Nitrification

Nitrogen plays an important role in the structure and make-up of all living organisms. In the aquarium environment, nitrogen exists in the inorganic forms of nitrate, nitrite, ammonia, and nitrogen gas and in many forms of organic nitrogen. The nitrogen cycle in aquarium systems is depicted in the nitrogen cycle (Figure 1). Aquatic animals excrete nitrogen in the form of ammonia, amino acids, urea, and uric acid. Also, nitrogen is released through decomposition of dead animals and plants, uneaten food, bacteria and wastes. Ammonia, nitrite and nitrate are all highly soluble in water.

Figure 1. Nitrogen Cycle
The two processes in the nitrogen cycle that is of major importance in aquarium systems are nitrification (shown in green) and denitrification (shown in red). Ammonia is oxidised to nitrite and then to nitrate through a series of biochemical reactions called nitrification. Denitrification is primarily a reduction of nitrate to nitrogen gas by anaerobic bacteria.

When fish are maintained in an aquarium and fed protein-rich feeds, uneaten food particles and some by-products of digested food, namely amino acids and proteins, become major sources of organic compounds that accumulate on the gravel bed and in the filters. These wastes are ultimately metabolised by heterotrophic bacteria to ammonia. Heterotrophic bacteria use solubilised organic sources of carbon from proteins, fats, and carbohydrates to build their body components. Bioconversion of dissolved organic material by heterotrophic bacteria is a precursor to nitrification as high levels of soluble organic products can inhibit nitrification.

Scientific study has clearly proven that wastes excreted by fish are in the form of ammonia and urea. At a pH range of 6-8 approximately 90% of the total nitrogenous waste is excreted across the gills, with ammonia accounting for approximately 85% of this total. Excretion of urea usually makes up the remaining 10-20%. The amount of ammonia excreted by fish varies with the amount of food put into the aquarium, accelerating as stocking and feeding rates increase. In well-planted tanks most of the ammonia is taken up directly by the plants (including algae), as most plants prefer ammonia if given a choice. When nitrate is used, the nitrate must first be reduced to ammonia (the reverse of nitrification). In the average aquarium most of the ammonia will be converted by nitrifying bacteria to nitrite, and then nitrate. However, these micro-organisms have certain environmental preferences which must be routinely satisfied, which included elevated dissolved oxygen, neutral to slightly alkaline pH and moderate temperature.

Ammonia is toxic to fish and must be removed or converted into benign substances before it builds up to lethal levels. In water, ammonia exists as a mixture of two forms, un-ionised ammonia (NH3) and ionised ammonia (NH4) in equilibrium. This does not mean that they are present in equal proportions, but that they are converted from one to the other at an equal rate depending upon the pH and temperature of the water. The higher the temperature and pH, the higher the concentration of un-ionised ammonia. At a pH of 7.0 most of the ammonia is in the ionised form while at a pH of 8.0 the majority is in the un-ionised form. As the pH falls, the ammonia equilibrium shifts in the direction of ionised ammonia (ammonium) - the total ammonia does not change.

The interactions of pH, nitrification, and water quality can be quite complex. In general, nitrification is most efficient at pH levels ranging from about 7.5 to 8.2. pH levels above 9.0 or below 6 must be avoided since either extreme may harm the nitrifying bacteria. At the higher pH ranges (8.5 - 9.0), nitrification rates are fastest given sufficient ammonia. However, at the low ammonia concentrations usually found in aquarium systems, operating at a pH of about 7.0 can be more efficient. Because pH also effects the relative concentration of ionised and un-ionised ammonia in water and nitrifying bacteria use the ionised form, operating at a pH of about 7.0 usually increases the efficiency of the aquarium biological filtration. Another positive effect of operating at the lower pH is that the toxicity of ammonia to fish increases with increasing pH, so operating in the lower range also reduces ammonia toxicity. Therefore, the pH level should be routinely monitored.

Total ammonia (NH3 plus NH4) in mg/L or ppm is most commonly measured with a test kit. Most test kits measure the sum of both forms of ammonia. The only way to know how much ammonia is in the toxic form is by determining the pH and temperature and then calculating the percentage in toxic form. Unionised ammonia can be mathematically calculated from Table 1, based on water temperature, pH, and total ammonia levels. High temperatures and high pH levels can cause lethal concentrations of unionised ammonia, which is probably responsible for more unexplained fish losses in aquariums than anything else.

