Ammonia toxicity in fish

Ammonia is an unusual toxicant in that it is produced by, as well as being poisonous to, animals. In aqueous solution ammonia has two species, NH3 and NH4+, total ammonia is the sum of [NH3] + [NH4+] and the pK of this ammonia/ammonium ion reaction is around 9.5. The NH3/NH4+ equilibrium both internally in animals and in ambient water depends on temperature, pressure, ionic strength, and pH; pH is most often of greatest significance to animals. Elevated ammonia levels in the environment are toxic. Temperature has only minor effects on ammonia toxicity expressed as total ammonia in water, and ionic strength of the water can influence ammonia toxicity, but pH has a very marked effect on toxicity. Acid waters ameliorate, whereas alkaline waters exacerbate ammonia toxicity. The threshold concentration of total ammonia ([NH3] + [NH4+]) resulting in unacceptable biological effects in freshwater, promulgated by the EPA (1998), is 3.48 mg N/liter at pH 6.5 and 0.25 mg N/liter at pH 9.0. There is only a relatively small saltwater data set, and a paucity of data on ammonia toxicity in marine environments, particularly chronic toxicity. The national criteria promulgated in the EPA (1989) saltwater document is a criterion continuous concentration (chronic value) of 0.99 mg N/liter total ammonia and a criterion maximum concentration (half the mean acute value) of 6.58 mg N/liter total ammonia, somewhat less than the equivalent freshwater pH 8.0 values of 1.27 and 8.4 mg N/liter total ammonia, respectively. This is consistent with marine species being somewhat more sensitive to ammonia than freshwater species. Toxicity studies are usually carried out on unfed, resting fish in order to facilitate comparison of results. Based on recent studies, however, environmental stresses, including swimming, can have dramatic effects on ammonia toxicity. It is also clear that feeding results in elevated postprandial body ammonia levels. Thus, feeding will probably also exacerbate ammonia toxicity. Fish may be more susceptible to elevated ammonia levels during and following feeding or when swimming. Thus, present ammonia criteria may fail to protect migrating fish and may be inappropriate for fish fed on a regular basis. Most teleost fish are ammonotelic, producing and excreting ammonia by diffusion of NH3 across the gills. They are very susceptible to elevated tissue ammonia levels under adverse conditions. Some fish avoid ammonia toxicity by utilizing several physiologic mechanisms. Suppression of proteolysis and/or amino acid catabolism may be a general mechanism adopted by some fishes during aerial exposure or ammonia loading. Others, like the mudskipper, can undergo partial amino acid catabolism and use amino acids as an energy source, leading to the accumulation of alanine, while active on land. Some fish convert excess ammonia to less toxic compounds including glutamine and other amino acids for storage. A few species have active ornithine—urea cycles and convert ammonia to urea for both storage and excretion. Under conditions of elevated ambient ammonia, the mudskipper P. schlosseri can continue to excrete ammonia by active transport of ammonium ions. There are indications that some fish may be able to manipulate the pH of the body surface to facilitate NH3 volatilization during aerial exposure, or that of the external medium to lower the toxicity of ammonia during ammonia loading. Future investigation of these aspects of “environmental ammonia detoxification” may produce new information on how fish avoid ammonia intoxication.

Ammonia toxicity, tolerance, and excretion

Many fishes are ammonotelic but some species can detoxify ammonia to glutamine or urea. Certain fish species can accumulate high levels of ammonia in the brain or defense against ammonia toxicity by enhancing the effectiveness of ammonia excretion through active NH4+ transport, manipulation of ambient pH, or reduction in ammonia permeability through the branchial and cutaneous epithelia. Recent reports on ammonia toxicity in mammalian brain reveal the importance of permeation of ammonia through the blood-brain barrier and passages of ammonia and water through transporters in the plasmalemma of brain cells. Additionally, brain ammonia toxicity could be related to the passage of glutamine through the mitochondrial membranes into the mitochondrial matrix. On the other hand, recent reports on ammonia excretion in fish confirm the involvement of Rhesus glycoproteins in the branchial and cutaneous epithelia. Therefore, this review focuses on both the earlier literature and the up-to-date information on the problems and mechanisms concerning the permeation of ammonia, as NH3, NH4+ or proton-neutral nitrogenous compounds, across mitochondrial membranes, the blood-brain barrier, the plasmalemma of neurons, and the branchial and cutaneous epithelia of fish. It also addresses how certain fishes with high ammonia tolerance defend against ammonia toxicity through the regulation of the permeation of ammonia and related nitrogenous compounds through various types of membranes. It is hoped that this review would revive the interests in investigations on the passage of ammonia through the mitochondrial membranes and the blood-brain barrier of ammonotelic fishes and fishes with high brain ammonia-tolerance, respectively.

