# A Simple Alternative Method for Preservation of 2-Methylisoborneol in Water Samples
## 3 Results and Discussion
### Impact of Chlorine on 2-MIB Concentration in Natural Water  
Similar to permanganate, chlorine was further tested as a preservation agent for 2-MIB in natural water using the same natural water samples and the same temperatures. However, for the chlorination experiments, three chlorine doses, 2, 5, and 10 mg/L, were tested. In addition, before the analysis, all the samples were separated into two subsamples, one being filtered through a 0.3 \(\upmu\)m-fiberglass filter to represent the extracellular 2-MIB, and the other being used for total 2-MIB (cell bound plus extracellular). Figures 4 and 3 show the effect of temperature and chlorine dosage on preservation of 2-MIB in the three tested natural water samples at 25 \({}^{\circ}\)C and 4 \({}^{\circ}\)C, respectively. The figures clearly demonstrate that for the three natural water samples tested, the 2-MIB concentration was reduced by more than 20% after 14 days at both temperatures if chlorine dosages were at or below 2 mg/L. For those with chlorine dosages of 5 or 10 mg/L, 2-MIB concentrations only changed by \(<\)13% during the 14 days of experiments for all the cases tested. As shown in Figures 4D and 3D, when the chlorine dosage was at 2 mg/L, the residual chlorine concentrations were all below 1 mg/L after the first day of the experiment and were almost equal to 0 after 3-5 days. However, for chlorine dosages at 5 and 10 mg/L, in all cases, residual chlorine was observed to be \(>\)1.5-2.0 mg/L in all the tested cases. Chlorine is known to be a very good bactericide, and reports [38] have shown that if the residual concentration is higher than 0.5 mg/L, bacterial growth is significantly suppressed. Therefore, it is reasonable to find that in these tested natural water samples, 2-MIB concentrations remained almost constant if the chlorine dosages were enough to maintain a residual chlorine concentration \(>\)1.5 mg/L. Figures 4 and 3 also demonstrate that a higher preservation temperature (25 \({}^{\circ}\)C) (Figure 4) led to higher reduction in the 2-MIB concentration, i.e., 37.9%-55.0% after 14 days, as compared with those at 4 \({}^{\circ}\)C (Figure 3), which ranged from 20.6% to 25.3%. Because bacteria prefers to grow at approximately 25 \({}^{\circ}\)C [39], it is reasonable to observe higher biodegradation of 2-MIB at that temperature. In the figure, it can also be seen that there are some differences between the total and extracellular 2-MIB concentrations for the three tested reservoir waters at day 0. Because the differences between the total and extracellular samples represent those bound with cells, it can be seen that at day 0, about 26.1%, 13.3%, and 23.9% of the 2-MIB was cell bound as calculated from the samples with no addition of  
Figure 3: Change of 2-MIB concentration (100 ng/L) in deionized water with presence of manganese dioxide (1 mg/L). chlorine. When the chlorine dosage was increased, the ratio of cell-bound 2-MIB decreased at day 0. [PERSON] et al. [30] studied the interaction of chlorine and cyanobacteria cells and showed that cells may be damaged after contact with chlorine, causing the release of intracellular metabolites. Our observation of increasing chlorine dosages causing greater release of 2-MIB is consistent with the finding of their study. After 5 days of reaction, as shown in the figure, the total 2-MIB concentrations were almost the same as their corresponding extracellular 2-MIB concentrations, suggesting that all the cyanobacteria cells were lysed and that all the 2-MIB was released into the water. To further quantify the degradation of 2-MIB in the preservation experiments, the data of the three natural water cases (Figure 4) were simulated with a first-order reaction model. As shown in Table 1, the rate constants decreased with increasing chlorine concentrations added to the samples. Although the data may not be perfectly fitted by the model, the rate constants may provide a quick estimation of the degrees of degradation.