Identification, formation and control of polar brominated disinfection byproducts during cooking with edible salt, organic matter and simulated tap water

Journal Pre-proof Identification, formation and control of polar brominated disinfection byproducts during cooking with edible salt, organic matter and simulated tap water Dan Zhang, Yun Wu, Xiangru Zhang, Wenbin Li, Yan Li, Aimin Li, Yang Pan PII:

S0043-1354(20)30062-2

DOI:

https://doi.org/10.1016/j.watres.2020.115526

Reference:

WR 115526

To appear in:

Water Research

Received Date: 9 November 2019 Revised Date:

15 January 2020

Accepted Date: 19 January 2020

Please cite this article as: Zhang, D., Wu, Y., Zhang, X., Li, W., Li, Y., Li, A., Pan, Y., Identification, formation and control of polar brominated disinfection byproducts during cooking with edible salt, organic matter and simulated tap water, Water Research (2020), doi: https://doi.org/10.1016/ j.watres.2020.115526. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Graphical abstract

1 2 3 4 5

Identification, formation and control of polar brominated disinfection byproducts during cooking with edible salt, organic matter and simulated tap water Dan Zhang a, Yun Wu a, Xiangru Zhang b, Wenbin Li a, Yan Li a, Aimin Li a, Yang Pan a,*

6 7 8 9 10 11 12 13 14 15 16 17 18 19

a

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China b Department of Civil and Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, China *Corresponding author e-mail: [email protected]

Abstract Edible salt is essential to the health of humans and serves as a seasoning universally. Besides chloride, edible salt also contains other anions such as bromide, fluoride, sulfate, and carbonate due to incomplete removal during raw salt refinement. In a household cooking (e.g., soup making) process, a chlorine/monochloramine residual in tap water could react with bromide in edible salt and organic matter in food (e.g., rice, wheat) to form numerous brominated

21

disinfection byproducts (Br-DBPs) at significant levels, which might induce adverse health effects to human beings. In this study, we solicited 20 edible salts of different types (i.e., sea salts,

22

well and rock salts, lake salts, and bamboo salts) from nine countries and determined their

23

bromide levels to be 67–375 mg/kg, with an average level of 173 mg/kg. A total of 25 polar

24

Br-DBPs were detected and identified with structures/formulae in cooking water samples using ultra performance liquid chromatography/electrospray ionization-triple quadruple mass

20

25 26 27 28 29 30 31 32 33

spectrometry (UPLC/ESI-tqMS) and high-resolution mass spectrometry. Effects of cooking conditions (e.g., disinfectant type and level, edible salt dose, organic matter type and dose, sequence and time interval of adding organic matter and salt, etc.) on the formation of polar Br-DBPs were investigated, and optimized cooking conditions with minimized formation of polar Br-DBPs were determined. Further aided with an Hep G2 cell cytotoxicity assay, it was found that the overall cytotoxicity of chlorinated and chloraminated cooking water samples prepared after cooking condition optimization was reduced by 57% and 22%, respectively, compared with those prepared before cooking condition optimization.

34 35

Keywords: tap water; disinfection byproducts; edible salt; cytotoxicity; cooking

36

1

37

1. Introduction

38

Chlorine has been widely used as a disinfectant for the 20th century due to its relatively low

39

cost and high performance in disinfection. However, many drinking water utilities have switched

40

to combinations of primary disinfectants with chloramines as secondary disinfectants to meet

41

more stringent regulations of disinfection byproducts (DBPs) (McGuire, 2006; Dotson et al.,

42

2012). In a drinking water supply system, maintaining a disinfectant residual (usually a chlorine

43

or monochloramine residual) provides an effective barrier against regrowth/contamination of

44

microorganisms (Kumpel and Nelson, 2014; Li and Mitch, 2018; Li et al., 2019). The U.S.

45

Environmental Protection Agency (EPA) guidelines regulated that either chlorine or chloramines

46

must be maintained and not exceed 4.0 mg/L as Cl2 in the drinking water distribution systems

47

(U.S. EPA, 2007). In China, the levels of residual chlorine and monochloramine in the end of

48

pipelines should be maintained at 0.05–4.0 and 0.05–3.0 mg/L as Cl2, respectively (GB

49

5749-2006).

