Predominant N-haloacetamide and haloacetonitrile formation in

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Predominant N-haloacetamide and haloacetonitrile formation in drinking water via the aldehyde reaction pathway Trang Nha Vu, Susana Y. Kimura, Michael J Plewa, Susan D. Richardson, and Benito J. Marinas Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02862 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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Environmental Science & Technology

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Predominant N-haloacetamide and haloacetonitrile formation in drinking water via the aldehyde reaction pathway

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Trang N. Vu1, Susana Y. Kimura2, Michael J. Plewa3,4, Susan D. Richardson2, Benito J. Mariñas1,4*

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1Department

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of Civil and Environmental Engineering, 3Department of Crop Science, 4Safe Global Water Institute, University of Illinois at Urbana – Champaign, Urbana, Illinois 61801, United States 2Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States* Corresponding author. Address: 1114A NCEL 205 N Mathews Ave. Urbana, IL 61801; Tel: +1-217-333-6961; Fax: +1-217-333-9464; E-mail: [email protected]

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ABSTRACT

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In drinking water disinfection, switching from free chlorine to alternative chemical disinfectants

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such as monochloramine may result in the formation of different classes of toxic disinfection

14

byproducts (DBPs). Haloacetonitriles (HANs) and haloacetamides (HAMs) are two currently

15

unregulated nitrogen-containing DBP (N-DBP) groups commonly found in water disinfected

16

with monochloramine that have been shown to be more cyto- and genotoxic than regulated

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DBPs. For the first time, this study confirms the formation of HAN and HAM dominant species

18

found in disinfected water, dichloroacetonitrile and dichloroacetamide, from the reaction

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between monochloramine and dichloroacetaldehyde via the aldehyde reaction pathway.

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Results from experiments with natural water treated with labelled 15N-monochloramine

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confirmed the relevance of the aldehyde pathway. Monochloramine reacted quickly with

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dichloroacetaldehyde reaching equilibrium with the carbinolamine 2,2-dichloro-1-

23

(chloroamino)ethanol (K1=1.87x104 M-1s-1). Then, 2,2-dichloro-1-(chloroamino)ethanol

24

underwent two parallel reactions where, (1) it slowly dehydrated to 1,1-dichloro-2-

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(chloroimino)ethane (k2=1.09x10-5 s-1) and further decomposed to dichloroacetonitrile, and (2)

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it was oxidized by monochloramine (k3= 4.87x10-2 M-1s-1) to form a recently reported N-DBP,

ACS Paragon Plus Environment


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the N-haloacetamide N,2,2-trichloroacetamide. At high pH, dichloroacetonitrile hydrolyzed to

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-1 -1 dichloroacetamide (𝑘04=3.12x10-7 s-1, 𝑘OH 4 =3.54 M s ). Additionally, trichloroacetaldehyde was

29

also produced from the reaction of dichloracetaldehyde and monochloramine (k5=2.12x10-2 M-

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1s-1)

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protonation. Within the N-haloacetamide family, N,2,2-trichloroacetamide (LC50=3.90x10-4 M)

32

was found to be more cytotoxic than N-chloroacetamide but slightly less potent than N,2-

33

dichloroacetamide.

under the presence of monochlorammonium ion, a product of monochloramine

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INTRODUCTION

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Chlorination has been widely used since the last century protecting against waterborne

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pathogens making water safe to drink.1 However, chlorine reacts with organic matter to

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unintentionally form disinfection by-products (DBPs), such as trihalomethanes (THMs) and

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haloacetic acids (HAAs).2-4 Because THMs and HAAs were shown to be carcinogenic, chronic

40

exposure to DBPs can potentially increase human health risk.5, 6 As a result, maximum

41

concentrations of these DBP groups in drinking water were established to minimize health risks

42

for consumers.7-9 For this reason, alternative disinfectants such as chloramines have been

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increasingly applied by water utilities to reduce the formation of regulated DBPs. A switch of

44

disinfectant however, may increase the formation of other unregulated DBP classes, including

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haloacetonitriles (HANs) and haloacetamides (HAMs)10-13 that are more toxic than DBPs

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predominantly produced by chlorination.4, 14


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According to occurrence studies, nitrogen-containing DBPs (N-DBPs) form at slightly higher

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levels in drinking water samples collected from water utilities that use chloramines compared

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to chlorine.10-12 Among HANs and HAMs, dichloroacetonitrile (DCAN) and dichloroacetamide

50

(DCAM) were reported as predominant species with median of 1 µg/L and 1.3 µg/L and up to

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maximum concentrations of 12 µg/L and 5.6 µg/L, respectively.10, 11 Although HANs and HAMs

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are found at low levels, they were demonstrated to be more cyto- and genotoxic compared to

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regulated DBPs, and could contribute significantly to the overall toxicity of disinfected waters.4,

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13, 15-17

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Because of the occurrence and elevated toxicity of HANs and HAMs,18 there is an emphasis on

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research efforts to characterize their formation mechanism relevant in drinking waters.19-29 The

57

formation of HANs and HAMs in drinking water has been hypothesized based on the origin of

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nitrogen atom in their molecule. Nitrogen-containing organic matter (i.e., amino acids, algal

59

matter) could serve as a major precursor to form N-DBPs when it reacts with chlorine or

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chloramine via the decarboxylation pathway.19-21 On the other hand, the nitrogen atom of the

61

nitrile group or the amide group of HANs or HAMs could also come from monochloramine, an

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inorganic source, reacting with aldehydes25-27 or aromatic compounds.22, 24, 30, 31 All of these

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formation pathways may occur simultaneously and one pathway may become dominant at

64

certain conditions. In two previous studies using 15N-labelled-monochloramine, one reported

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>70% DCAN produced contained 15N atom21 while the other demonstrated >92% of DCAN

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produced contained 15N atom28. Furthermore, most of the 15N from the labelled

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monochloramine transferred to DCAN. These results supported the importance of the



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aldehyde pathway on the formation of these N-DBPs under drinking water disinfection

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conditions.

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The reaction of monochloramine with aldehydes, commonly formed as byproducts from ozone

71

and chlorine disinfection,32-35 that lead to the formation of HANs, HAMs, and N-haloacetamides

72

(N-HAMs), a recently discovered group, is referred to as the aldehyde pathway. The formation

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mechanism of chloroacetonitrile, chloroacetamide, and N,2-dichloroacetamide was

74

characterized in a recent study from the reaction between monochloramine and

75

chloroacetaldehyde.26 Monochloramine reacted with chloroacetaldehyde by nucleophilic

76

addition, to form the carbinolamine 2-chloro-(1-chloroamino)ethanol in a reversible reaction.

