Most aquarists have some familiarity with nitrite. It is part of the "nitrogen cycle" that takes place in most aquariums, and so is one of the first encounters that many aquarists have with their aquariums' chemistry. The marine aquarium hobby is replete with commentary about nitrite, some of which is, unfortunately, incorrect or misleading. Its toxicity in marine systems is far lower than in freshwater systems. Nevertheless, many aquarists incorrectly extrapolate this toxicity to reef aquariums and suggest that any measurable amount of nitrite is a concern.

In reality, nitrite probably is not toxic enough to warrant measuring in most marine systems. This article serves to provide a backdrop for that opinion by addressing what nitrite is, where it comes from, where it goes, the mechanisms by which it can be toxic and the evidence for its toxicity (or lack thereof) in typical reef aquariums.

Contents

• Nitrite in the Ocean
• Where Does Nitrite Come From?
• Where Does Nitrite Go?
• Effects of Nitric Oxide
• How is Nitrite Toxic?
• How Toxic is Nitrite to Fish?
• How Toxic is Nitrite to Invertebrates?
• Summary
• References

Nitrite in the Ocean

Nitrite (NO2^-) is a fairly small ion, consisting of a central nitrogen atom with two attached oxygen atoms in a bent configuration (Figure 1). One of the oxygen atoms carries a negative charge. More correctly, the nitrite ion has two oxygen atoms that are capable of carrying the negative charge, and in reality, the solvated ion in solution probably has two identical oxygen atoms, each with a partial negative charge. Nitrite is a fairly strong acid, and becomes protonated to nitrous acid (HNO2) only as the pH drops below 4 (pKa = 3.35).

In the ocean, nitrite typically varies in concentration from very low levels to about 0.2 ppm.^1,2 The higher end of this range is typically found only in anoxic layers deep below the surface. Nitrite in surface Atlantic and Caribbean seawater has been reported to range from 0.000005 to 0.00002 ppm,³ and a series of measurements in the South China Sea and the Philippine Sea showed an average of 0.00002 ppm.⁴

Nitrite is sometimes elevated in the water buried in sediments due to decomposing organic material, and the fact that such pore water is often anoxic. In natural coral reef sediments, however, nitrite can still be very low (much lower than ammonia (NH3) and nitrate (NO3^-), which can rise to as high as 0.7 ppm).⁵

Where Does Nitrite Come From?

Most aquarists associate nitrite with the traditional "nitrogen cycle." In this process, bacteria convert ammonia into nitrite and then into nitrate by oxidizing it. The bacteria gain chemical energy in this fashion, just as other organisms (from bacteria to people) gain energy by oxidizing carbon compounds (such as ethanol, CH3CH2OH) into more oxidized versions, such as carbon dioxide (CO2).

This process can be described as starting with ammonia (NH3) excreted by animals or by bacteria and other organisms that are consuming organic compounds containing nitrogen, such as proteins. The ammonia from the water column is taken up by bacteria and is oxidized in a step-wise fashion, first to nitrite:

NH₃ + ³/₂ O₂  à  NO₂^-  +  H⁺   +  H₂O

And then to nitrate (possibly in bacteria species other than those that produce the nitrite):

NO₂^-   +  ½ O₂  à  NO₃^-

During an aquarium's initial setup, few of these ammonia- and nitrite-oxidizing bacteria are present. As the ammonia accumulates, bacteria that utilize it increase in population. As that occurs, they consume the initial ammonia spike, and a nitrite spike results. Then, the nitrite-oxidizers take advantage of the nitrite spike, increase in population, and consume the nitrite, thereby producing nitrate.

After some period of time (often a few weeks), the bacterial action begins to equilibrate, and neither ammonia nor nitrite is present in high concentrations. This doesn't mean that a lot of each is no longer being produced, only that they are consumed as fast as they are produced, leaving a low steady-state concentration. In most reef aquaria, the steady-state concentrations of both ammonia and nitrite are quite low (less than 0.1 ppm), and often are below the detection limits of many test kits.