50

Edible salt is essential to the health of humans and serves as a seasoning universally. Raw

51

salt for edible salt production comes from brine and land salt. Brine, with seawater as the

52

predominant source, is water containing a high concentration of salt. Land salt mainly includes

53

rock salt, well salt, and lake salt, which originally comes from the sea, and due to billions of

54

years of crustal movement, the salt of the sea has remained on the land or in the stratum. Either

55

in natural brine or land salt, various anions such as bromide, fluoride, sulfate, and carbonate are

56

coexisting with chloride (the main component of edible salt), which are hard to be completely

57

removed during raw salt refinement. Previous studies have shown that, bromide presents in

58

natural waters (e.g., reservoirs, surface waters, ground waters) at levels of several µg/L to a few

59

mg/L, sea water contains 60–70 mg/L of bromide, and the bromide content in reagent grade

2

60

sodium chloride (NaCl) can be up to 100 mg per kg of salt (Xie and Rechow, 1996; Magazinovic

61

et al., 2004), suggesting that appreciable levels of bromide should also be present in edible salt.

62

Mishra et al. (2001) reported that the bromide contents in two Indian edible salts were 152 and

63

336 mg per kg of salt. Reddy-Noone et al. (2007) also found that the bromide levels in 10

64

commercial edible salts were 86–306 mg per kg of salt. Even so, concerns on bromide content in

65

edible salt have been quite limited, probably because of the relatively low toxicity of bromide on

66

mammals (Sangster et al., 1983) as well as the slow individual edible salt consumption rate

67

(suggested to be no more than 3.75–5.75 g per day on age) (U. S. DA and HHS, 2010). Food and

68

Agriculture Organization (FAO)/World Health Organization (WHO) has given an Acceptable

69

Daily Intake (ADI) value of bromide at 1.0 mg per kg body weight for humans (FAO/WHO,

70

1967) (i.e., an adult with a body weight of 70 kg might intake up to 70 mg of bromide per day),

71

indicating that the bromide intake through edible salt consumption was expected to be far below

72

the ADI value.

73

Although bromide is considered to be benign by itself in edible salt, its role playing in a

74

cooking process might not be neglected. During cooking, the bromide in edible salt may react

75

with the chlorine/monochloramine residual in tap water to form hypobromous acid

76

(HOBr)/monobromamine (NH2Br)/bromochloramine (NHBrCl), which would react with the

77

food organic matter (e.g., rice/wheat flour) or with the natural organic matter in tap water to form

78

brominated DBPs (Br-DBPs) (Cowman and Singer, 1996; Kristiana et al., 2009; Zhai et al., 2014;

79

Zhu and Zhang, 2016). The bromide content in the Indian edible salt was up to 336 mg per kg of

80

salt. Applying 1 g of such salt in 1 L of tap water (a relatively low dose according to human salty

81

acuity (Mitchell et al., 2013)) would produce water with a bromide concentration of 336 µg/L,

82

that is much higher than the average bromide level (i.e., 109 µg/L) in drinking water sources in

3

83

the U.S.

(Richardson

et al.,

2008)

and

the bromide

level

(i.e., 55

µg/L)

in

84

human-activity-impacted ground and surface waters in Switzerland (Soltermann et al., 2016).

85

Accordingly, Br-DBPs are expected to form at significant levels during cooking with edible salt.

86

Relative to drinking water Br-DBPs that have been studied for decades (Zhai and Zhang, 2011;

87

Zhai et al., 2014; Zhang and Yang, 2018), cooking water Br-DBPs are quite unfamiliar to

88

environmental researchers, and the only studies about this were focusing on formation and

89

control of trihalomethanes and haloacetonitriles under household water treatment processes and

90

simulated cooking conditions (Yan et al., 2016; Ma et al., 2017; Shi et al., 2017).

91

Accordingly, we designed this study to determine bromide contents in different types of

92

edible salts around the world, to disclose whole pictures of polar Br-DBPs generated in cooking

93

with edible salt and simulated chlorinated and chloraminated tap water, to explore formation of

94

polar Br-DBPs under different cooking conditions, and to evaluate the mixture cytotoxicity of

95

cooking water samples before/after cooking condition optimization with an Hep G2 cell

96

cytotoxicity assay.

