Chloride Enhances DNA Reactivity with Chlorine under Conditions Relevant to Water Treatment

Free available chlorine (FAC) is widely used to inactivate viruses by oxidizing viral components, including genomes. It is commonly assumed that hypochlorous acid (HOCl) is the chlorinating agent responsible for virus inactivation; however, recent studies have underscored that minor constituents of FAC existing in equilibrium with HOCl, such as molecular chlorine (Cl2), can influence FAC reactivity toward select organic compounds. This study measures the FAC reaction kinetics with dsDNA and ssDNA extracted from representative bacteriophages (T3 and ϕX174) in samples augmented with chloride. Herein, chloride enhances FAC reactivity toward dsDNA and, to a lesser extent, toward ssDNA, especially at pH < 7.5. The enhanced reactivity can be attributed to the formation of Cl2. Second-order rate constants were determined for reactions of ssDNA and dsDNA with HOCl and Cl2. DNA chlorination kinetics followed the reactivity-selectivity principle, where the more-reactive nucleophilic species (ssDNA, ∼100× more reactive than dsDNA) reacted less selectively with electrophilic FAC species. The addition of chloride was also shown to enhance the inactivation of bacteriophage T3 (dsDNA genome) by FAC but did not enhance the inactivation of bacteriophage ϕX174 (ssDNA genome). Overall, the results suggest that Cl2 is an important chlorinating agent of nucleic acids and viruses.

Text S2. Trace contaminant effect on dsDNA FAC reactivity We tested whether trace contaminants present in salts affect FAC reactivity with DNA. To do so, we measured FAC reactivity with dsDNA (pH = 9.0) in the presence of 10 mM trace metal grade perchlorate or nitrate salts. We compared the FAC reactivity to that observed with 10 mM ACS grade perchlorate salt used throughout the manuscript ( Figure S7). The observed second order rate constants were within 10% of each other regardless of salt used (Table S6). Statistical tests comparing the second order rate constants showed no differences between the conditions (Table  S13). When we used statistical tests to compare samples collected at the longest reaction time, we found that the Region A amplicon sample in the trace metal grade perchlorate condition was statistically different from the Region A amplicon sample in the ACS grade perchlorate condition (Table S14). However, given the lack of statistical significance in all other comparisons, it is highly unlikely that trace contaminants in salts affect FAC reactivity with DNA in our study.
We used the variable-chloride and variable-pH data sets (Table S6; 10 mM added salts) to estimate the contribution of chlorine species to DNA FAC reactivity.
Given that 5 mg-Cl2/L of free active chlorine (FAC) was added to each reaction, and less than 10% of FAC was lost throughout the reactions, we assumed a constant, 5 mg-Cl2/L (0.071 mM), FAC concentration. Four FAC species, HOCl, OCl -, Cl2O and Cl2 were considered as potential reactive chlorinating agents in our kinetic models We used the following equilibrium equations to construct a chlorine speciation diagram ( Fig S10): HOCl ⇌ OCl -+ H + pKa,HOCl = 7.54 (eq. S1) Our experiments were conducted under pseudo-first-order conditions, and the change in DNA concentration over time was expressed as: where [DNA] is the concentration of either ssDNA or dsDNA, and kobs is the observed pseudofirst-order rate constant calculated from the apparent second-order rate constants reported in Table S6. kobs can be rewritten as a function of second-order reaction rate constants: Scientist 3.0 was used to conduct all regression analyses based on the following algorithms:

dsDNA:
Using the variable-chloride data set (n = 8; Table S6), second-order rate constants for Cl2 and HOCl were fit simultaneously while assuming a negligible contribution from OCl -. These results gave good fits to the data collected as a function of pH (10 mM NaCl; Table S6), which serve as robust, independent validation of the calculated second-order rate constants. Inclusion of a second-order rate constant for OClor Cl2O did not improve model fits across conditions for dsDNA samples. When OClwas included for dsDNA data, the overall error, driven by error at S6 pH = 6.0-7.5 increased. Hence, we did not include OClas a reactant for dsDNA data. We assume that OCland Cl2O do not contribute appreciably to the reactivity of dsDNA in these systems.

ssDNA:
1. Using the variable-chloride data set (n = 8; Table S6), second-order rate constants for Cl2 and HOCl were fit simultaneously while assuming a negligible contribution from OCl -. 2. Using the variable pH data (10 mM added NaCl; Table S6), second-order rate constant for OClwas fit while assuming fixed values for the second-order rate constant for HOCl and Cl2 (determined in the previous step). 3. Using the variable-chloride data set (n = 8), second-order rate constants for Cl2 and HOCl were fit simultaneously while assuming a fixed value for the second-order rate constant for OCl -(determined in the previous step). 4. Steps 2 and 3 were repeated iteratively until second-order rate constants for all active chlorine species changed by <1% upon subsequent iteration.
Inclusion of a second-order rate constant for Cl2O did not improve model fits. Therefore, we assume that Cl2O does not contribute appreciably to the reactivity of ssDNA in these systems.
The resulting values for kHOCl, kOCl-, and kCl2 for ssDNA and dsDNA are listed in Table 1 in the main manuscript. Using these values, we calculated the contribution of HOCl, OCl -, and Cl2 to the reactivity of the ssDNA and dsDNA genomes ( Figure S11).