What happens when an aquarium is initially set up, however, is not necessarily what happens later. Many organisms in reef aquaria consume ammonia and nitrite directly, and metabolize it into organic matter. Macroalgae, for example, can take up ammonia directly, and many species actually take up ammonia preferentially to nitrate. Consequently, in a reef aquarium such as mine where most of the nitrogen export is via macroalgae, little nitrite may be produced in the first place. I have no way of knowing how much of the nitrogen added to my aquarium from foods enters the macroalgae as ammonia, and how much in other forms (such as nitrite or nitrate), but it is very likely that not all of the nitrogen added passes through a nitrite stage before becoming part of the macroalgae.

In addition to the standard nitrogen cycle, there are other ways that nitrite can be produced. One of these ways is by photolysis of nitrate. That is, nitrate can break apart when exposed to UV light, producing nitrite and hydroxyl radical (OH).^6,7

NO₃^-   +  H₂O +  UV  à  NO₂^-   +  2 OH

Another method of nitrite synthesis can occur inside organisms, although this nitrite may not be released back into the water. For example, nitrite can be produced from nitrate internally by corals (e.g., Pocillopora damicornis) and macroalgae (e.g., Ulva lactuca).⁸

Where Does Nitrite Go?

Nitrite can take a number of different pathways in the ocean. Many organisms can directly take up nitrite. Such uptake has been demonstrated in anemones (Condylactis sp., for example, take up nitrite, possibly for its symbionts),⁹ diatoms (Eucampia zodiacus)¹⁰ and zooxanthellae isolated from a variety of species (Zoanthus spp., Tridacna crocea, Seriatopora hystrix, Montastrea annularis, Porites furcata and Stylophora pistillata).¹¹

Nitrite can also be broken down by exposure to UV light, producing nitric oxide (NO), hydroxyl radical (OH) and hydroxide ion (OH^-).^6,7,12

NO₂^- + H₂O + UV à NO + OH + OH^-

In a laboratory situation with nitrate-free seawater with no organisms present, ambient sunlight can reduce the nitrite concentration by 2-15% per day.^12,13 The primary products of this reaction are nitric oxide (NO) and hydroxyl radical (OH). Both of these compounds are chemically and biologically active, so this reaction may be important to a number of biochemical pathways in the ocean and in various organisms. The effect of nitric oxide is discussed in more detail later in this article.

In the anammox process, bacteria use nitrite to oxidize ammonia, producing N2:

NH₃  +  NO₂^-  +  H⁺  à  N₂  + 2H₂O

The importance of this process in marine sediments has long been unknown. In recent studies, however, it has been shown to be important in some circumstances.^14-17 In two continental shelf sites, the conversion of ammonia to N2 by this pathway produced 24% and 67% of the total N2 produced. In a eutrophic bay, however, this process was negligible compared to ordinary denitrification (the conversion of nitrate into N2 when the nitrate is used as an electron acceptor for degradation of organic material in low oxygen situations). A different study showed that this process accounted for between 4% and 79% of the N2 produced in coastal sediments.

Finally, in a reef aquarium, nitrite can be removed by reaction with ozone, presumably to produce nitrate.¹⁸

NO₂^-  +  O₃   à  NO₃^-  +  O₂

Effects of Nitric Oxide

As described above, nitrite can break down under UV light to produce nitric oxide. Consistent with this process, nitric oxide is found to increase during the day and to decrease at night.¹² Nitric oxide itself has a variety of different biological effects. Exposure to different concentrations of supplemental nitric oxide was found to speed or inhibit the growth of four species of phytoplankton (Skeletonema costatum, Dicrateria zhanjiangensis nov. sp., Platymonas subcordiformis and Emiliania huxleyi) , consistent with its known role as a growth regulator in terrestrial plants.¹⁹

Nitric oxide also may play a role in the symbiosis of certain cnidarians with dinoflagellates. An enzyme that produces nitric oxide has been found in the cnidarian Aiptasia pallida. This enzyme is apparently downregulated when the organism goes into acute heat shock, and inhibitors of the enzyme cause retraction of the tentacles, as is observed under heat shock conditions.²⁰ Further, addition of nitric oxide donors to the system prevents this retraction of tentacles. Whether this process has anything to do with nitrite or nitric oxide in the water column is not clear.

The effects, if any, that nitric oxide and this reaction from nitrite in particular might have on reef aquaria is unclear. Nitric oxide effects on marine organisms is an active area of research, and a greater understanding of it is expected in the future. Whatever the effects are, however, any effect attributable to NO produced from nitrite may be most pronounced in a newly cycling reef aquarium (where nitrite is elevated) and when a UV system is in use.