97 98

2. Materials and methods

99

2.1. Materials

100

Suwannee River natural organic matter (SRNOM, 2R101N) was provided by the

101

International Humic Substances Society. Rather than purchasing only locally available edible

102

salts, we solicited salts from friends and colleagues from different parts of the world. A total of

103

20 edible salts (coded as A–T) were collected from nine countries (i.e., Australia, China,

104

Germany, Italy, Japan, Netherlands, Thailand, U.K., and U.S.), of which A and B were lake salts,

105

C was a bamboo salt, D−H were well and rock salts, and I−T were sea salts. Organic matter (i.e.,

4

106

rice flour, wheat flour, corn starch, and sweet potato starch) was purchased from a common local

107

supermarket. Soluble starch (reagent grade), D-(+)-glucose (≥99.5%), D-(+)-maltose

108

monohydrate (≥95%), and dimethyl sulfoxide (DMSO) (≥99.5%) were purchased from

109

Sigma-Aldrich. A stock solution of sodium hypochlorite (NaOCl) was purchased from Tokyo

110

Chemical Industry and measured according to the DPD ferrous titrimetric method (APHA et al.,

111

2012). Monochloramine (NH2Cl) was freshly prepared by reacting NaOCl and NH4Cl solutions

112

in a chlorine-to-ammonia molar ratio of 0.8 just before use. The human hepatoma cells Hep G2,

113

cell counting kit-8 (CCK-8), and phosphate buffered saline (PBS) were supplied by KeyGEN

114

Biotech (China). Dulbecco’s Modified Eagles Medium (DMEM) (containing 10% fetal bovine

115

serum) was offered by Thermo Scientific.

116 117

2.2. Determination of bromide contents in edible salts

118

The bromide content in each of the 20 edible salts was determined using a

119

spectrophotometric method according to a previous study (Chiu and Eubanks, 1989). Briefly, a

120

3.5 mL portion of an edible salt solution (prepared by dissolving 2 g of the edible salt in

121

ultrapure water to 1 L) was added to a 10 mL volumetric flask containing 1 mL of a

122

NaH2PO4·2H2O buffer solution (pH 6.3). The mixture in the flask was dosed with 0.25 mL of a

123

freshly prepared NaOCl solution (1 mol/L NaOCl in 0.1 mol/L NaOH), and immersed in a

124

boiling water bath for 10 min. The excessive NaOCl was destroyed to be sodium chloride and

125

sodium bicarbonate with sodium formate and the mixture was continued to be heated in the

126

boiling water bath for another 5 min. Then, the mixture was cooled to ~21 ºC with an ice bath,

127

added with 4.5 mL of 2 mol/L HBr, and diluted to 10 mL with ultrapure water. The ultraviolet

128

(UV) absorbance of the solution at 267 nm against ultrapure water was measured using a

5

129

Shimadzu UV2550 spectrophotometer. For each edible salt solution, triplicate aliquots were

130

analyzed to obtain an average bromide concentration. Calibration standard solutions were

131

prepared using KBr at concentrations from 0.02 to 0.50 mg/L as Br–.

132 133

2.3. Preparation of simulated cooking water

134

Preparation of simulated raw water was carried out with ultrapure water containing 3 mg/L

135

SRNOM as C and 90 mg/L NaHCO3 as CaCO3. To prepare simulated tap water, the simulated

136

raw water was chlorinated/chloraminated at room temperature with aimed disinfectant residual

137

levels of 1.0, 2.0, 3.0, 4.0 mg/L as Cl2 after a contact time of 12 h. For preparation of simulated

138

cooking water, a 1 L portion of the simulated tap water was poured to a 2-L glass beaker and

139

dosed with 0.5–4.0 g/L of edible salt (bromide content: 200 mg per kg salt) and 0–0.4 g/L of rice

140

flour. The mixture in the beaker was heated on a stirrer/ hot plate (PC-620D, CORNING) with an

141

aluminum foil cover and a thermometer to different temperatures (20–100 ºC) in 20 min, and

142

kept cooking at a given temperature for 5–60 min. During cooking, different sequences and

143

intervals (0–20 min) of adding the simulated tap water, edible salt, and rice flour were conducted.

144

Immediately after cooking, the simulated cooking water sample was brought back to ~ 20 ºC

145

with an ice bath. The disinfectant residual in the sample was quantified and quenched with

146

Na2SO3.

147 148

2.4. Pretreatment of simulated cooking water

149

The simulated cooking water samples were pretreated based on previous studies (Zhang et al.,

150

2008; Pan et al., 2016a). The process of pretreatment was detailed in the Supporting Information

151

(SI). For the Hep G2 cell cytotoxicity assay, the organic layer was dried under a gentle nitrogen

6

152

gas flow, and re-dissolved in 100 µL of DMSO (105× concentration of the original cooking water

153

sample).