Unionised ammonia is a dissolved gas in water that can pass unimpeded through the membranes of the fish's gills. Continuous exposure to more than 0.02 ppm of the unionised form can cause reduced growth, increased susceptibility to disease and premature death. Ionised ammonia (ammonium) does not exist as a gas and cannot pass through the gill membranes and is, therefore, relatively non-toxic. It can, however, in high concentrations, produce external burns that are identical to acid burns. This is often seen when fish are crowded in shipping bags.

If a test kit gives a reading of 1 ppm for total ammonia at 25°C, in fresh water, this means that at pH = 7.0 the toxic form is 0.6% of the total or 1 ppm x 0.006 = 0.006 ppm NH3, but at pH = 8.5 the toxic form is 15% of the total or 1 ppm x 0.15 = 0.05 ppm NH3. Another factor to consider is the units that the test kit measures. Ammonia contains nitrogen (N) and hydrogen (H). Some kits may report total ammonia, as described above, but other kits refer to the quantity of nitrogen in the ammonia molecule. In this case the test kits units are presented as total ammonia nitrogen, which is the sum of NH3 N + NH4 N. Results reported in the two forms, are not the same. It is necessary to know the percentage of nitrogen in the ammonia molecule to compare the two units. The atomic weight of nitrogen is 14 and that of hydrogen is 1; thus the molecular weight of NH3 is 14+3 = 17. Nitrogen is 14/17 or 82% of the weight of the ammonia molecule. The difference is much more important for nitrite and nitrate (see below). Thus 1 ppm of NH3 is the same thing as 0.82 ppm of NH3 N. It is essential to know what units are being measured with each test kit.

It is easy to eliminate the possibility of disaster when ammonia and its relationship to temperature and pH are understood. Higher levels of ammonia usually occur at the start-up of the system, again when the system approaches its maximum capacity, or when something has caused a setback to the bacteria. If necessary, a water exchange will reduce ammonia levels. Remember to detoxify chloramines from tap water before introducing it into the system.

Ammonia is consumed by bacteria that initially convert it into nitrite and subsequently into nitrate. Nitrite is toxic to fish and concentrations as low as 0.5 ppm can reduce growth and adversely affect fish health with resulting mortalities. As for ammonia, test kits give results in terms of nitrite or nitrite-nitrogen. The difference between the two units is greater than for ammonia. The atomic weight of oxygen is 16; thus the molecular weight of nitrite is 14+32=46. Nitrogen is 14/46 or 30% of the weight of the nitrite molecule. Thus a test kit reading of 1 ppm of nitrite is the same thing as 0.3 ppm of nitrite-nitrogen. It is essential to know what units are being measured with each test kit.

A 0.5% solution of salt (about 98% NaCl), can be added to the aquarium water to help reduce the effect of nitrite toxicity. However, this salt solution will adversely affect any aquatic plants in the system. Generally, water exchange is the best method of nitrite reduction. Nitrite levels peak during the initial start-up, during periods of overfeeding, and again when the system nears its maximum carrying capacity.

Nitrate is the end product of nitrification and is normally not toxic to fish even in high concentrations. Levels of up to 200 ppm are tolerated by some fish species however, ideally it should be maintained below 20 ppm. Nitrate accumulation in aquaria can be controlled by regular water changes and can also be converted to nitrogen gas under special conditions.

In properly balanced aquariums, ammonia, and nitrite levels should always be zero when using common test kits available from pet stores. Presence of either is an indication that something in the system is out of balance and should alert you to start corrective action. If low levels of these elements persist they will cause chronic problems for your fish and increase their susceptibility to other disease conditions.

Since the major source of ammonia/nitrite is feeding fish, the first thing to do when any quantity is present in the water is to reduce or stop feeding for a 48-hour period. Fish are not likely to eat during periods of ammonia/nitrite stress and the uneaten food will only make the situation worse. Overfeeding is a major cause of ammonia and nitrite problems, and stopping the feeding will allow the natural nitrogen cycle to catch-up with the nutrient load.

