Jessica M. Boyd

Characterization and determination of chloro- and bromo-benzoquinones as new chlorination disinfection byproducts in drinking water.

We report the characterization and determination of 2,6-dichloro-1,4-benzoquinone and three new disinfection byproducts (DBPs): 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,3,6-trichloro-1,4-benzoquinone, and 2,6-dibromo-1,4-benzoquinone. These haloquinones are suspected bladder carcinogens and are likely produced during drinking water disinfection treatment. However, detection of these haloquinones is challenging, and consequently, they have not been characterized as DBPs until recently. We have developed an electrospray ionization tandem mass spectrometry technique based on our observation of unique ionization processes. These chloro- and bromo-quinones were ionized through a reduction step to form [M + H](-) under negative electrospray ionization. Tandem mass spectra and accurate mass measurements of these compounds showed major product ions, [M + H - HX](-), [M + H - HX - CO](-), [M + H - CO](-), and/or X(-) (where X represents Cl or Br). The addition of 0.25% formic acid to water samples was found to effectively stabilize the haloquinones in water and to improve the ionization for analysis. These improvements were rationalized from the estimates of pK(a) values (5.8-6.3) of these haloquinones. The method of tandem mass spectrometry detection, combined with sample preservation, solid phase extraction, and liquid chromatography separation, enabled the detection of haloquinones in chlorinated water samples collected from a drinking water treatment plant. The four haloquinones were detected only in drinking water after chlorination treatment, with concentrations ranging from 0.5 to 165 ng/L, but were not detectable in the untreated water. This method will be useful for future studies of occurrence, formation pathways, toxicity, and control of these new halogenated DBPs.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC(50)) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC(50) values for the well-known carcinogen NDMA (15-95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24>CHO>A549>HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC(50) concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC(50). Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPhA causing cell-cycle arrest. This study demonstrates a new approach to comprehensive testing of chemical toxicity.

A toxic disinfection by-product, 2,6-dichloro-1,4-benzoquinone, identified in drinking water.

Disinfection of drinking water is a critical public health measure to inactivate pathogens and eliminate waterborne disease outbreaks. However, disinfection of water unintentionally results in the formation of disinfection by-products (DBPs) from the reactions between disinfectants (e.g., chlorine, chloramines, and ultraviolet irradiation) and natural organic matter (NOM) in water. Epidemiological studies have found associations between the consumption of chlorinated water and an increased risk of bladder cancer, and have suggested associations with adverse reproductive effects. Safe drinking water requires deactivation of pathogens, while the risks possibly associated with DBP formation have required scientific research and precautionary regulatory attention. Currently, trihalomethanes (THMs) and haloacetic acids (HAAs), the major DBPs that are readily detectable, are regulated. 8] However, accumulating evidence suggests that they are not likely causes of the increased bladder cancer risk or adverse reproductive effects. Other as yet unidentified, but more toxic, DBPs produced at much lower levels are more likely contributors. Drinking water is a pervasive exposure route for the public, which makes an understanding of potential health risks from DBPs vitally important. Quantitative structure–toxicity relationship (QSTR) analysis has predicted that haloquinones are highly toxic and may form during water disinfection. The potential toxicity of haloquinones is demonstrated by benzoquinones, which are redox-active, toxicologically important metabolites of other organic molecules that interact with a variety of biologically active molecules (proteins and DNA), thus resulting in various hazardous effects. The chronic lowest observed adverse effect levels (LOAELs) of haloquinones are predicted to be in the low mgkg 1 body weight per day range, which is 1000 times lower than most of the regulated DBPs except for bromate. To date, the formation or occurrence of haloquinones as DBPs in drinking water has not been reported. Consequently, we aimed at determining whether haloquinones are present in treated drinking water. This knowledge is critical because current water utility practices aimed at complying with the regulated, but largely surrogate, DBPs have resulted in increased production of more toxic, but previously unidentified, DBPs. Effective management of DBP health risks requires better knowledge of disinfection chemistry combined with DBP toxicology. We initially tested four relevant haloquinones (see the Supporting Information, Figure S1, for their structures): 2,6dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TCBQ), and 2,6-dibromo-1,4-benzoquinone (DBBQ). However, it is difficult to generate stable ionization of [M+1] and [M 1] ions from these compounds by using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). To address this issue, we examined the negative-ionization pathway of haloquinones. We consistently observed [M+H] ions, which can be explained based on the electrochemistry of chloroquinones on the ESI tip (Scheme 1). During negative ESI, chloroqui-