154 155

2.5. (UPLC/)ESI-tqMS analysis

156

The pretreated samples were analyzed by a Waters Acquity I-Class UPLC system coupled to

157

an Xevo TQ-S Micro ESI-tqMS (UPLC/ESI-tqMS). The specific MS parameters were in SI. By

158

setting ESI-tqMS PIS of m/z 79/81, nearly all electrospray-ionizable Br-DBPs should be detected

159

(Zhang et al., 2008; Yang et al., 2019). Aided with the UPLC system, the UPLC/ESI-tqMS was

160

achieved to provide multiple reaction monitoring (MRM) and product ion scan analyses, via

161

which structural information of a molecular ion detected by the PIS could be obtained.

162

Parameters of the UPLC were detailed in SI.

163 164

2.6. High-Resolution MS analysis

165

For some unknown detected ion clusters, a high resolution hybrid quadrupole time-of-flight

166

mass spectrometer (AB SCIEX 5600) was used to obtain their exact m/z values for formulae

167

determination. The instrument parameters were set as follows: full scan mode, ESI negative, ion

168

spray voltage −4500 V, curtain gas pressure 35 psi, ion source gas 1 pressure 55 psi, ion source

169

gas 2 pressure 55 psi, temperature 550 ºC.

170 171

2.7. Cytotoxicity assay with Hep G2 Cells

172

An Hep G2 cell cytotoxicity assay was performed to compare the overall cytotoxicity of

173

simulated cooking water samples prepared before and after cooking condition optimization.

174

Procedures of the assay were basically followed previous studies and described specifically in SI

7

175

(Marabini et al., 2006; Gong et al., 2017). Cell viability was measured by relative absorbance of

176

six replicates for each concentrated sample. The 50% maximal effect concentration (EC50) value

177

was calculated according to the curve of cell viability versus sample concentration factor, which

178

was plotted with SigmaPlot 12.5 (Systat Software Inc., San Jose, CA). The coefficient of

179

determination (r2), in regression analysis, evaluates the degree of correlation between the sets of

180

data. For multiple comparisons among treatment and control groups, a one-way analysis of

181

variance (ANOVA, followed by Holm-Sidak Multiple Comparisons) test was performed.

182

Differences were considered statistically significant at P < 0.05.

183 184

3. Results and discussion

185

3.1. Occurrence of bromide in edible salt

186

As shown in Fig. 1, bromide was ubiquitously detected in the 20 edible salts at levels from

187

67 to 375 mg per kg of salt, with an average level of 173 mg per kg of salt. The bromide contents

188

in different sources of edible salts varied substantially: the sea salts contained the highest levels

189

of bromide (averagely 212 mg per kg salt), followed by the well and rock salts (averagely 137

190

mg per kg salt), whereas the lake salts and bamboo salt contained relatively low levels of

191

bromide (below 100 mg per kg of salt). During cooking, applying 1 g of an edible salt containing

192

the average level of bromide (i.e., 173 mg per kg of salt) in 1-L tap water would produce water

193

with a bromide concentration of 173 µg/L, that was much higher than the average bromide

194

concentration (i.e., 109 µg/L) in drinking water sources in the U.S. (Richardson et al., 2008) as

195

well as the bromide level (i.e., 55 µg/L) in human-activity-impacted ground and surface waters

196

in Switzerland (Soltermann et al., 2016).

197

8

198

3.2. Detection of new/unknown polar Br-DBPs in simulated cooking water

199

A representative chlorinated cooking water sample (prepared using simulated tap water with

200

3.0 mg/L NaOCl as Cl2, added simultaneously with 0.2 g/L rice flour and 2 g/L edible salt,

201

heated to 100 ºC in 20 min and cooked at 100 ºC for 30 min) and a representative chloraminated

202

cooking water sample (prepared using simulated tap water with 3.0 mg/L NH2Cl as Cl2, added

203

simultaneously with 0.2 g/L rice flour and 2 g/L edible salt, heated to 100 ºC in 20 min and

204

cooked at 100 ºC for 30 min) were used for detection and identification of cooking water polar

205

Br-DBPs. As shown in Fig. 2, numerous new/unknown polar Br-DBPs were detected in the two

206

cooking water samples at various levels. Many high-molecular-weight (m/z > 300) polar

207

Br-DBPs were detected in the two representative cooking samples (especially in the

208

chloraminated sample). Further aided with the UPLC, some overlapping bromine-containing

209

homologues were differentiated and 24 ion clusters were detected as summarized in Table S1.