77

The carbinolamine dehydrated and produced the imine 1-chloro-(2-chloroimino)ethane that

78

underwent subsequent acid and base catalyzed decomposition to chloroacetonitrile. In a

79

parallel reaction, the carbinolamine was oxidized by monochloramine to form N,2-

80

dichloroacetamide, which was identified for the first time as a new N-DBP subgroup, N-HAMs

81

family. A similar mechanism was observed for the formation of acetonitrile, acetamide, and N-

82

chloroacetamide from the reaction of monochloramine with acetaldehyde.27

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Although several formation mechanisms for HAN, HAM, and N-HAM formation were

84

proposed,20, 21, 25-28 the predominance of a specific pathway has not been identified in natural

85

waters. The formation of DCAN and DCAM via the aldehyde pathway has also not been

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determined. The possible formation of the N-HAM N,2,2-trichloroacetamide (N,2,2-TCAM) has

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not been shown. It is possible that DCAN, N,2,2-TCAM and DCAM may be produced from the

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reaction between dichloroacetaldehyde (DCAL) and monochloramine. This hypothesis is


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supported by the fact that DCAL and chloral hydrate are two species with the highest

90

concentration amongst the haloaldehyde group quantified in drinking water.11

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The main objective of this study was to determine the predominance of DCAN and DCAM

92

formation from the reaction between DCAL and monochloramine via the aldehyde pathway,

93

and the cytotoxicity of new N-DBPs. To achieve this, the scope of work included the following

94

tasks: (1) confirmation of the aldehyde reaction pathway through the identification of

95

intermediates and products, including N,2,2-TCAM; (2) determination of the cytotoxicity of

96

N,2,2-TCAM and comparison to other species from the N-HAM and HAM groups; (3) treatment

97

of natural waters with 15N-labeled monochloramine to evaluate the relevance of the aldehyde

98

pathway on the formation of DCAN, N,2,2-TCAM, and DCAM under drinking water conditions;

99

and (4) determination of reaction rate constants for the proposed pathway via batch

100

experiments using synthetic buffered water. A key novelty of this study was that for the first

101

time a kinetic model developed from experiments performed with synthetic solutions was used

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to simulate the formation of DCAN, N,2,2-TCAM, and DCAM in a real water. The model was

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validated by comparing experimental and simulated data and used to quantify the formation of

104

DCAN, N,2,2-TCAM, and DCAM through the aldehyde pathway.

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EXPERIMENTAL METHODS

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Reagents and Solutions

108

Reagents of the highest purity grade commercially available were used in this study. Potassium

109

biphosphate (99%), sodium perchlorate (>98%), sodium hydroxide (97%), perchloric acid (70%),



110

ammonium chloride (99.9%), sodium bicarbonate (99%), sodium hypochlorite (5-6%), chloral

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hydrate (>97%), 2,2-dichloroacetonitrile (>99.5%), sodium sulfate (99%), methyl tertiary butyl

112

ether (HPLC grade 99.9%), hexane (>98.5%) and ethyl acetate (HPLC grade 99.9%) were

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purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA). Commercial

114

DCAL hydrate solid stock (>95%) was purchased from Tokyo Chemical Industry (TCI) – America

115

(Portland, OR). Acetamide 2,2-dichloroacetamide (>98%) was purchased from Alfa Aesar (Ward

116

Hill, MA).

117

All solutions were prepared with MilliQ water (>18 MΩ.cm, Millipore, Billerica, MA) with a total

118

phosphate buffer concentration of 0.02 M and ionic strength of 0.1 M. Phosphate buffer

119

solution was prepared daily by dissolving potassium biphosphate in pure water. The pH was

120

adjusted with perchloric acid and a sodium hydroxide solution that was prepared every month.

121

Ionic strength was adjusted with a 1 M sodium perchlorate solution, shown not to affect the

122

kinetics of similar reactions,25-27 also prepared monthly.

123

Monochloramine stock solution was prepared daily for each set of experiments by a slow drop-

124

wise addition of sodium hypochlorite solution into an ammonium chloride solution (N/Cl molar

125

ratio = 1.1) under fast stirring. Both solutions were of equal volumes and pH of 8.5.

126

Monochloramine (molar extinction coefficient at 243 nm ε243 = 461 M-1cm-1)36 and sodium

127

hypochlorite (ε292 = 362 M-1cm-1)37 were standardized with a spectrophotometer.

128

A 1-2 M DCAL stock solution was freshly prepared for each experimental set by diluting DCAL

129

hydrate in oxygen-free pure water. The aldehyde solution was stored at 4oC for several hours

130

before use. Chloral hydrate and 2,2-dichloroacetonitrile were prepared by dilution in


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acetonitrile and were used to prepare calibration curves for gas chromatography (GC)-mass

132

spectrometry (MS) analysis.

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N,2,2-Trichloroacetamide was prepared following the procedures described in Text S1 of SI.

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Additional details about this synthetic procedure were described elsewhere.26, 27, 38

135

Controlled Reactions

136

Reaction intermediates and products were identified under pseudo-first order conditions by

137

reacting excess DCAL (10 and 50 mM) with monochloramine (1 and 15 mM) at pH 9.5. Sample

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aliquots without quenching were extracted by LLE, and analyzed by GC-MS and GC-HRMS.

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Tentative intermediate and products were confirmed by analysis of pure standards or by mass

140

spectral interpretation.

141

Experiments conducted for this study are shown in Table 1. Experiments under drinking water

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conditions (GC-1) were tested in triplicate to evaluate the relevance of the aldehyde pathway to

143

form N,2,2-trichloroacetamide and dichloroacetonitrile.

144

added to treated surface waters from Bloomington, IL collected after filtration and before

145

disinfection. Kinetic constants of the proposed reaction pathway were determined using batch

146

reactors for experiments GC-2, GC-3, GC-4, UV-1, UV-2, HR-1, and HR-2. Fast reactions,

147

including the reversible reaction of DCAL with monochloramine to form the corresponding

148

carbinolamine, were studied under experimental conditions SF-1 with stop flow analysis. All

149

experiments were maintained at 25.0 ± 0.1oC throughout the whole reaction.

