BENCH SCALE CHLORINATION EXPERIMENTS

Stock solutions of studied BACs (Table 2.1) at a target concentration of 1000mg/L of each were prepared in HPLC grade methanol from neat analytical standards stored in a freezer at approximately -20°C, and used within five months in HPLC grade methanol. On the day of sample preparation, 100µL of the stock solution was spiked into 100mL of LGW to achieve a target concentration of 1mg/L for subsequent dosing with chlorine. In order to facilitate analysis of reaction products without excessive sample processing, the target initial concentration of each BAC was 1mg/L and a chlorine dose of about 25mg/L as Cl2 was used.

Although this chlorine dose is larger than what is typically employed at a DWTP, the objective of the experiment was to simulate disinfection where an excess of chlorine is added relative to the concentration of organic microcontaminants, and using high enough doses of BACs that are well above limits of instrument detection. The 100mL sample reactions took place in sealed 125mL amber glass jars (no mixing) with headspace in order to more accurately simulate DWTP conditions over 24 hours at 20°C in the dark.

The pH of the reaction was not controlled during the course of the experiment in order to avoid adding interfering ions that would cause ion suppression during MS analysis but was in the range of 6-8. After the 24 hour contact time, a 1mL aliquot of the chlorinated samples (diluted 1:10 in LGW) was removed for measurement of free chlorine residual using a HACH test kit pocket colorimeter based on Standard Method 4500-Cl G. (APHA, 1999), and the remaining sample quenched with a 40mg/mL sodium sulfite solution in LGW for TOX analysis or a 40mg/mL ascorbic acid solution in LGW for MS/MS analysis. The

solutions mentioned above used for quenching the chlorine reaction were prepared immediately before addition into the sample. Separate sets of samples were prepared for the different analyses because each analysis was completed on a different day.

The control samples included LGW blanks, free chlorine solutions prepared in LGW at a target concentration of 25mg/L and quenched with sodium sulfite, and BAC solutions in LGW at the target initial concentration of 1mg/L (same as the chlorinated samples). All control samples were stored for the same reaction time and under the same conditions as the chlorinated BAC samples. After the 24 hour contact time, a 10mL aliquot of the control samples was removed for measurement of free chlorine residual (typically a 1:10 or 1:20 dilution) using a HACH test kit pocket colorimeter based on Standard Method 4500-Cl G. (APHA, 1999), and quenching agent was added to the remaining sample as described previously to ensure uniform conditions between the chlorinated and non-chlorinated samples. Chlorine demand was calculated by subtracting the free chlorine residual from the concentration of the chorine dose applied (APHA, 1999), and since chlorine demand indicates that chlorine has reacted with species in the water, this value provides insight into the extent of the reaction that occurred. The chlorine demand of the LGW control sample was determined to be negligible during the experiment. Chlorine demand was used to determine the relative reactivity of the BACs with chlorine, TOX analysis was used to study the incorporation of chlorine onto the parent BAC molecule during the reaction, and MS/MS analysis was used to confirm transformation of select BACs. The net chlorine demand was calculated by subtracting the chlorine demand of the relevant controls from the chlorine demand of the chlorinated samples.

Chlorine Demand Determination

The chlorine residual was determined for all chlorinated samples using a HACH test kit pocket colorimeter based on Standard Method 4500-Cl G (APHA, 1999) and N,N- Diethyl-p-Phenylenediamine (DPD) packets. If needed, samples were diluted to ensure the free chlorine residual was in the range of the colorimeter. The free chlorine residual was then subtracted from the initial chlorine dose to calculate the chlorine demand of the sample.

Total Organic Halide (TOX) Analysis

TOX analysis was used to determine the extent to which chlorine had become incorporated into the parent BAC molecule during reaction. After quenching the residual chlorine as described above, the samples were analyzed for TOX using a procedure adapted from Standard Method 5320 (APHA, 1999). 100mL samples were acidified using 20 drops of concentrated sulfuric acid to ensure optimal adsorption onto one glass-packed activated carbon (GAC) column using a Tekmar-Dohrmann AD-2000 Adsorption Module (Cincinnati, OH). While the standard method calls for two carbon columns, preliminary experimental results showed that the amount of breakthrough of the analytes onto the second column was less than 5% and, therefore, in order to increase productivity, only one glass-packed carbon column was used (see Table 3.3). The sample adsorption rate was 3mL/min and the sample volume adsorbed was 50mL. Removal of inorganic halides adsorbed on the carbon was achieved by flowing 2mL of a ~5g/L NO3-N solution prepared from potassium nitrate at a

After sample adsorption and removal of halides, the carbon was pyrolyzed using a Rosemount Dohrmann DX-2000 Organic Halide Analyzer (Cincinnati, OH). The carbon columns were completely combusted at 850°C and the organohalogen component of the molecule converted to hydrogen halide which was then transported with the carrier gas, helium, to the coulometric cell containing silver electrodes. As the current in the cell increases due to the presence of the halide ions the change in voltage is recorded. The average TOX recovery of the performance check standard, 500ng Cl/µL trichlorophenol, was 88.5% (n = 5; standard deviation: 18%). The net increase in concentration of organic halogen was calculated by taking the difference between the unchlorinated BAC and chlorinated BAC samples.

Ultraviolet-Visible (UV-Vis) Analysis

The fate of the aromaticity of the reacted tetracycline was monitored using a Hitachi U-3300 Spectrophotometer. First the wavelength of maximum absorption of tetracycline was determined and then the parent compound and products in solution were scanned from 200- 750nm to provide some additional characterization of the reaction products. Additionally, for a chlorinated sample a wider wavelength range was scanned, 200-750nm, to determine if new λmax associated with transformation products were formed.

Tandem Mass Spectrometry Analysis (MS/MS)

A separate aliquot was prepared for MS/MS analysis by quenching residual chlorine in a 100mL sample using a 40mg/mL solution of ascorbic acid in LGW and then infusing directly into a Varian 1200L mass spectrometer (Palo Alto, CA) using a Harvard Apparatus syringe pump (Holliston, MA) at a flow rate of 20µL/min. Liquid nitrogen was used as the drying gas, argon was the collision induced dissociation (CID) gas in positive electrospray ionization mode (+ESI), while zero grade air was used as the CID gas in negative electrospray ionization mode (-ESI). The API housing was at 50°C, and the drying gas was at 300°C. The detector was set at 1300V, the shield at 600V, and the capillary between 40- 60V. In order to increase analyte ionization in the electrospray interface, the quenched chlorinated samples was mixed with HPLC grade methanol at a volume ratio of sample:methanol of 9:1. All samples were filtered through 0.45µm Laboratory Supply Distributors (Millville, NJ) syringe tip filters 0.45µm prior to MS/MS analysis. All control samples were analyzed first followed by the chlorinated and unchlorinated BAC samples. While infusing the sample into the mass spectrometer, data was collected using Varian MSWS Software Version 6.8., and the full scan spectra ranging from m/z 50-700 was observed in real time in quadrupole 1 (Q1). The full scan spectra were scanned in order to locate prominent ions. The chlorinated tetracycline samples were compared to the unchlorinated tetracycline samples in order to identify new ions that may be associated with transformation products. During infusion experiments it is possible to distinguish characteristic chlorine isotope ratios. These chlorine isotope ratios are helpful searching for unknown chlorination by-products of BACs because they show, through characteristic patterns, the number of chlorine atoms on a molecule. Prominent peaks observed in Q1 were

optimized and daughter ions were scanned in quadrupole 2 (Q2) by applying argon, the collision gas, at 2.00mTorr and increasing the collision energy from 5-50eV.

2.2.2 Occurrence Sampling