210

Among the 24 ion clusters, m/z 293/295/297 showed up at two retention times, indicating that it

211

corresponded to two Br-DBPs. Accordingly, a total of 25 polar Br-DBPs were detected in the

212

two representative cooking samples: 11 were detected in the chlorinated cooking water sample,

213

and 17 were detected in the chloraminated cooking water sample. Among these detected polar

214

Br-DBPs, 11 have been previously identified in drinking water (Pan and Zhang, 2013; Huang et

215

al., 2018), whereas it is the first time that they were reported as cooking water polar Br-DBPs.

216

All the new/unknown cooking water polar Br-DBPs were either analyzed with the

217

UPLC/ESI-tqMS MRM and product ion scans to obtain structural information or analyzed with

218

the high-resolution MS to obtain accurate m/z values.

219 220

3.3. Structure/formula identification of cooking water polar Br-DBPs

9

221

As displayed in Fig. 2, the isotopic abundance ratio of ion cluster m/z 277/279/281 was 1:2:1,

222

showing that this compound should contain 2 Br. As shown in Fig. 3a–f, the retention time (RT)

223

and product ion scan spectra of this compound suggested that this compound was probably

224

3,5-dibromo-4-hydroxybenzaldehyde (Huang et al., 2018). Accordingly, we proved the

225

compound to be 3,5-dibromo-4-hydroxybenzaldehyde through standard compound spiking.

226

Similarly, ion clusters 127/129/131, 171/173/175, 193/195, 215/127/129, 233/235/237,

227

249/251/253, 283/285/287/289, 293/295/297, and 327/329/331/333 were confirmed to be

228

chlorobromoacetic acid

229

dibromoacetic acid, 3-bromo-5-chloro-4-hydroxybenzaldehyde, 3-bromo-5-chlorosalicylic acid,

230

2,6-dibromo-4-chlorophenol, 3,5-dibromo-4-hydroxybenzoic acid, 3,5-dibromosalicylic acid,

231

and 2,4,6-tribromophenol in the simulated cooking water samples, respectively, as displayed in

232

Table S1 (Figs. S1–S10).

(decarboxylated),

chlorobromoacetic acid,

bromomaletic acid,

233

For the other 14 polar Br-DBPs, formulae identification was performed using high-resolution

234

MS analyses, and identification of ion cluster m/z 533/535/537/539 was shown here as an

235

example. The isotopic abundance ratio of ion cluster m/z 533/535/537/539 was 1:3:3:1 (Fig. 2),

236

indicating that the compound should contain 3 Br. The exact m/z values of the ion cluster were

237

determined to be 532.9894/534.9877/536.9857/538.9836 (Fig. 3g), which should correspond to

238

C18H32O3Br3– (with an m/z value error of –2.4 ppm). Similarly, ion clusters m/z 257/259/261/263,

239

301/303/305/307, 345/347/349/351, 377/379, 391/393, 407/409, 413/415/417, 427/429/431,

240

453/455/457, 471/473/475, 489/491/493/495, 507/509/511/513, and 551/553/555 were identified

241

to

242

C18H35O3BrCl–, C18H33O4BrCl–, C18H31O3Br2–, C18H33O4Br2–, C18H32O3Br2Cl–, C18H34O4Br2Cl–,

243

and C21H29O7Br2–, respectively (SI and Figs. S11–S23). Among these ion clusters, m/z

be

C5O3BrCl2–,

C5O3Br2Cl–,

C5O3Br3–,

C18H34O3Br–,

C18H32O4Br–,

C18H32O5Br–,

10

244

257/259/261/263, 301/303/305/307, and 345/347/349/351 have been reported to be a group of

245

emerging DBPs in drinking water (Zhai and Zhang, 2011; Gonsior et al., 2014), and their

246

structures were identified to be trihalo-5-hydroxy-4-cyclopentene-1,3-diones (Pan et al., 2016b).

247

According to the molecular formulae of the other 11 ion clusters, they were suggested to be a

248

group of interrelated polar Br-DBPs with similar carbon skeletons and chain structures. Since

249

formulae of ion clusters m/z 413/415/417 and 507/509/511/513 were saturated, we speculated

250

their

251

CH3(CH2)10(CHOH)3(CHBr)2CHClCH2O–, respectively. Accordingly, structures of ion clusters

252

m/z 377/379, 391/393, 407/409, 427/429/431, 453/455/457, 471/473/475, 489/491/493/495,

253

533/535/537/539, and 551/553/555 were further proposed as summarized in Table S1. These ion

254

clusters could be converted to one another via oxidation, substitution, hydrolysis, elimination,