150

Instrumentation and Methods

15N-labeled


monochloramine was


151

A Thermo Electron Orion ROSS Ultra pH electrode connected to an Accumet AB15 Plus pH

152

meter were used to measure pH. Measured pH values were adjusted to actual hydrogen ion

153

concentration using the Davis equation (μ=0.1 M). A water bath re-circulator (PolyScience,

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Niles, IL) was used to maintain a constant temperature (25.0 ± 0.1oC) for all reactions.

155

A 2550 UV-Vis spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) was used

156

to standardize solutions and monitor reactions. Samples were placed in 10 mm quartz cuvettes

157

and absorbance spectra were taken at wavelengths between 200 – 400 nm.

158

A SX20 stopped flow spectrophotometer (Applied Photophysics, Surrey, UK) was used to study

159

fast reactions of experimental set SF-1. Equal volumes of each reactant were mixed and

160

monitored at λmax=243 nm, the wavelength for maximum absorbance of monochloramine. Five

161

replicates were taken for each experiment. The resulting data was analyzed by the chemical

162

relaxation method presented in Text S2 of SI.27 Micromath Scientist 3.0 (St. Louis, MO) was

163

used to fit the experimental data to the kinetic model.

164

GC-MS (Agilent Technologies GC 6850-MSD 5975C) was employed to identify and quantify

165

intermediates and products from experimental set GC-1 to GC-4 shown in Table 1. Aliquots 7

166

mL in volume were sampled from the reactor over time and extracted by LLE using 1 mL of

167

ethyl acetate and 2 g of sodium sulfate. Extracts were injected under split mode (2:1 split ratio)

168

at 230oC. Compounds were separated with a 30 m DB-624 column (J&W Scientific) by initially

169

holding the oven temperature at 35oC for 3 min, then ramping at 10oC/min to 90oC, holding at

170

90oC for 1 min, and ramping again at 10oC/min to 240oC, and finally holding at 240oC for 5 min.

171

Helium was used as carrier gas at a flow rate of 1.0 mL/min. Electron ionization (EI) of the

172

compounds in the sample was performed under full scan mode at m/z 30-350. With this LLE-


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GC/EI/MS technique we were able to identify DCAN, DCAM and 1,1-dichloro-2-

174

(chloroimino)ethane but not N,2,2-TCAM.

175

Labeled 15N-monochloramine and GC-MS analysis was also applied in drinking water

176

experiments to verify the dominance of the aldehyde pathway in the competition with other

177

pathways under the chloramination conditions investigated. Surface water from the city of

178

Bloomington, IL treated with conventional treatment after filtration and before disinfection was

179

used for these experiments. Monochloramine was added to a reactor with a dose of 10 mg/L

180

as Cl2. Aliquots of 200 mL were quenched with sodium thiosulfate and analyzed for HANs,

181

HAMs, and HALs over time. HANs and HAMs were extracted from an aliquot of 100 mL by LLE

182

with 2 mL of methyl tert-butyl ether (MTBE), 30 g of sodium sulfate, and 1,2-dibromopropane

183

as an internal standard.39 The mixture was shaken for 30 min and the top layer was transferred

184

to a vial after a 5 min rest. HALs were derivatized with O-2,3,4,5,6-

185

pentafluorobenzylhydroxylamine (PFBHA) and extracted from another 100 mL aliquot

186

according to a procedure described elsewhere.40 The GC-MS program was slightly modified to

187

analyze both extracts with a 30 m Rtx-200 (Restek Corporation, Bellefonte, PA). A 2 µL extract

188

was injected into the inlet under split mode (2:1 split ratio) at 230oC. The oven was held at

189

40oC for 3 min, increased to 70oC (ramped 10oC/min), held at 70oC for 2 min, increased to 240oC

190

(ramped 10oC/min) and held at 240oC for 5 min. EI was used and the scan range was m/z 30-

191

450. For DCAN, the fragments m/z 74 and 75 were used to distinguish DCAN containing 14N and

192

15N,

193

and 15N, respectively. Calibration curves were developed for the quantification of these DBPs by

194

preparing increasing concentrations (0.1, 0.25, 0.5, 1, 2, 5, 10 and 25 µg/L). HALs, HANs and

respectively. Similarly, m/z 127 and 128 were applied to differentiate DCAM containing 14N



195

HAMs calibration curve correlation coefficient were > 0.99 and their linear ranges were 0.1 – 5

196

µg/L for HALs, HAMs and 0.1 – 10 µg/L for HANs, respectively. The detection limits of these

197

compounds were 0.05 µg/L.

198

For the main purpose of identifying N,2,2-TCAM, GC-HRMS analysis was done with a HP 5890

199

GC (Agilent Technologies, Santa Clara, CA) coupled to a Synapt G2-Si high resolution time-of-

200

flight mass spectrometer with an atmospheric pressure chemical ionization (APCI) source

201

(Waters Cooperation, Milford, MA). The resolution of this instrument was 50,000, and it was

202

operated in full scan mode. The GC oven program was as follows: hold for 2 min at 50oC, ramp

203

at 15oC/min to 280oC, and hold at 280oC for 5 min. A 30 m DB-5MS GC column with 0.25 mm ID

204

and 1 µm film thickness (J&W Scientific) was used. The ion source and transfer line were 150

205

and 200oC, respectively.

206

Mammalian Cell Cytotoxicity

207

Chinese Hamster Ovary (CHO) cells used in this study were derived from line K141-43. A detailed

208

description of the CHO cells was published44. CHO cells were maintained on glass culture plates

209

in Ham's F12 medium containing 1% antibiotics (100 U/mL sodium penicillin G, 100 μg/mL

210

streptomycin sulfate, 0.25 μg/mL amphotericin B in 0.85% saline), 5% fetal bovine serum (FBS),

211

and 1% glutamine at 37 °C in a humidified atmosphere of 5% CO2.

