An additional factor that should be considered in regard to nitrite 

 toxicity is the pH of the solution. Nitrite ion establishes the following 

 aqueous equilibrium. 



NO2" + H+ t HNO2 



The concentration of nitrous acid (HNO?) is 4-5 orders of magnitude less 

 than the concentration of nitrite ion (N02~) within the pH range 7.5 to 

 8.5; in going from pH 7.5 to 8.5, the N02~ concentration stays essentially 

 constant, whereas the HNO2 concentration decreases tenfold. Because this 

 equilibrium is pH-dependent, we studied the toxicity of nitrite to rainbow 

 trout over the pH range 6.4 to 9.0, to examine the effect of pH on nitrite 

 toxicity and to see whether toxicity could be attributed to one or the other 

 of the chemical species. 



The results for a series of these experiments are shown in Figures 5 

 and 6. The first figure is a plot of 96-hour LC50 vs. pH for total NO2-N. 

 It shows that the toxicity of nitrite decreases with increasing pH. If the 

 toxicity of nitrite were solely due to the N02~ ion, this plot would be a 

 horizontal line. The second figure shows a plot of LC50 vs. pH for nitrous 

 acid (as N). If all the toxicity were attributable to this nitrite species, 

 this plot would be horizontal. Neither plot is horizontal, suggesting that 

 neither chemical species alone is responsible for the entire toxicity. Over 

 the pH range studied, both species are significantly, although not neces- 

 sarily equally, toxic. It is not possible to separate the toxicity into its 

 components without additional data, but in order to obtain these data by the 

 design we chose, experiments would have to be carried out beyond the pH 

 range acceptable for fishes. 



The question of mode of toxic action of nitrite on fishes has also been 

 studied. Oxygen is transported in fish blood by the respiratory blood pig- 

 ment hemoglobin. The iron in hemoglobin is present in the ferrous, Fe(II), 

 state. Hemoglobin combines loosely with oxygen to form the easily disso- 

 ciated compound oxyhemoglobin, in which iron is still in the Fe(II) state. 

 The transport of oxygen by blood is dependent on the ease with which hemo- 

 globin unites with oxygen and with which oxyhemoglobin gives up oxygen. If 

 the iron in hemoglobin is oxidized to the ferric, Fe(III), state, methemo- 

 globin is formed. Methemoglobin is not capable of combining reversibly with 

 oxygen, and thus sufficiently high concentrations can cause hypoxia and 

 death. Nitrite in the blood oxidizes hemoglobin to methemoglobin, thereby 

 increasing the amount of methemoglobin present and impairing the ability of 

 the blood to transport oxygen. 



It has been established that increased nitrite concentrations produce 

 increased methemoglobin levels in fish blood (Smith and Williams 1974; Smith 

 and Russo 1975; Brown and McLeay 1975; Crawford and Allen 1977; Perrone and 

 Meade 1977; Bortz 1977). The presence of high levels of methemoglobin in 

 fish blood is visually apparent in that the blood becomes brown-colored. 

 Different levels of methemoglobin have been reported as the concentrations 

 causing mortality in fishes. Species differences and differences in overall 

 physical condition may influence fishes' tolerance to different methemoglo- 

 bin levels. 



235 



