, though this may vary slightly with species of fish.
The fact that low pH reduces ammonia toxicity is fortuitous for fish culturists. Fish produce both carbon dioxide and ammonia as waste products. Carbon dioxide reacts with water to form carbonic acid. In the absence of buffering this lowers the pH of the water lowering the toxicity of the ammonia that is also being produced. While recirculation aquaculturists may count on low pH to reduce ammonia toxicity during hauling and other times of brief confinement, they cannot use this effect in their systems because of the negative effect of low pH on nitrification as explained below.
Nitrification is the biological process in which bacteria use reduced nitrogen compounds, such as ammonia, as food by oxidizing them. This process drives the earth's nitrogen cycle, so it is widespread in the biosphere. It also is used by sanitary engineers in municipal waste treatment plants to remove ammonia in sewage. In the part of the cycle of interest in recirculation aquaculture, ammonia (NH3 or NH4+) is oxidized by bacteria to partially reduced nitrite (NO2) which is toxic to fish, but less so than ammonia. Nitrosomonas spp. are the bacteria that biology textbooks always give credit to for this reaction, though in the aquatic environment other bacteria are likely more important. It is probably best just to refer to them as ammonia-nitrite conversion bacteria. Nitrite is then further oxidized to nitrate (NO3) by bacteria (again Nitroacter spp. is the common example though nitrite-nitrate conversion bacteria is better). Nitrate is relatively non-toxic to fish and may safely accumulate in the tank until it is flushed out by replacement water or converted to gaseous nitrogen (N2) by anaerobic heterotrophs and lost to the atmosphere in a process known as denitrification. Since the conversion of ammonia is a biological process, time is required for the bacterial population to develop sufficient biomass to remove the toxic nitrogen load. Also, the bacteria that oxidize ammonia must develop first and produce the nitrite before the bacteria that use nitrite for food can grow. Nitrification bacteria do not grow well below a pH of 7 and will cease to provide nitrification if system pH falls into the acidic range. This puts the recirculation aquaculturist in a balancing act. If pH rises to near 8 or above, even tiny amounts of ammonia that may be left by the bacteria will be toxic to the fish, but if pH falls below 7 the bacteria will quit and ammonia will soar to levels dangerous even at the relatively safe low pH. As explained previously carbonate buffering provided by sodium bicarbonate makes it relatively easy to maintain pH in the ideal range of between 7 and 8.
Measuring Nitrogen Compounds
Ammonia is usually measured by the nessler or the salicylate method, both of which are available in kit form. These tests are colorimetric. The nessler method has a greater range while the salicylate method is more sensitive. Since ammonia is a byproduct of the catabolism of protein, it is strongly influenced by feeding and subsequent metabolism of the feed by fish and bacteria. Ammonia typically peaks about 90 minutes after feeding.
When measuring ammonia, you must also measure pH because the toxicity of any given amount of ammonia is relative to the pH of the water. The pH determines how much of the ammonia is ionized and how much is unionized. The test kits yield total ammonia which is both ionized and unionized combined.
Test kits can give total ammonia readings two ways, simply total ammonia or total ammonia-nitrogen (TAN). When total ammonia is measured the weight expressed (usually mg/L) is the weight of the whole ammonia molecule, the nitrogen atom and the hydrogen atoms. When TAN is measured the weight is only the nitrogen atom. Thus, the same amount of ammonia would be heavier expressed as total ammonia than as TAN. Ionized ammonia is a little heavier than unionized because of the extra hydrogen atom, but the difference is slight, and most of the ammonia is ionized, so we can just assume the ionized weight to convert between total ammonia and TAN. To convert from total ammonia to TAN multiply the total ammonia by 0.775. To convert from TAN to total ammonia multiply TAN by 1.29.
Nitrite is a toxic compound produced from ammonia during the first step of nitrification. The toxicity of nitrite is dependent on chloride concentration and the acutely toxic concentration varies from less than 1 mg/L to 10 or more, depending on chloride. Under certain conditions it can be elevated even when ammonia is not, so it should be measured, as well. Nitrite is measured by a colorimetric assay that is altered by chloride.
When significant concentrations of nitrite exist in a tank the fish develop a blood disease termed methemoglobinemia. When nitrite ions come into contact with hemoglobin they oxidize the iron atoms in the hemoglobin molecule and the hemoglobin can no longer reversibly bind oxygen. With the hemoglobin poisoned, the blood cannot carry sufficient oxygen and the fish dies of tissue suffocation. This is very similar to the way carbon monoxide poisoning kills. Methemoglobinemia is called "brown blood disease" because the oxidized hemoglobin is brown, not red, giving the blood a rusty color. Ironically, the osmoregulatory mechanisim in freshwater fish causes the fish to poison themselves in the presence of nitrite. All freshwater fish are excellent ion scavengers, but if the concentration of chloride ions (Cl-) is low and the concentration of NO2- is high, the Cl--HCO3- pump in the chloride cell will exchange HCO3- for NO2- instead of Cl-, actually pumping the toxic NO2- into their body. Therefore, an antidote to NO2- poisoning is to put salt (or calcium chloride) in the water. This works by raising the Cl- content relative to the NO2- concentration, allowing the Cl--HCO3- pump to snag the right molecule. As an example, for catfish, a ratio of Cl- to NO2- of 16:1 or greater is protective. In practical terms, raising the Cl- concentration of water to 40 mg/L decreases the toxicity of nitrite by a factor of 30.
Nitrate is the end product of nitrification. It is not terribly toxic to fish and can safely build up in tanks to 50 mg/L or more. The nitrate assay is sometimes used to signal the need to conduct a partial water change. Most nitrate tests first reduce nitrate to nitrite and then test for nitrite, so if there is significant nitrite in the tank, that amount needs to be subtracted from the nitrate value to get an accurate result, however, if you have enough nitrite in the water to alter the nitrate test your fish are dying from nitrite toxicity!