212

CHO cells were used to determine the chronic cytotoxicity of N,2,2-trichloroacetamide. A 20

213

mM N,2,2-trichloroacetamide stock solution was buffered with 14 mM NaHCO3 and this

214

solution was diluted with F12 +FBS culture medium into a series of concentrations within the

215

range from 10 to 1000 µM. The experimental design consisted of a concurrent negative control


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plus 12 concentrations of N,2,2-trichloroacetamide that covered the entire concentration-

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response from no cytotoxicity to complete killing. Eight replicate clones of cells per

218

concentration were treated and after the 72 h exposure period the cell density per well was

219

measured. The results were converted into the mean percentage of the cell density of the

220

negative control. Data analysis and nonlinear regression fitting were obtained by Sigmaplot

221

(Sytat Software Inc., San Jose, CA). The LC50 value was calculated from the regression analyses

222

of the concentration response curve. The LC50 value is the concentration of N,2,2-

223

trichloroacetamide induced a reduction of 50% of the cell density as compared to the

224

concurrent negative control. Details about the experimental procedure of this test quantitative

225

comparative biological assay were published.18, 45

226 227

RESULTS AND DISCUSSION

228

N-haloacetamide and haloacetonitrile formation via the aldehyde reaction pathway

229

Similar to other aldehydes previously reported,26, 27 DCAL is proposed to react with

230

monochloramine in a fast and reversible reaction to form the carbinolamine 2,2-dichloro-1-

231

(chloroamino)ethanol (Figure 1). Monochloramine attacks the carbonyl carbon in DCAL via

232

nucleophilic addition. The carbinolamine subsequently follows two parallel reactions. In the

233

first reaction, the carbinolamine dehydrates to form the imine 1,1-dichloro-2-

234

(chloroimino)ethane that further decomposes to DCAN. Intermediate 1,1-dichloro-2-

235

(chloroimino)ethane and product DCAN were observed in GC-MS results for samples extracted

236

at a reaction time of 30 min as shown in Figure S3 of SI. The identity of 1,1-dichloro-2-



237

(chloroimino)ethane was confirmed from the m/z fragmentation pattern shown in Figure S4 of

238

SI. A three chlorine isotope pattern was observed from the molecular ion cluster

239

145(M)/147(M+2)/149(M+4) of 1,1-dichloro-2-(chloroimino)ethane. Additionally, isotope

240

patterns from clusters 110/112/114 and 83/85/87 pertain to fragments of the imine that have

241

two chlorines. Intermediate carbinolamine 2,2-dichloro-1-(chloroamino)ethanol was not

242

observed, possibly because it could have dehydrated to the imine at the GC inlet (230°C).

243

Reaction products, including DCAN, DCAM, and choral hydrate, were identified by comparing

244

their retention times and mass spectra from those obtained from pure standards.

245

In the second reaction, carbinolamine 2,2-dichloro-1-(chloroamino)ethanol was oxidized by

246

monochloramine to form the N-HAM N,2,2-trichloroacetamide (N,2,2-TCAM). To confirm this

247

reaction, a pure N,2,2-TCAM standard was prepared in this study and compared to reaction

248

products extracted at reaction time of 24 h (N,2,2-TCAM peaks in samples extracted at shorter

249

reaction times were difficult to resolve above background noise). Both extracts were analyzed

250

by GC-APCI-HRMS. The N,2,2-TCAM standard had a [M+H]+ ion of 161.9283 (100% relative

251

abundance) with isotopes of 163.9250(96%), 165.9224(31%) and 167.9202(3%) as shown in

252

Figure S5 in SI. The abundance ratio indicated the presence of 3 chlorine atoms in the

253

molecular structure of N,2,2-TCAM. The mass error of the observed molecular ion was 1.2

254

ppm. Total ion chromatograms from the reaction extracts and pure standards are shown in

255

Figure S6 of SI. The [M+H]+ ion cluster from the N,2,2-TCAM standard was also observed in the

256

reaction products with a retention time of 12.1 min. A possible isomer of N,2,2-TCAM, 2,2,2-

257

trichloroacetamide, was also analyzed as a pure standard. This isomer had an later retention

258

time of 16.21 min and was not observed in the reaction mixture, therefore, eliminating the


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possibility of 2,2,2-trichloroacetamide as a product from the aldehyde reaction pathway. These

260

results confirmed that N,2,2-TCAM was produced from the reaction between monochloramine

261

and DCAL. The formation of N-HAMs via the aldehyde pathway has been reported in recent

262

studies.26, 27

263

Additionally, GC-MS analyses also suggested that DCAM was a product from the reaction

264

between DCAL and monochloramine as shown in Figure S4 and S6 in SI. One possible reaction

265

could be the hydrolysis of DCAN to DCAM. Previous studies have shown that haloacetonitriles

266

hydrolyze to haloamides and haloacetic acids.23, 46 Control experiments (HR-1 in Table 1, Figure

267

S7 of SI) of DCAN at different pH conditions showed that DCAN quickly hydrolyzed

268

corresponding to the increasing formation of DCAM from the solution at high pH (pH > 9). Even

269

though DCAN hydrolysis was negligible at pH 7 – 8, DCAM was observed in experiments

270

conducted at this pH range (GC-1, GC-2, GC-4, and HR-1 in Table 1). Another reaction that has

271

been shown in previous studies26, 27 is that the addition of a quencher to reduce

272

monochloramine and stop the reaction will also reduce N-HAMs to HAMs. Sodium thiosulfate,

273

quencher used in this study, reduced the chlorine attached to the amide nitrogen, and N,2,2-

274

TCAM was identified and quantified as DCAM. N,2,2-TCAM was quantified as DCAM in GC-MS

275

experiments conducted in this study.

276

High levels of trichloroacetaldehyde (TCAL) were also found as a product from the reaction

277

between DCAL and monochloramine, especially at neutral pH. However, the formation of TCAL

278

at high pH (pH> 9) was negligible compared to the formation of DCAN and N,2,2-TCAM. The

279

formation of more substituted haloaldehydes found in the reaction between DCAL and

280

monochloramine was not observed with other aldehydes.26, 27 Experimental set GC-4 showed



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increasing formation rate of TCAL with decreasing pH (data not shown). Some studies have

282

hypothesized that the halogenation of DCAL leading to the formation of TCAL involved

283

monochlorammonium ion (NH3Cl+) resulting from monochloramine protonation.47, 48 At low

284

pH, monochlorammonium ion would occur at a higher concentration and thereby enhance the

285

formation of TCAL. Monochlorammonium ion has also been suggested as a possible

286

halogenation agent at low pH.49 It is also possible that NH2Cl reacts directly with DCAL and that

287

the reaction undergoes acid catalysis, which could be mathematically be represented with the

288

same expressions at the pH range investigated.

289

Relevance of the aldehyde reaction pathway under drinking water conditions

290

The relevance of the aldehyde reaction pathway in Figure 1 that leads to the formation of

291

DCAN, N,2,2-TCAM, and DCAM was tested in drinking water.

292

(15NH2Cl) can react with natural organic matter and can be incorporated into 15N-labeled DCAN

293

and 15N-labeled DCAM through the aldehyde reaction pathway.