Water changes of 10-25% per day for a week or so is also beneficial, as it will help to remove some of the ammonia and nitrite. Reduce the number of fish in the aquarium and check the filtration system for possible malfunction or improper size. By testing the water on a regular basis, most problems can be minimised, since corrective measures can be taken before fish start to die. Once fish have started to die, it is difficult to correct the problem without losing more fish.

Denitrification

In the absence of oxygen (< 1 mg/L) denitrification can occur in which certain anaerobic bacteria obtain their oxygen by removing it from the nitrate ion, finally leaving nitrogen gas or organic nitrogen compounds. Several intermediates are involved:

NO₃^- > NO₂^- > NO > N₂O > N₂ gas.

Nitrogen gas is then either volatilised to the atmosphere or converted by biological fixation to ammonia. Aquatic plants are able to do assimilative nitrate reduction; that is to say, they use nitrate as a nitrogen source by reducing it and incorporating the nitrogen atoms into organic molecules. Denitrification generally requires anoxic conditions and adequate soluble organic carbon. This combination of conditions is usually lacking in an aquarium, and this prevents the complete removal of nitrate.

Other Water Chemistry Factors

Many chemicals have been found to be inhibitory or toxic to nitrifying bacteria. A general rule is if a substance is toxic to fish, then it is probably toxic to the bacteria. Chemicals used to treat fish for a variety of diseases and parasites can be toxic to nitrifying bacteria at therapeutic levels for fish. Antibiotics are generally toxic. It is advisable to take biofilters off-line during short treatments and to do a 50% waterchange prior to re-establishing the filtration system.

Alkalinity (as CaCO3)
Nitrification produces acid and reduces alkalinity, so alkalinity levels in aquarium systems must be continually monitored and adjusted. Many basic solutions can be used to buffer, or add alkalinity, to aquarium systems, including sodium bicarbonate, calcium carbonate, etc. Care is needed in the selection of the buffer, as too much of a strong buffer can lead to wide swings in pH, which is stressful to the fish and nitrifying bacteria. Buffers that contain calcium (e.g. calcium carbonate) can lead to excessive calcium build-up in the aquarium. It's probably best to use one of the many commercially available products to increase the alkalinity. However, many aquarists prefer to use sodium bicarbonate (baking soda) as an alkalinity supplement because it produces small changes in system pH, is readily available, and inexpensive. Alkalinity levels may be infrequently checked as a complement to the aforementioned pH measurements. Bicarbonate alkalinity levels of ~ 50 to 200 mg/L as CaCO3 should be adequate.

Temperature
Temperature directly affects growth and nitrification rates of nitrifying bacteria. Optimal growth has been ascertained to occur at 22 - 30°C with lethal temperatures above 38°C. Basically, research on temperature and its effects on nitrification show that nitrification occurs and can be acclimated to conditions that are also favourable to aquatic species. Nitrification rates are slower at lower temperatures and increase linearly through the range of temperatures found in most aquarium systems.

Dissolved Oxygen
Dissolved oxygen is critical for nitrification to occur. As dissolved oxygen levels decrease to 1.0 mg/L in biological filters, dissolved oxygen rather than ammonia becomes the growth limiting factor. To prevent dissolved oxygen from becoming a limiting factor, water entering a biofilter should have minimum oxygen levels of 2.0 mg/L. Assumedly, the dissolved oxygen in the aquarium will be much higher, and with reduced loading levels on the attached nitrifying bacteria, oxygen depletion should not be much of a concern. Biofiltration using trickling or rotating biological filters benefit from natural oxygenation occurring as air flows past media covered with biofilms.

Light
Biological filters designs should prevent too much light from contacting the bacterial surfaces. Research has found that light intensities of less than 1% of sunlight intensities were inhibitory to nitrifying bacteria. Ammonia-oxidising bacteria were found to be more sensitive to light than nitrite-oxidising bacteria. Ideally, the nitrifying bacteria should neither be exposed to sunlight or room light of a colour other than red (e.g. darkroom lighting). When grown as an attached biofilm, though, considerable shading provided by bacterial layering will shelter the lower organisms from stressful light. Hence, this recommendation against light exposure is somewhat conservative.

© Copyright Adrian R. Tappin
Updated October, 1999.