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Ammonia production, excretion, toxicity, and defense in fish: a review.

The branchial epithelium of the mudskipper Periophthalmodon schlosseri is densely packed with mitochondria-rich (MR) cells. This species of mudskipper is also able to eliminate ammonia against large inward gradients and to tolerate extremely high environmental ammonia concentrations. To test whether these branchial MR cells are the sites of active ammonia elimination, we used an immunological approach to localize ion-transport proteins that have been shown pharmacologically to be involved in the elimination of NH(4)(+) (Na(+)/NH(4)(+) exchanger and Na(+)/NH(4)(+)-ATPase). We also investigated the role of carbonic anhydrase and boundary-layer pH effects in ammonia elimination by using the carbonic anhydrase inhibitor acetazolamide and by buffering the bath water with Hepes, respectively. In the branchial epithelium, Na(+)/H(+) exchangers (both NHE2- and NHE3-like isoforms), a cystic fibrosis transmembrane regulator (CFTR)-like anion channel, a vacuolar-type H(+)-ATPase (V-ATPase) and carbonic anhydrase immunoreactivity are associated with the apical crypt region of MR cells. Associated with the MR cell basolateral membrane and tubular system are the Na(+)/K(+)-ATPase and a Na(+)/K(+)/2Cl(-) cotransporter. A proportion of the ammonia eliminated by P. schlosseri involves carbonic anhydrase activity and is not dependent on boundary-layer pH effects. The apical CFTR-like anion channel may be serving as a HCO(3)(-) channel accounting for the acid-base neutral effects observed with net ammonia efflux inhibition.

Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri)

Mudskippers are amphibious fishes that have adapted to live on mudflats. Here, the authors sequence the genomes of four different mudskipper species and highlight genetic changes that may have had an evolutionary role in the water-to-land transition of vertebrates.

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Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes

The aim of this study was to elucidate if the mudskipper Periophthalmodon schlosseri, in relation to its capability to survive on land, has acquired a greater capacity to detoxify ammonia than more aquatic species. The tolerance of P. schlosseri to environmental ammonia was much higher than that of another mudskipper, Boleophthalmus boddaerti, and those of other fishes. The 24, 48, and 96 h median lethal concentrations (LC50) of unionized ammonia (NH3) for P. schlosseri were 643, 556 and 536 µM, respectively. The corresponding LC50 values for B. boddaerti were 77.1, 64.0, and 60.2 µM. The relatively high tolerance of P. schlosseri to ammonia could be partially due to the presence of high activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH, aminating) in its brain. When P. schlosseri and B. boddaerti were exposed to their sublethal NH3 concentrations of 446 and 36 µM, respectively, both mudskippers detoxified ammonia by converting it to free amino acids (FAA). This led to increases in concentrations of total FAA (TFAA) in the brain, liver and muscle. Increases in TFAA concentrations in the brain were mainly due to increases in glutamine concentrations. The activities of GS and GDH in the brain of both mudskippers increased significantly after they were exposed to their respective sublethal concentrations of NH3. Urea production and excretion were not utilized as a means for environmental ammonia detoxification in these mudskippers. The most intriguing results obtained were the lack of effect on any of the parameters studied when P. schlosseri was exposed to 36 µM of environmental NH3. These results suggest that P. schlosseri might be able to maintain a low steady state level of internal ammonia during ammonia loading at a concentration which is lethal to other fishes.

W.K. Kok

Papers

The mudskippers Periophthalmodon schlosseri and Boleophthalmus boddaerti can tolerate environmental NH3 concentrations of 446 and 36µM, respectively