Halobenzoquinones in Swimming Pool Waters and Their Formation from Personal Care Products

Halobenzoquinones (HBQs) are a class of disinfection byproducts (DBPs) of health relevance. In this study, we aimed to uncover which HBQs are present in swimming pools. To achieve this goal, we developed a new method capable of determining eight HBQs while overcoming matrix effects to achieve reliable quantification. The method provided reproducible and quantitative recovery (67-102%) and detection limits of 0.03-1.2 ng/L for all eight HBQs. Using this new method, we investigated water samples from 10 swimming pools and found 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) in all the pools at concentrations of 19-299 ng/L, which was as much as 100 times higher than its concentration in the input tap water (1-6 ng/L). We also identified 2,3,6-trichloro-(1,4)benzoquinone (TriCBQ), 2,3-dibromo-5,6-dimethyl-(1,4)benzoquinone (DMDBBQ), and 2,6-dibromo-(1,4)benzoquinone (2,6-DBBQ) in some swimming pools at concentrations of <0.1-11.3, <0.05-0.7, and <0.05-3.9 ng/L, respectively, but not in the input tap water. We examined several factors to determine why HBQ concentrations in pools were much higher than in the input tap water. Higher dissolved organic carbon (DOC), higher doses of chlorine and higher temperatures enhanced the formation of HBQs in the pools. In addition, we conducted laboratory disinfection experiments and discovered that personal care products (PCPs) such as lotions and sunscreens can serve as precursors to form additional HBQs, such as TriCBQ, 2,6-dichloro-3-methyl-(1,4)benzoquinone (DCMBQ), and 2,3,5,6-tetrabromo-(1,4)benzoquinone (TetraB-1,4-BQ). These results explained why some HBQs existed in swimming pools but not in the input water. This study presents the first set of occurrence data, identification of new HBQ DBPs, and the factors for their enhanced formation in the swimming pools.

Formation of N-nitrosodiphenylamine and two new N-containing disinfection byproducts from chloramination of water containing diphenylamine.

N-nitrosodiphenylamine (NDPhA) is a disinfection byproduct (DBP) in drinking water. However, it is not known what governs the formation of NDPhA and which precursor(s) in the raw water is responsible for its formation. We report here diphenylamine (DPhA) as a key precursor of NDPhA, and we describe the effect of water pH and chloramination conditions on the formation of NDPhA. To identify precursors of NDPhA, raw water samples were collected from the same drinking water system in which NDPhA was previously detected. Analysis of the raw water samples showed the presence of 1.3 ng/L of DPhA and no detectable NDPhA. Seven hours after the treatment of the raw water with chloramines, the concentration of DPhA decreased to 0.4 ng/L with corresponding formation of NDPhA (0.4 ng/L). Controlled experiments involving chloramination of DPhA in water showed that chloramines were essential to the formation of NDPhA, and that increasing the pH from 4 to 10 resulted in 64-fold enhancement in NDPhA formation. Removal of DPhA and formation of NDPhA was found by mass imbalance, which led to the identification of two new DBPs, phenazine (MW 180 Da) and a chlorinated phenazine derivative (MW 216 Da), using liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry. Both new DBPs were detected only in the treated water and not in the raw water. Phenazine and N-chlorophenazine have never been reported as DBPs and neither their occurrence in drinking water nor their health effects are known.