294

Bloomington lake water treated by sedimentation and recarbonation with no disinfection (total

295

organic carbon - TOC = 1.8 mg/L, pH= 7.8-8.1) and analyzed by GC-MS over the course of 5 days,

296

representing an average residence time in a distribution system. Results are shown in Figure 2.

297

In the first sample taken at 4 h, 15N-DCAN concentration was 44% of the total DCAN

298

concentration of 0.24 μg/L. At 95 h, total DCAN increased to a maximum concentration of 1.05

299

μg/L with 64% attributed to 15N-DCAN. Similarly, total DCAM was relatively low (0.32 μg/L) at 4

300

h with 51% as 15N-DCAM. Total DCAM reached a maximum concentration of 4.6 μg/L at 5 days

301

with 67% as 15N-DCAM. Our findings show that about 60-70% of the total DCAN and DCAM

302

produced from the chloramination of the feed water contain a 15N atom in their structures


15N-labeled

15NH Cl 2

monochloramine

was added to

Page 15 of 34


303

which can only be provided by the 15NH2Cl spike. These findings are also consistent with other

304

studies21, 28 that used 15NH2Cl in natural waters and model solutions with dissolved natural

305

organic matter (NOM) and observed greater than 50% formation of 15N-DCAN and 15N-DCAM of

306

the total DCAN and DCAM.

307

The concentration of N,2,2-TCAM, quantified as 15N-DCAM, was also found to be higher than

308

15N-DCAN

309

maximum concentration (3.0 µg/L) after 5 days which was 4.7 times higher than 15N-DCAN

310

concentration (0.63 µg/L). The predominant formation of DCAM over DCAN suggests that

311

DCAM was produced independently from the hydrolysis of DCAN, which was insignificant at

312

neutral pH range in this experiment. These results are consistent with a previous study21 that

313

showed independent formation of DCAM and DCAN during chloramination of drinking water

314

and wastewater effluents.

315

A kinetic model that predicts the formation of N,2,2-TCAM, DCAM, and DCAN under drinking

316

water conditions can validate the proposed mechanism in Figure 1. In this experiment set, the

317

feed water initially did not contain any detectable amount of DCAL (Figure S8 in SI), an initial

318

reactant in the aldehyde pathway. However, reactions between 15NH2Cl and NOM during

319

chloramination produced DCAL, which then reacted with 15NH2Cl via the aldehyde pathway

320

consequently forming 15N-DCAN and 15N-DCAM. The apparent formation rate of DCAL was

321

experimentally determined from the quantified DCAL residue and its products (i.e., DCAN and

322

DCAM) assuming that DCAL was only consumed by the aldehyde pathway reactions proposed in

323

this study (Figure 1). To predict the formation of 15N-DCAN and 15N-DCAM in drinking water,

324

equilibrium and reaction rate constants were determined as shown in the following sections.

throughout the chloramination experiment.

15N-DCAM


(or N,2,2-TCAM) reached a


Page 16 of 34

325

Kinetic rate constant determination

326

Equilibrium constant and formation of carbinolamine 2,2-dichloro-1-(chloroamino)ethanol.

327

In aqueous solution, DCAL exists as two species in equilibrium: the anhydrous aldehyde and its

328

hydrated form. Total DCAL concentration (𝐶𝑇, Cl2CHCHO) is expressed as the sum of these two

329

compounds. The dissociation constant for hydrated species of DCAL, Kd, was estimated

330

according to a predictive model specific for aldehydes and ketones50 and is equal to: 𝐾𝑑 =

[Cl2CHCHO] [Cl2CHCH(OH)2]

= 4.46 × 10 ―4

(1)

331

The hydration constant reveals that DCAL will predominantly exist as the hydrated species (Cl2

332

CHCH(OH)2) in solution. The fractions of DCAL and its hydrated form of the total concentration

333

𝐶𝑇, Cl2CHCHO were determined as 4.45 x 10-4 and 0.99955, respectively.

334

The reversible reaction of DCAL with monochloramine was fast and reached equilibrium with

335

carbinolamine 2,2-dichloro-1-(chloroamino)ethanol within 10–20 s. The equilibrium expression

336

for this reaction is expressed as

𝐾1 =

𝑘1 𝑘 ―1

=

[Cl2CHCH(OH)NHCl]𝑒

[NH2Cl]𝑒[Cl2CHCHO]𝑒

(2)

337

where, K1 is the equilibrium constant, k1 and k-1 are the forward and reverse reaction rate

338

constants.

339

(chloroamino)ethanol, monochloramine and DCAL concentrations at equilibrium, respectively.

340

Experimental set SF-1 in Table 1 was performed to study this fast reaction. The chemical

341

relaxation method27, 51 was used to determine the forward and reverse reaction rate constants

342

described in Text S2 of SI. The reaction was monitored at absorbance 243 nm until it reached

[Cl2CHCH(OH)NHCl]𝑒, [NH2Cl]𝑒, and [Cl2CHCHO]𝑒 are the 2,2-dichloro-1-


Page 17 of 34


343

equilibrium. Results from each experimental condition were fitted to eq 3 to obtain the values

344

of Abse, Abs0 and 1/τ.

( 𝜏𝑡) + 𝐴𝑏𝑠

𝐴𝑏𝑠𝑡 = (𝐴𝑏𝑠0 ― 𝐴𝑏𝑠𝑒)exp ―

345

(3) 𝑒

Abse and Abs0 are the absorbance values at equilibrium and at t = 0, and (1/τ) is defined by eq 4. 1 = 𝑘′1 + 𝑘 ―1 = 𝑘1[Cl2CHCHO]0 + 𝑘 ―1 𝜏

(4)

346

For each initial DCAL concentration used, a (1/τ) value was obtained from data fitting. Results

347

are plotted in Figure S9 of SI as (1/τ) versus initial unhydrated DCAL concentration [Cl2CHCHO]0.

348

Consistent with eq 4, a linear relationship was observed. The slope was k1=1.73±0.08x104 M-1s-1

349

and the intercept at [Cl2CHCHO]0=0 was k-1=0.922±0.016 s-1.

