Activity and safety of Bronopol Table VI. Stability of Bronopol after storage at room temperature in the dark Initial assay Assay after Bronopol storage, Bronopol •o Time of storage • 99.2 24 months 98.2 99.2 101.1 22 months 101.5 102-2 100.2 21 months 101.4 102.4 99.9 18 months 99.7 99.5 Stability in Aqueous Solution Bryce and Smart (14) have shown that aqueous solutions of Bronopol are reasonably stable when acid. To investigate the stability of the compound in more detail, aqueous solutions of Bronopol (0.2• w/v) were prepared at pH 4 and 6 (McIlvaine buffer) and pH 8 (phosphate buffer). The solution at pH 4 was stored in the dark at 50øC, the solu- tion at pH 8 at 30øC, and the solution at pH 6 at 30, 40 and 50øC. At appropriate time intervals aliquots were removed and examined microbiologically, polarographically, gas- chromatographically and for bromide ion, nitrate ion, nitrite ion and formaldehyde. Aqueous solutions of Bronopol (10• w/v) at about pH 6 were stored at temperatures ranging from 40 to 100øC, the pH being maintained by the addition of 5N sodium hydroxide. Aliquots were removed and examined by thin-layer chromatography (t.l.c.) and bioautography. Attempts were made to isolate the decomposition products from partially decomposed solutions by preparative layer and Sephadex column chromato- graphy. The decomposition of Bronopol was found to be accelerated by increasing the pH or the temperature of the solution. These effects are shown graphically in Figs. I and 2 using a factor of approximately 4 as the increase or decrease in the rate of decomposition per 10øC temperature change, the times for 50• decomposition extrapolated to 20øC, and based on the g.l.c. assay results, are as shown below: Time for 50Yo pH decomposition 4 • 5 years 6 1« years 8 2 months The initial process in the decomposition of Bronopol appears to be a retroaldol reaction with the liberation of formaldehyde and the formation of bromonitroethanol' NO• NO, I I HOCH•-- C -- CH•OH •CH• + HOCH•-- CH I I Br Br

10 D.M. Bryce et al. I00 PH 4 (m•rob•ologi•al assay._..•) IO[ PH • (G LC)•og,½al assoy) I 2 5 4 5 6 7 8 9 I0 Time of storoge (weeks) Figure 1. Effect of pH on stability of aqueous solutions of Bronopol at 40øC (initially 0'2•o w/v) 100 8O 2O 10 ß . .-'•--•_ 500C (G/C) • I • I I I• I I I I 2 5 4 5 6 7 8 9 I0 Time of storage (weeks) Figure 2. Effect of temperature on stability of aqueous solutions of Bronopol at pH 6 (initially 0'2•o w/v)