Liquid chromatography tandem mass spectrometry determination of free and conjugated estrogens in breast cancer patients before and after exemestane treatment.

We report liquid chromatographic separation with tandem mass spectrometry determination of 12 endogenous estrogens and their intact conjugates in blood and urine and its application to study effects of exemestane treatment on estrogen generation and metabolism in postmenopausal women with estrogen-dependent breast cancer. A 0.5 mL aliquot of each urine or serum sample is fractionated with solid phase extraction to a fraction of free estrogen and another fraction of their conjugates. The reversed phase LC/MS/MS determines dansylated estrogens with positive ionization and intact conjugates with negative ionization. The method provides reproducible separation and limit of detection as low as 1 pg mL(-1) for free estrogens and 0.03 ng mg(-1) creatinine for the conjugates in serum and urine samples. The method enabled us to acquire unique concentration profiles of 12 endogenous estrogens and their intact conjugates in 30 breast cancer patients before and after one month of exemestane treatment. Exemestane suppressed total serum and urinary estrogens by 11-97% (P<0.0001) and 8.7-91% (P<0.0001), respectively. Specifically, these data show that exemestane preferentially suppressed E1, E1-3S, E1-3G, and E2-17G more than other estrogens. Linear regression analysis of estrogen concentrations before and after treatment showed correlation coefficients of 0.8385 (n=289, P<0.0001) and 0.8863 (n=360, P<0.0001). This study provides urinary and blood estrogen concentration profiles in breast cancer patients to demonstrate the effect of exemestane on estrogen generation, supporting inhibition of aromatase activity.

UV-induced transformation of four halobenzoquinones in drinking water.

Halobenzoquinones (HBQs) are a group of emerging disinfection byproducts (DBPs) found in treated drinking water. Because the use of UV treatment for disinfection is becoming more widespread, it is important to understand how the HBQs may be removed or changed due to UV irradiation. Water samples containing four HBQs, 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,3,6-trichloro-1,4-benzoquinone (TCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), and 2,6-dichloro-1,4-benzoquinone (DBBQ), were treated using a modified bench scale collimated beam device, mimicking UV treatment. Water samples before and after UV irradiation were analyzed for the parent compounds and products using a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method. As much as 90% of HBQs (0.25 nmol L(-1)) in both pure water and tap water were transformed to other products after UV254 irradiation at 1000 mJ cm(-2). The major products of the four HBQs were identified as 3-hydroxyl-2,6-dichloro-1,4-benzoquinone (OH-DCBQ) from DCBQ, 5-hydroxyl-2,6-dichloro-3-methyl-1,4-benzoquinone (OH-DCMBQ) from DCMBQ, 5-hydroxyl-2,3,6-trichloro-1,4-benzoquinone (OH-TCBQ) from TCBQ, and 3-hydroxyl-2,6-dibromo-1,4-benzoquinone (OH-DBBQ) from DBBQ. These four OH-HBQs were further modified to monohalogenated benzoquinones when the UV dose was higher than 200 mJ cm(-2). These results suggested possible pathways of UV-induced transformation of HBQs to other compounds. Under the UV dose commonly used in water treatment plants, it is likely that HBQs are partially converted to other halo-DBPs. The occurrence and toxicity of these mixed DBPs warrant further investigation to understand whether they pose a health risk.

Ultra Pressure Liquid Chromatography−Negative Electrospray Ionization Mass Spectrometry Determination of Twelve Halobenzoquinones at ng/L Levels in Drinking Water