350

Equilibrium constant K1=1.87x104 M-1 was calculated with eq 2. In the presence of

351

monochloramine and DCAL, K1 predicts that carbinolamine will exist as the predominant

352

species. Equilibrium constant K1 was also found to be one to two orders of magnitude higher

353

compared to the equilibrium constants 109 M-1 and 1468 M-1 reported for the respective

354

reactions of acetaldehyde and chloroacetaldehyde with monochloramine.26, 27 An increased

355

substitution of electron-withdrawing chlorine on the beta carbon of aldehydes will create a

356

partially positive carbonyl carbon making it more electrophilic and susceptible to nucleophilic

357

attack by monochloramine. As a result, the equilibrium constant K1 of DCAL is significantly

358

higher than that for chloroacetaldehyde, which in turn is higher than that for acetaldehyde.

359

Consistently, the corresponding dissociation rate constants k-1 of 6.71x10-1, 2.70x10-2, and

360

4.46x10-4 s-1 becomes slower with increasing chlorine substitution.26, 27



Page 18 of 34

361

Dichloroacetonitrile, dichloroacetamide, N,2,2-trichloroacetamide, and trichloroacetaldehyde

362

formation

363

Carbinolamine 2,2-dichloro-1-(chloroamino)ethanol quickly reached equilibrium and slowly

364

decomposed through parallel dehydration and oxidation reactions as shown in Figure 1. The

365

total monochloramine concentration (𝐶𝑇,NCl) is expressed as the sum of monochloramine and

366

carbinolamine concentration at a given time. 𝐶𝑇,NCl = [NH2Cl] + [Cl2CHCH(OH)NHCl]

(5)

367

Monochloramine and carbinolamine concentrations are expressed as fractions of CT,NCl, or α0 and

368

α1 defined as. 𝛼0 =

𝛼1 =

[NH2Cl] 𝐶𝑇,NCl

=

1 1 + 𝐾1[Cl2CHCHO]

[Cl2CHCH(OH)NHCl] 𝐶𝑇,NCl

=

𝐾1[Cl2CHCHO] 1 + 𝐾1[Cl2CHCHO]

(6a)

(6b)

369

The 𝐶𝑇,NCl decomposition rate is composed by the loss of carbinolamine by dehydration and

370

oxidation and the reacted monochloramine to produce TCAL expressed as:

―

𝑑𝐶𝑇,NCl 𝑑𝑡

= 𝑘2[Cl2CHCH(OH)NHCl] + 𝑘3[Cl2CHCH(OH)NHCl][NH2Cl] + 𝑘5[Cl2CHCHO][NH2Cl] (7 )

371

The individual rate expressions for the formation of products DCAN, DCAM, N,2,2-TCAM, and

372

TCAL are: 𝑑[Cl2CHCN] 𝑑𝑡

= 𝑘2𝛼1𝐶𝑇,NCl ― 𝑘4[Cl2CHCN]


(8)

Page 19 of 34


𝑑[Cl2CHC(O)NH2]

= 𝑘4[Cl2CHCN]

(9)

= 𝑘3𝛼1𝛼0(𝐶𝑇,NCl)2

(10)

= 𝑘5[Cl2CHCHO]𝛼0𝐶𝑇,NCl

(11)

𝑑𝑡 𝑑[Cl2CHC(O)NHCl] 𝑑𝑡 𝑑[Cl3CCHO] 𝑑𝑡 373

Because excess aldehyde compared to monochloramine was used, reaction rate is 𝑘′5 = 𝑘5

374

[Cl2CHCHO]≅𝑘5[Cl2CHCHO]0 and eq 11 can be simplified to a pseudo-first order equation.

375

Experimental sets UV-1 and UV-2 (Table 1) were used to determine concentrations at time t of

376

monochloramine ([NH2Cl]𝑡), carbinolamine 2,2-dichloro-1-(chloroamino)ethanol (

377

[Cl2CHCH(OH)NHCl]) and N,2,2-TCAM ([Cl2CHC(O)NHCl]𝑡). These concentrations will be

378

used to determine reaction rate constants. For neutral pH condition (UV-1), DCAN hydrolysis to

379

DCAM was negligible and thus it was not included in the kinetic model. Reactions were

380

monitored at two wavelengths, 210 and 243 nm, and the measured absorbance is expressed as: 𝐴𝑏𝑠𝜆,𝑡 = (𝜀𝜆,NH2Cl + 𝜀𝜆,Cl2CHCH(OH)NHCl𝐾1[Cl2CHCHO]𝑜)[NH2Cl]𝑡 + 𝜀𝜆,Cl2CHC(O)NHCl[Cl2CHC(O)NHCl]𝑡 (12 )

381

where, 𝜀𝜆,NH2Cl, 𝜀𝜆,Cl2CHCH(OH)NHCl, and 𝜀𝜆,Cl2CHC(O)NHCl are the molar extinction coefficients

382

previously determined from the absorbance of the pure standards at varying concentration

383

levels (Table S1 of SI). Concentrations for [Cl2CHCH(OH)NHCl] and 𝐶𝑇,NCl at time t were

384

determined according to eqs 2 and 5.

385

DCAN and TCAL were not included in eq 12 because their individual absorbance values at the

386

concentration levels that were produced in the reaction were negligible compared to the



387

measured absorbance. Instead, DCAN and TCAL concentrations were determined by GC-MS

388

(GC-2) at the exact times samples were collected and analyzed with UV-Vis. To ensure samples

389

where taken at the same time for both method, reaction aliquots were quenched with sodium

390

thiosulfate.

391

Experimental data obtained from UV-Vis and GC-MS were fitted to the kinetic model to

392

determine the kinetic rate constants k2, k3, and k5. N,2,2-TCAM was quantified by GC-MS

393

indirectly as DCAM. N,2,2-TCAM was also quantified by UV-Vis and results showed good

394

agreement between both measurements as shown in Figure S10 of SI. The fitted rate constants

395

at neutral pH 7.8 were k2=(1.09±0.06)x10-5 s-1, k3=(4.87±0.25)x10-2 M-1s-1 and k5=(2.12±0.15)x10-

396

2

397

At pH >9, the hydrolysis of DCAN to DCAM was dominant and the formation of TCAL was not

398

significant. In this case, DCAN, DCAM and N,2,2-TCAM were the main products from the

399

reaction between DCAL and monochloramine. However, carbonate buffer caused an

400

interference that affected the UV-VIS data analysis. In future work, we will employ different

401

buffers and reaction conditions that will minimize its interference with our data analysis. This

402

will enhance the applicability of our kinetic model as a predictive tool for the formation of HANs

403

and HAMs at a wider pH range.

