THE EFFECTS OF PERMANGANATE PRETREATMENT ON THE MUTAGENIC ACTIVITY AND CHLOROFORM CONCENTRATIONS OF CHLORINATED HUMIC ACID SOLUTIONS

The objective of this research was to measure changes in mutagen production and chloroform levels in solutions of humic acid that were treated with 0-, 1-, 2-, and 4-g/L permanganate at pH 3, 7, and 9, without buffer, and at pH 3 with added buffer, before being chlorinated. Optimal conditions for the production of mutagens by chlorination of humic acid were determined after which humic acid samples were prepared, pretreated with permanganate, and chlorinated. Chloroform production and mutagenic activity were measured as were total oxidant levels and the pH values of the samples at each stage of treatment. Results showed that increasing concentrations of permanganate lowered mutagenic activity in chlorinated humic acid samples that were started at pH values of 7 and 9, but no definite conclusion could be reached for the samples started at pH value of 3, with or without added buffer. Chloroform levels did not correlate with changes in permanganate concentration except in samples started at the pH value of 3 with added buffer. Attempts were made to label, quantitate, and inactivate mutagens by derivatizing them with a chromophoric nucleophile, 4-nitrothiophenol (NTP), after which the decrease in mutagenic activity of the sample was correlated with a change in the HPLC absorbance profile at 345 nm. Several methods for removing residual chlorine from the chlorinated samples were tested and compared with respect to changes in mutagenic activity, production of mutagenic artifacts, and efficiency of residual chlorine removal. It was found that residual chlorine can be removed from samples by rotary evaporation or speed vacuum centrifugation to dryness without loss of mutagenic activity providing the pH of the sample is kept below 4 during treatment and reconstitution is with phosphate buffer at pH 3. These evacuation methods are preferable to the use of a chemical quenching agent such as ferrous sulfate. Because mutagenic activity was detected in chlorinated MilliQ water treated with ferrous sulfate, studies were undertaken to characterize and quantitate these results.