How is Nitrite Toxic?

Nitrite can be toxic in a number of ways.²¹ Freshwater fish rapidly take up nitrite through their gills, leading to high levels in their bodies. In freshwater fish, nitrite taken up through the gills can compete with chloride for the same uptake proteins, so in some cases of elevated nitrite the fish can suffer from chloride depletion. It has been observed that some freshwater fish (e.g., bluegill; Centrarchidae: Lepomis macrochirus) do not take up chloride via their gills, and these species are notably resistant to nitrite toxicity.²²

The internalized nitrite then causes a number of internal disturbances, including loss of potassium from certain tissues (such as skeletal muscle) and the oxidation of hemoglobin into methemoglobin, which reduces the blood's oxygen carrying capacity. This can cause reduced tissue oxygenation, hyperventilation and heart rate increases. Many other biochemical pathways become altered as well, including steroid synthesis, vasodilation (blood vessel enlargement) and changes in internal levels of ammonia and urea. Nitrite detoxification in freshwater fish is accomplished by direct nitrite excretion and by internal conversion of nitrite into nitrate.²³

Marine species are less susceptible to nitrite toxicity because chloride (at 19,350 ppm in seawater) outcompetes nitrite for the same uptake mechanisms. Nevertheless, it is possible for some marine fish to take up nitrite via both their gills and their intestines after swallowing seawater. For example, when exposed to 46 ppm nitrite in seawater, the European flounder (Platichthys flesus) takes up 66% of its nitrite via intestinal routes.²⁴ Further, its internal nitrite concentration was found to remain below the ambient nitrite level in the water. At these concentrations, there was some alteration of internal biochemical parameters (such as an increase in methemoglobin levels from 4% in nonexposed fish to 18% of hemoglobin in exposed fish). Nevertheless, there were no mortalities under these conditions, and the difference between this result and what is often observed in freshwater fish at similar nitrite concentrations is attributed to differences in their internal nitrite concentrations.

How Toxic is Nitrite to Fish?

For the reason described above, nitrite is considerably more toxic to many freshwater fish (Table 1) than it is to most marine species (Table 2). The data in these tables are primarily the LC50, which is the concentration at which 50% of the test organisms die (24-h LC50 is the concentration that kills half of the tested organisms within 24 hours). As Table 1 shows, some freshwater fish can die at nitrite levels below 1 ppm. This toxicity is the reason many aquarists worry about nitrite in aquaria. It can be a significant problem in freshwater aquaria. Tests in marine species, however, showed the toxicity to be much lower. None of the thirteen marine fish species for which I could find nitrite toxicity data had LC50 values below 100 ppm, and half had LC50 values of 1,000 - 3,000 ppm or more.

Death is, of course, a very crude indicator of toxicity. An aquarium's nitrite level should not come anywhere close to the LC50 value, because less severe toxicity can occur even at levels below that. In the previous section, I showed data on one marine species in which biochemical effects could be detected at levels well below concentrations that caused death. We saw, for example, a rise in methemoglobin at values as low as 46 ppm nitrite. However, the point remains valid that marine species are orders of magnitude less susceptible to the effects of nitrite than are many freshwater species. The marine aquaculture industry often uses a rough guideline that the safe rearing level of many compounds is a factor of 10 or less than their LC50.³⁰

In examining ammonia, nitrite and nitrate toxicity in marine species, one might think to look at the effects on larval fish to see if they are more sensitive. In examining the incidence of the larvae's first feeding after hatching, and the 24-h LC50, it was found that for seven different marine species, only ammonia was found to be toxic at concentrations that might possibly be encountered in aquaculture facilities.²⁵

Table 3 brings out the distinction between freshwater and seawater organisms most clearly. In these tests, two fish and one shrimp species that are able to live in both freshwater (or brackish water) and seawater were tested for toxicity at different salinities. At least for these three species, it is clearly shown that nitrite is much more toxic in freshwater (or at lower salinity) than in seawater, even to the same species.