255

addition, and dehydration, and their specific transformation pathways were depicted in Fig. 4.

structures

to

be

CH3(CH2)12(CHOH)2CHClCHBrCH2O–

and

256 257

3.4. Formation of polar Br-DBPs in simulated cooking water

258

3.4.1. Effects of disinfectant type and level, and edible salt dose

259

Notably, as displayed in Fig. S24, high-molecular-weight polar Br-DBPs (m/z > 300) were

260

preferably generated in chloraminated samples. According to previous studies, HOCl and HOBr

261

are strong oxidants that are responsible for the formation and decomposition of polar Br-DBPs

262

during chlorination, whereas NHBrCl and NH2Br are main oxidants in chloramination that are

263

too weak to cause decomposition of Br-DBPs (Zhai et al., 2014; Zhu and Zhang, 2016).

264

Therefore, high-molecular-weight polar Br-DBPs could accumulate in chloramination but

265

quickly decompose in chlorination. Furthermore, the highest formation of polar Br-DBPs was

266

detected at the disinfectant dose of 3 mg/L as Cl2 in chlorinated and chloraminated samples. As

11

267

presented in Fig. S25, with the increasing dose of edible salt from 0.5 to 4 g/L, formation of

268

polar Br-DBPs arrived maximum at the dose of 2 g/L in chlorinated and chloraminated samples.

269

Since high-molecular-weight polar Br-DBPs might be intermediate compounds, they could be

270

degraded under a relatively high bromide concentration (Pan and Zhang, 2013).

271 272

3.4.2. Effects of type and dose of organic matter

273

As shown in Fig. 5a–f, levels and species of polar Br-DBPs generated in the chlor(am)inated

274

cooking water samples made with only SRNOM and only rice flour were quite different:

275

low-molecular-weight polar Br-DBPs were at higher levels in samples made with only SRNOM,

276

while high-molecular-weight polar Br-DBPs were at higher levels in samples made with only

277

rice flour. This might be due to relatively lower molecular weights of DBP precursors in

278

SRNOM than in rice flour (Kwon et al., 2005; Jobling et al., 2002). Polar Br-DBPs generated in

279

the sample made with both SRNOM and rice flour were a compromise and combination of those

280

formed in the samples made with only SRNOM and only rice flour.

281

Besides rice flour, the formation of polar Br-DBPs in cooking water samples prepared with

282

other three types of organic matter (wheat flour, corn starch, and sweet potato starch) was also

283

investigated. As shown in Fig. S26, similar species but different levels of polar Br-DBPs were

284

formed in these samples. The four types of organic matter are mainly composed of carbohydrates

285

(~80%), proteins (~6%), fat (~1%), moisture, ash, and vitamins (Shih and Daigle, 1997;

286

Bhattacharya et al., 1999; Okoye et al., 2008; Yadav et al., 2006). Amino acids (hydrolysates of

287

proteins) and vitamins were confirmed to be major precursors of a few DBPs (Krasner et al.,

288

2009; Zhang et al., 2019). Carbohydrates are predominant components of these organic matters,

289

and can be hydrolyzed into monosaccharides (e.g., glucose), disaccharides (e.g., maltose), and

12

290

polysaccharides (e.g., starch). As displayed by Fig. 5g–n, polar Br-DBPs were also generated in

291

cooking water samples made with only starch, only maltose, and only glucose, suggesting that

292

these carbohydrates were important precursors of cooking water polar Br-DBPs. This is because

293

that hydroxyl groups on these hydrolysates could be oxidized to aldehydes, ketones and

294

carboxylic groups accompanying glucosidic bonds cleavage by disinfectants during cooking

295

(Hebeish et al., 1989; Sangseethong et al., 2010). In addition, the highest formation of polar

296

Br-DBPs in both chlorinated and chloraminated cooking water samples was detected at a rice

297

flour dose of 0.2 g/L (Fig. S27).

298 299

3.4.3. Effects of sequence and time interval of adding edible salt and rice flour

300

Formation of polar Br-DBPs in chlor(am)inated cooking water samples prepared with three

301

different processes were compared: (i) adding edible salt 5 min earlier than adding rice flour, (ii)

302

adding edible salt and rice flour together, and (iii) adding rice flour 5 min earlier than edible salt.