S7
Text S4. Analysis of salts and solutions for bromide Six nominally bromide-free salts were analyzed for bromide following the method of Dias et al. 3 The six examined salts included NaCl (ACS grade), NaCl (trace-metal grade), NaClO4 (ACS grade), NaClO4 (trace-metal grade), NaNO3 (ACS grade), and NaH2PO4 (ACS grade). Solutions containing ~1 M of each salt were prepared in ultrahigh-purity (Miliq) water. Solutions of phosphate-buffered saline (PBS) and ultrahigh-purity water (solvent blank) were also analyzed. NaOCl was added to aliquots of each salt solutions and reagent blank to oxidize any bromide present into HOBr. NaOCl-amended aliquots were allowed to stand at room temperature for 2 min prior to addition of 1,3,5-trimethoxybenzene (TMB, methanolic spike) to derivatize HOBr into 1-bromo-2,4,6-TMB (Br-TMB). Quenched samples were extracted into toluene and were analyzed for Br-TMB by gas chromatography with a quadrupole mass analyzer. For complete methodological details, see Dias et al. 3 Bromide was detected in ACS and trace-metal grade NaCl at 58 and 11.3 mg/kg (Table S15). These levels of bromide as an impurity in NaCl samples are on par with those reported by Dias et al. 3 All other examined salts had concentrations of bromide less than 2 mg/kg. Table S1. qPCR assay parameters for T3 and X174 genomes.
S9 Table S2. p-values comparing T3 dsDNA genome degradation at elevated chloride concentrations. Statistical differences between conditions were determined by comparing observed second order rate constants modelled from the pseudo-first order portions of the reactions. p-values < 0.05 are bolded.   Table S6. Based normalized apparent second order reaction rate constants for the reaction of T3 dsDNA genome and X174 with FAC (5 mg-Cl2/L; 0.071 mM). Reaction rate constants were calculated using data where pseudo-first order kinetics held (where the r 2 for the log-linear fit was greater than 0.97). Asterixis mark reaction rate constants that were modelled through a subset of the collected data set (where the r 2 for the log-linear fit was greater than 0.97). For rate constants that are not marked with an asterisk, pseudo-first-order kinetics held through the entire collected data set (the r 2 was greater than 0.97 for the log-linear fit for all the data points collected). Error bars indicate the range of experimental duplicates.    Table S10. p-values comparing X174 ssDNA genome degradation at varying pHs. Given the deviation from linear kinetics for select reaction conditions, statistical differences between conditions were determined by comparing samples collected at the longest reaction times. pvalues < 0.05 are bolded. Table S11. p-values comparing T3 dsDNA genome degradation at varying pHs. Statistical differences between conditions were determined by comparing observed second order rate constants modelled from the pseudo-first order portions of the reactions. p-values < 0.05 are bolded. Table S12. p-values comparing T3 dsDNA genome degradation at varying pHs. Given the deviation from linear kinetics for select reaction conditions, statistical differences between conditions were determined by comparing samples collected at the longest reaction times. pvalues < 0.05 are bolded. Table S13. p-values comparing T3 dsDNA genome degradation when ACS grade ClO4 -, trace metal grade ClO4 -, ACS grade NO3 -, and ACS grade Clwere added to samples at pH = 9.0. Statistical differences between conditions were determined by comparing observed second order rate constants modelled from the pseudo-first order portions of the reactions. p-values < 0.05 are bolded. Table S14. p-values comparing T3 dsDNA genome degradation when ACS grade ClO4 -, trace metal grade ClO4 -, ACS grade NO3 -, and ACS grade Clwere added to samples at pH = 9.0. Given the deviation from linear kinetics for select reaction conditions, statistical differences between conditions were determined by comparing samples collected at the longest reaction times. p-values < 0.05 are bolded. Table S15. Measured concentrations of bromide as an impurity in salts and solutions. Uncertainties denote 95% confidence intervals determined from triplicate samples. Method limit of detection = 0.0014 mg Br -/kg salt. Method limit of quantitation = 0.005 mg Br -/kg salt.           S28 Figure S12. Inactivation of (A) T3 dsDNA bacteriophage or (B) X174 ssDNA bacteriophage with FAC (2 mg-Cl2/L, pH = 7.5) at increasing chloride (Cl -) concentrations. Unfilled markers with downward arrows represent data that was below detection limit. Experimental duplicates/triplicates are shown for each condition.