In the only published article²⁶ that I could find showing toxicity tests to typical reef aquarium fish, Tom Frakes and Bob Studt exposed tank-raised clownfish (Amphiprion ocellaris; Figure 2) to nitrite concentrations ranging from 0 to 330 ppm in artificial seawater. Two of five fish died after a few days at 330 ppm, giving an LC50 not appreciably different from the other species listed in Table 1. At 33 ppm (the next dose down from 330 ppm), the fish were lethargic and breathing with difficulty, but otherwise experienced no lasting problems. At 3.3 ppm nitrite no effects were observed.

One of the difficulties with interpreting toxicity issues, as related by hobbyists who claim to have seen nitrite toxicity in marine fish, is the possible presence of ammonia. In any aquarium with elevated nitrite, the ammonia level also may be elevated. Since ammonia is known to be very toxic to marine fish (LC50 value below 1 ppm), on the aquarist must ensure that the observations are not flawed by such contaminants. In all of the toxicity tests described above, nitrite is added directly to the seawater, and ammonia would not be expected to be present at significant concentrations, whereas in aquariums the levels of the two materials are not independent of one another.

How Toxic is Nitrite to Invertebrates?

Reef aquaria, obviously, contain far more organisms than just fish. Unfortunately, however, the number of organisms that have been examined for nitrite toxicity is fairly low. Those selected for study are most often those for which there is a significant aquaculture industry, such as prawns. It turns out that most invertebrates studied are fairly insensitive to nitrite (Tables 4-6). Because of the nature of these studies, many endpoints besides death were examined. These other endpoints include growth rates, "intoxication" and feeding. Like fish, the invertebrates showed a wide range of susceptibility to nitrite toxicity. Some showed little effect at hundreds of ppm nitrite. The lowest noted effect was a slowing of gonadal development in a sea urchin at 1.6 ppm, although it was feeding normally and surviving at 33 ppm nitrite.

Summary

As far as has been studied, nitrite is not as toxic to marine fish as it is to freshwater fish. This difference is striking, with some of the most sensitive freshwater species being more than a thousand times more sensitive than typical marine species. Does this apply to all fish that might be kept in reef aquaria? I have no way of knowing. I have seen nitrite toxicity test results on only one marine species that we normally maintain in reef aquaria (Figure 3). Nevertheless, there is no reason to think them more or less susceptible.

Figure 3. Like most reef aquarium fish, the susceptibility to nitrite toxicity of this longhorn cowfish (Lactoria cornuta) from my aquarium has not been established.

The same can be said of nitrite toxicity to marine invertebrates; few studies have been done. Those that have been tested suggest that lethal doses of nitrite are fairly high (i.e., tens to thousands of ppm nitrite). Biochemical and developmental effects have been observed at lower doses, however, and there is no reason to suspect that the most sensitive invertebrates have actually been tested.

Aquarists obviously can decide for themselves, based on the data provided above, what level of nitrite to allow in a reef aquarium, or alternatively, at what level to take some action to reduce it (e.g., a water change). Fortunately, nitrite levels in reef aquaria are almost always far lower than would cause concern from a toxicity perspective. While a new aquarium's nitrite levels might reach up toward 10 ppm or more, such an aquarium should not yet contain sensitive organisms. Once the nitrite is below 1 ppm, there is little reason to be concerned.

From my perspective, nitrite and the nitrogen cycle may be interesting phenomena to monitor during a reef aquarium's initial set up, but nitrite does not warrant routine testing. I haven't tested any aquaria for nitrite in years, despite setting up several reef aquaria in that time. I simply do not think that nitrite is worth the time and expense of testing, unlike ammonia, phosphate, alkalinity, pH, calcium, etc. It is my opinion that many things other than nitrite concentration are more worthy of reef aquarists' attention.

Happy Reefing!

References:

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http://www.pesticideinfo.org/Detail_Chemical.jsp?Rec_Id=PC34406

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The details of the Rural King ad and in-ad sales will be interesting for harvesting time.

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39. Effects of nitrite on growth and molting of Penaeus monodon juveniles. Chen, Jiann Chu; Chen, Sheue Feng. Dep. Aquac., Natl. Taiwan Ocean Univ., Keelung, Taiwan. Comparative Biochemistry and Physiology, Part C: Pharmacology, Toxicology & Endocrinology (1992), 101c(3), 453-8.