303

In the chlorinated cooking water samples, polar Br-DBPs were most preferentially generated in

304

process (i), followed by process (ii) and (iii) (Fig. 6a–c). This is because in process (i), the

305

reaction rates of HOCl/OCl– and Br– (k=1.4×102 M–1s–1) as well as HOBr/OBr– and natural

306

organic matter (NOM) (k=1.0×106 M–1s–1) were much faster than that of HOCl/OCl– and NOM

307

(k=41 M–1s–1), leading to the accumulation of HOBr/OBr– and the enhanced formation of

308

Br-DBPs. Different from that in the chlorinated cooking water samples, polar Br-DBPs’

309

formation in the chloraminated samples was the highest in process (iii), followed by process (ii)

310

and (i) (Fig. 6d–f). It is because that addition of bromide before rice flour initiated reactions

311

between NH2Cl and Br– to form NH2Br, NHBr2 and NHBrCl, which could auto-decompose to

312

cause loss of chloramines (Zhu and Zhang, 2016). On the contrary, addition of rice flour before

13

313

edible salt allowed formation of polar Cl-DBPs, which could further react with bromide in edible

314

salt to form Br-DBPs. Notably, it was found that increasing the time interval between the

315

addition of edible salt and rice flour in the chlorinated samples and increasing the time interval

316

between the addition of rice flour and edible salt in the chloraminated samples from 0 to 20 min

317

decreased polar Br-DBPs’ formation by 64% and 47%, respectively (Fig. 6g–n).

318 319

3.4.4. Effects of cooking temperature and time length

320

As shown in Fig. S28, when cooking temperature was elevated from 20 to 100 ºC, polar

321

Br-DBPs’ formation gradually decreased in the chlorinated sample, whereas presented a first

322

rising and then falling trend in the chloraminated sample. Different from chlorine (depleted at 80

323

ºC), monochloramine persisted during heating from 20 to 80 ºC, and thus polar Br-DBPs’

324

formation in the chloraminated sample was still in process at a rate higher than their degradation

325

rate. Furthermore, with cooking time length accumulating from 5 to 60 min at 100 ºC, the levels

326

of the polar Br-DBPs kept relatively stable in chlorinated samples, but substantially decreased in

327

chloraminated samples (Fig. S29). This is because that decomposition of polar Br-DBPs in the

328

chlorinated sample might occur before the temperature reaching 100 ºC, and thus the decrease of

329

polar Br-DBPs in the chloraminated samples was observed after depletion of monochloramine.

330 331

3.5. Comparative cytotoxicity of simulated cooking water

332

According to the aforementioned experiment results, another two simulated cooking water

333

samples were prepared with the optimized cooking condition as follows: cooking time length, 60

334

min; cooking temperature, 100 ºC; addition sequence and interval, adding rice flour 20 min

335

earlier than edible salt (chlorination) or adding edible salt 20 min earlier than rice flour

14

336

(chloramination). Cytotoxicity of the cooking water samples prepared before and after

337

optimization of cooking conditions was evaluated with an Hep G2 cell cytotoxicity assay, which

338

has been performed in examining comparative cytotoxicity of a few DBPs and real water

339

samples (Gong et al., 2017; Yin et al., 2020). Fig. 7 displays the concentration factor–response

340

curve for the cytotoxicity of chlor(am)inated cooking water samples prepared before and after

341

cooking condition optimization. For each curve, a regression analysis was performed to

342

determine its r2 and EC50 values. ANOVA test results showed that all the analyses were

343

statistically significant with P ≤ 0.001. For the chlorinated cooking water sample, optimization of

344

cooking conditions substantially reduced the cytotoxicity by 57% (with EC50 increasing from

345

125× to 288×). Similarly, cooking condition optimization reduced the cytotoxicity of the

346

chloraminated cooking water sample by 22% (with EC50 increasing from 220× to 282×). It was

347

noted that before cooking condition optimization, the cytotoxicity of the chlorinated cooking

348

water sample was higher than that of the chloraminated cooking water sample, which should

349

mainly result from the higher total organic halogen level (positively correlated with the toxicity

350

of a disinfected water sample) in the chlorinated cooking water sample (Han and Zhang, 2018).