404

Figure 3 represents the mass balance of NH2Cl as a function of reaction time t. The initial NH2Cl

405

concentration is equal to the sum of the concentrations at time t of: NH2Cl, carbinolamine

406

(Cl2CHCH(OH)NHCl), the products trichloroacetaldehyde (Cl3CCHO), dichloroacetonitrile

407

(Cl2CHCN), and N-chloro-dichloroacetamide (Cl2CHC(O)NHCl):

M-1s-1 for 25oC.


Page 20 of 34

Page 21 of 34


[𝑁𝐻2𝐶𝑙]0 = [𝑁𝐻2𝐶𝑙]𝑡 + [𝐶𝑙2𝐶𝐻𝐶𝐻(𝑂𝐻)𝑁𝐻𝐶𝑙]𝑡 + [𝐶𝑙2𝐶𝐻𝐶𝑁]𝑡 + [𝐶𝑙2𝐶𝐻𝐶(𝑂)𝑁𝐻𝐶𝑙]𝑡 + [𝐶𝑙3𝐶𝐶𝐻𝑂]𝑡 (13) 408

and combining eq 13 with eq 5:

[𝑁𝐻2𝐶𝑙]0 = [𝐶𝑇,𝑁𝐶𝑙]𝑡 + [𝐶𝑙2𝐶𝐻𝐶𝑁]𝑡 + [𝐶𝑙2𝐶𝐻𝐶(𝑂)𝑁𝐻𝐶𝑙]𝑡 + [𝐶𝑙3𝐶𝐶𝐻𝑂]𝑡

(14)

409

Note that in Figure 3 data was not shown for carbinolamine because it was low.

410

Dichloroacetonitrile hydrolysis to dichloroacetamide

411

DCAN hydrolysis was found to be significant at high pH values. For this reason, DCAN’s

412

hydrolysis rates at different pH values were determined. The reaction was monitored at

413

wavelength 210 nm where the absorbance is equal to DCAN and DCAM concentration over

414

time (HR-1 in Table 1). Absorbance is expressed as 𝐴𝑏𝑠210,𝑡 ― 𝐴𝑏𝑠210,0 = (𝜀210,DCAM ― 𝜀210,DCAN)([DCAN]0 ― [DCAN]𝑡)

(15)

415

where, 𝐴𝑏𝑠210,0 is the absorbance of the DCAN sample at t=0, 𝐴𝑏𝑠210,𝑡 is the absorbance of

416

DCAN and DCAM at time t, and 𝜀210,DCAM and 𝜀210,DCAN are the molar extinction coefficients of

417

DCAM and DCAN that were previously determined (Table S1 of SI). [DCAN]0 and [DCAN]𝑡 are

418

the concentration of DCAN at t=0 and at time t.

419

The rate expression for DCAN hydrolysis is ―

𝑑[DCAN] ― = 𝑘4[DCAN] = {𝑘04 + 𝑘OH 4 [OH ]}[DCAN] 𝑑𝑡

(16)

420

The concentration of DCAN over time from each experiment at different pH values were fitted

421

to eq 16 and plotted the observed reaction rate k4 versus [OH ― ] as shown in Figure S11 of SI.



422

Results show a linear relationship between with a coefficient of determination R2=0.998 that

423

― indicates a base catalyzed reaction. Observed k4 values were fitted to 𝑘04 + 𝑘OH 4 [OH ] to obtain

424

-1 -1 o rate constants 𝑘04 = (2.86±0.90)x10-7 s-1 and 𝑘OH 4 =3.69±0.09 M s for 25 C. These rate constant

425

results are consistent and in good agreement with the hydrolysis rate constants of DCAN

426

-1 -1 o 52 The effect reported in literature (𝑘04=(1.78±0.35)x10-7 s-1 and 𝑘OH 4 =3.42±0.31 M s for 20 C).

427

of monochloramine on the hydrolysis of DCAN (HR-2) was tested and found not to be significant

428

(data not shown).

429

N,2,2-trichloroacetamide cytotoxicity

430

A mammalian cell cytotoxicity analysis was performed to determine the in vitro toxicity

431

characteristics of N,2,2-TCAM. After 72 h of exposure, N,2,2-TCAM induced a concentration-

432

response curve. The data were analyzed with an ANOVA test statistic with a Holm-Sidak

433

comparison test with the control group (1–β≥0.8, α=0.05). The lowest significant cytotoxic

434

concentration of N,2,2-TCAM was 250 μM. The concentration, which reduced 50% of the cell

435

density (LC50) compared to the negative control group, was estimated by nonlinear regression

436

analysis (Figure 5).

437

Among the N-HAM family, mammalian cell cytotoxicity of N,2,2-TCAM (LC50=3.90×10-4 M) was

438

found to be more toxic than N-chloroacetamide (LC50=1.78×10-3 M) but shown slightly less

439

potent than N,2-dichloroacetamide (LC50=2.56×10-4 M). In comparison with other HAMs, N,2,2-

440

TCAM was more toxic than dichloroacetamide (LC50=1.92×10-3 M) and trichloroacetamide

441

(LC50=2.05×10-3 M) but less potent than chloroacetamide (LC50=1.48×10-4 M).

442

Kinetic model validation and predominance of the aldehyde reaction pathway


Page 22 of 34

Page 23 of 34


443

The kinetic model and reaction rate constants determined in this study were used to predict

444

the formation of N,2,2-TCAM and DCAN from experiment GC-1. Quantified DCAL formation

445

and NH2Cl initial dose of 10 mg/L as Cl2 were used as inputs for the kinetic model. Modeled and

446

experimental data for a period of 5 days are plotted in Figure 4. Results show good agreement

447

between the measured DCAN-15N and DCAM-15N (indirect measurement for N,2,2-TCAM-15N)

448

and their prediction provided by the kinetic model. The model accounted for 81-99% of the

449

experimental data suggesting that these compounds predominantly formed through the

450

aldehyde reaction pathway proposed in this study. Additionally, these results also validated our

451

initial assumption that DCAL is produced from the reaction of NH2Cl with NOM in a water that

452

do not contain DCAL.

453

Although previous studies have shown that DCAN and DCAM are the most commonly found N-

454

DBP species among HANs and HAMs group in chloraminated drinking water, our study has

455

shown quantitatively for the first time that the aldehyde reaction pathway is a major pathway

456

for the formation of DCAN, N,2,2-TCAM and DCAM in a real water. Additionally, our kinetic

457

model could be applied as a useful tool to predict the formation of DCAN, N,2,2-TCAM and

458

DCAM under drinking water conditions. This reaction pathway could also extend to our

459

preceding work with other aldehydes.26, 27 Although the data was collected and the model was

460

based on one water, it could be applicable to other waters for which the water-specific

461

formation of DCAL from monochloramine has been determined, especially similar water

462

sources which have low ammonia concentration and low organic nitrogen concentration.