We report here the characterization of twelve halobenzoquinones (HBQs) using electrospray ionization (ESI) high resolution quadrupole time-of-flight mass spectrometry. The high resolution negative ESI spectra of the twelve HBQs formed two parent ions, [M + H(+) + 2e(-)], and the radical M(-•). The intensities of these two parent ions are dependent on their chemical structures and on instrumental parameters such as the source temperature and flow rate. The characteristic ions of the HBQs were used to develop an ultra pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. At the UPLC flow rate (400 μL/min) and under the optimized ESI conditions, eleven HBQs showed the stable and abundant transitions [M + H(+) + 2e(-)] → X(-) (X(-) representing Cl(-), Br(-), or I(-)), while dibromo-dimethyl-benzoquinone (DBDMBQ) showed only the transition of M(-•) → Br(-). The UPLC efficiently separates all HBQs including some HBQ isomers, while the MS/MS offers exquisite limits of detection (LODs) at subng/mL levels for all HBQs except DBDMBQ. Combined with solid phase extraction (SPE), the method LOD is down to ng/L. The results from analysis of authentic samples demonstrated that the SPE-UPLC-MS/MS method is reliable, fast, and sensitive for the identification and quantification of the twelve HBQs in drinking water.

Identification of Tobacco-Specific Nitrosamines as Disinfection Byproducts in Chloraminated Water

Tobacco-specific nitrosamines (TSNAs) exist in environmental waters; however, it is unknown whether TSNAs can be produced during water disinfection. Here we report on the investigation and evidence of TSNAs as a new class of disinfection byproducts (DBPs). Using five common TSNAs, including (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as the targets, we first developed a solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of rapidly determining these TSNAs at levels as low as 0.02 ng/L in treated water. Using this highly sensitive method, we investigated the occurrence and formation potential (FP) (precursor test conducted in the presence of chloramines) of TSNAs in treated water from two wastewater treatment plants (WWTPs) and seven drinking water treatment plants (DWTPs). NNAL was detected in the FP samples, but not in the samples before the FP test, confirming NNAL as a DBP. NNK was detected in the treated wastewater before the FP test, but its concentration increased significantly after chloramination in two of three tests. Thus, NNK could be a DBP and/or a contaminant in wastewater. Moreover, these TSNAs were detected in FP tests of wastewater-impacted DWTP plant influents in 9 of 11 samples. However, TSNAs were not detected at full-scale DWTPs, except for at one DWTP with high ammonia where breakpoint chlorination was not achieved. The concentration of the sum of five TSNAs (0.3 ng/L) was 100-fold lower than NDMA, suggesting that TSNAs have a minor contribution to total nitrosamines in water. We examined several factors in the treatment process and found that chlorine or ozone may destroy TSNA precursors and granular activated carbon (GAC) treatment may remove the precursors. Further research is warranted into the efficiency of these processes at different DWTPs using sources of varying water quality.

Direct large volume injection ultra-high performance liquid chromatography-tandem mass spectrometry determination of artificial sweeteners sucralose and acesulfame in well water

Acesulfame (ACE) and sucralose (SUC) have become recognized as ideal domestic wastewater contamination indicators. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis is commonly used; however, the sensitivity of SUC is more than two orders of magnitude lower than that of ACE, limiting the routine monitoring of SUC. To address this issue, we examined the ESI behavior of both ACE and SUC under various conditions. ACE is ionic in aqueous solution and efficiently produces simple [M-H](-) ions, but SUC produces multiple adduct ions, limiting its sensitivity. The formic acid (FA) adducts of SUC [M+HCOO](-) are sensitively and reproducibly generated under the LC-MS conditions. When [M+HCOO](-) is used as the precursor ion for SUC detection, the sensitivity increases approximately 20-fold compared to when [M-H](-) is the precursor ion. To further improve the limit of detection (LOD), we integrated the large volume injection approach (500μL injection) with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which reduced the method detection limit (MDL) to 0.2ng/L for ACE and 5ng/L for SUC. To demonstrate the applicability of this method, we analyzed 100 well water samples collected in Alberta. ACE was detected in 24 wells at concentrations of 1-1534ng/L and SUC in 8 wells at concentrations of 65-541ng/L. These results suggest that wastewater is the most likely source of ACE and SUC impacts in these wells, suggesting the need for monitoring the quality of domestic well water.