463

However, it is also important to point out that our model is not a universal tool for predicting

464

the formation of DCAN, N,2,2-TCAM and DCAM in any drinking water because reactions



465

between monochloramine and reactive intermediates (e.g., carbinolamines) of the aldehyde

466

pathways could take place with species such as for example nitrite and various forms of iron

467

and manganese not taken into account in our model, and thus discrepancies between predicted

468

and experimental formation of DCAN, N,2,2-TCAM and DCAM could be observed in waters

469

containing these reactive constituents.

470

Quenchers and Practical Implications

471

The use of quenchers, such as ascorbic acid and sodium thiosulfate, brings into question how

472

much HAMs and HANs are actually present in disinfected drinking water versus an artifact of

473

sample and preservation methods. For example, in occurrence studies, N,2,2-TCAM would

474

mistakenly be quantified as DCAM because grab samples will often use quenchers to reduce the

475

main disinfectant but they will also quench N,2,2-TCAM to DCAM. These new findings indicate

476

that drinking water consumers are predominantly exposed to N,2,2-TCAM, which was found to

477

be more toxic than DCAM and trichloroacetamide and has similar toxicity as other N-

478

haloacetamides tested in our preceding work. 12,13

479

Ammonium chloride, another commonly used quencher for free chlorine, will react with free

480

chlorine to form chloramines which will then continue to react with organic matter present in

481

samples to potentially form HANs and HAMs while being held for several days to weeks before

482

extraction. While quenchers might be appropriate for the detection and quantification of

483

certain DBPs it might not be appropriate for some N-DBPs. For this reason, sampling and

484

preservation methods used for DBP analysis should be carefully designed to minimize artifacts

485

caused by such methods.


Page 24 of 34

Page 25 of 34


486 487

ASSOCIATED CONTENT

488

Supporting Information

489

Chemical relaxation analysis to obtain equilibrium rate constant K1; GC-MS chromatograms and

490

mass spectra of reaction extracts; GC-API-MS chromatograms and mass spectra of N,2,2-TCAM

491

standard and reaction extracts; DCAN hydrolysis rate constant k4 at different pH conditions;

492

extinction molar coefficients of compounds analyzed in this study; and CHO cell cytotoxicity

493

data.

494 495

ACKNOWLEDGMENTS

496

The authors would like to acknowledge Furong Sun, Alex Ulanov, Anna Yang, and Shaoying Qi

497

from the University of Illinois at Urbana-Champaign for their help on GC-MS, GC-HRMS and 1H

498

NMR. We would also thank Amy Cuthbertson from the University of South Carolina for her

499

advice and help on analytical method development.

500 501



502

Page 26 of 34

Tables and Figures

503 504

Table 1. Experimental conditions and instrument used to monitor the studied reactions Experiment pHc

Reactant(s)

Instrument

GC-1

7.8 – 8.1

10 mg/L as Cl2 of 15NH2Cl in treated surface water before disinfection

GC/MS

GC-2

7.8

1 mM NH2Cl and 10 mM CT,aldehyde

GC/MS

GC-3

9.5


GC/MS

GC-4

5.5 – 7.0


GC/MS

UV-1

7.8


UV-VIS

UV-2

9.5


UV-VIS

HR-1

7.0 – 9.9

1 mM DCAN

UV-VIS

HR-2

7.5

1 mM DCAN and 0.4-1.2 mM NH2Cl

UV-VIS

SF-1

7.8

1 mM NH2Cl and 10-90 mM CT,aldehyde

Stopped flow

505 506 507 508 509


Page 27 of 34


Cl

k2 dichloroacetaldehyde (DCAL) Cl

O CH CH

+

Cl

Cl

NH2

- H2O

K1

Cl

OH

H CH C

N

k5

Cl

1,1-dichloro-2(chloroimino)ethane

Cl

Cl

HN

CH

Cl

Cl

trichloroacetaldehyde

Cl

(TCAL)

(DCAN) OHCl

O

k4

O CH

CH Cl

N

2,2-dichloroacetonitrile

NH2Cl

CH Cl

Cl

CH CH

k3 O

- HCl

Cl

2,2-dichloro-1(chloroamino)ethanol

H+

Cl

HN

Cl

N,2,2-trichloroacetamide

(N,2,2-TCAM)

Na2S2O3 quenched

Cl

NH2

2,2-dichloroacetamide

(DCAM)

510 511 512 513

Figure 1. Proposed formation pathway of 2,2-dichloroacetonitrile, N,2,2-trichloroacetamide and TCAL

514 515 516 517



518

519 520 521

Figure 2. Formation of 15N labelled and unlabeled DCAN and DCAM during chloramination of Bloomington water at pH 7.8 – 8.1.

522


Page 28 of 34

Page 29 of 34


523 524 525 526 527

Figure 3. Experimental and modeled concentrations of monochloramine, dichloroacetonitrile, N,2,2-trichloroacetamide and TCAL over time (UV-1 and GC-1). Symbols are experimental data and lines are kinetic model fitting curves. Experimental conditions: [NH2Cl] = 1 mM, [CT,Cl2CHCHO] = 10 mM, total phosphate buffer 20 mM, pH 7.8 ± 0.1, µ = 0.1 M, Temp = 25 ± 0.1 oC.

528 529 530



531 532 533 534 535

Figure 4. Experimental (GC-1) and modeled concentrations of dichloroacetonitrile (DCAN) and dichloroacetamide (DCAM) over time. Symbols are experimental data and lines are the simulation from the kinetic model.


Page 30 of 34

Page 31 of 34


538 539 540 541 542 543 544 545 546 547 548 549 550 551 552

CHO Cell Cytotoxicity: Mean Cell Density as the Percent of the Negative Control (±SE)

536 537

100 LC50 = 389.86 µM r2 = 0.99

80

60

40

20

0 0

200

400

600

800

1000

N,2,2-Trichloroacetamide (µM)

553 554 555 556

Figure 5. N,2,2-trichloroacetamide concentration response curve and regression curve for chronic CHO cell cytotoxicity. The lowest concentration that induced a significant reduction in cell viability was 250 µM.

557



558 559

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Predominant N-haloacetamide and haloacetonitrile formation in

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