351

Moreover, as shown in Fig. 2, in the chlorinated cooking water sample, the dominating Br-DBPs

352

were of low-molecular-weights, whereas high-molecular-weight Br-DBPs were predominant in

353

the chloraminated cooking water sample. Previous studies have pointed out that the toxicity

354

effects of organic compounds were achieved by narcosis of cell membrane followed by

355

transferring across the cell membrane and reacting with cellular macromolecules and organelles,

356

or accumulation in the membrane that hinders its normal function (Plewa et al., 2002; Schultz et

357

al., 2003; Liu and Zhang, 2014). Therefore, low-molecular-weight Br-DBPs might be of higher

358

ability to transfer across the cell membrane and react with cellular macromolecules and

15

359

organelles more easily, and thus exhibited higher cytotoxicity. Similar results were also found

360

during chlorination of iopamidol that low-molecular-weight DBPs were of higher cytotoxicity

361

than high-molecular-weight DBPs (Wendel et al., 2016).

362 363

4. Conclusions

364

Our study proved that bromide was ubiquitously present at significant levels in different

365

types of edible salts, especially in sea salts. Since bromide in edible salts was considered to be

366

safe at most cases, it did not attract much attention from researchers. However, problems

367

occurred when bromide in edible salts react with disinfectants in tap water and organic matter in

368

food in a household cooking process, during which formation of polar Br-DBPs was detected

369

and identified. With regard to the adverse health effects of polar Br-DBPs, practical protocols to

370

minimize their formation during cooking were raised, e.g., a comparatively long time interval

371

between the addition of rice flour and edible salt for chlorinated tap water, a comparatively high

372

cooking temperature, and a comparatively long cooking time length. Under optimized cooking

373

conditions, the overall cytotoxicity of the chlor(am)inated cooking water samples could be

374

substantially reduced. Last but not the least, with possible presence of iodine fortifiers in edible

375

salts, mixed Br-/I-DBPs or even mixed Cl-/Br-/I-DBPs might also be generated during cooking

376

process, and thus future studies are suggested to focus on a more comprehensive evaluation of

377

halogenated DBPs formed under various cooking conditions.

378 379

Acknowledgments

380

We acknowledge the research grants from National Key R&D Program of China (No.

381

2016YFE0112300), National Natural Science Foundation of China (No. 51778280), Natural

16

382

Science Foundation of Jiangsu Province, China (No. BK20180058), and Fundamental Research

383

Funds for the Central Universities.

384 385

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22

T S R Q P O N M L K J I H G F E D

lake salt bamboo salt well and rock salt sea salt

C B A 0

100 200 300 bromide content (mg per kg of salt）

400

Fig. 1. Bromide contents in 20 edible salts (n=3).

Br2CHCOO−

Br

COO-

C C H

HOOC

ClBrCHCOO−

Confirmed/ Proposed structures/formulae

171.0

193.1

x0.1

x0.2 321.3

259.0

375.3 409.4

153.2

x0.1

173.0

(b)

217.0 279.0 261.0

%

0 100

Br Cl

x0.0 215.0

(a)

%

100

OBr

333.3

393.5

153.2

Br2CHCOO−

200

250

300

350

400

Cl

x3

x0.1

409.2

450

465.2

497.3

500

550

m/z 600

O -

O

209.3

x5

(c)

553.1

455.2 471.2 489.2 265.3 302.9 321.2 391.3 409.3

0 x3 100

x5

%

(d)

0 100

523.7

Cl Br O

%

100

150

ClBrCHCOO−

Confirmed/ Proposed structures/formulae

0 100

440.5 476.7

304.9 153.2

150

200

250

377.4 349.0

225.1 291.4

300

350

555.0

457.2 473.2 511.0 411.3

400

450

500

550

m/z 600

Fig. 2. (a,b) ESI-tqMS PIS spectra of m/z 79 and 81 of the representative chlorinated cooking water sample, respectively; (c,d) ESI-tqMS PIS spectra of m/z 79 and 81 of the representative chloraminated cooking water sample, respectively. The y-axes of charts (a,b) and (c,d) were on the same scales with maximum intensities of 6.97×107 and 6.05×108, respectively.

3.27

%

100 (a) 0

5.00

%

100 (b) 0

%

15.00

10.00

15.00

2.67

5.00 3.83

100 (c) 0

10.00

3.84

2.68

5.00

Time 15.00

10.00 –80(H79Br)

78.9

100 (d)

x10

– 28 (CO)

277.0

%

168.9

196.8

–82(H81Br) – 28 (CO)

%

80.9

– 44 (CO2)

278.9

x10 120.9

169.0

196.9

0

235.2 –82(H81Br)

– 28 (CO)

80.9

100 (f) 50

– 44 (CO2)

x10

280.9

136.9 171.0

% 0

258.9

107.0

0

100 (e)

– 44 (CO2)

100

150

199.1

200

237.0

250

m/z 300

Fig. 3. (a–c) UPLC/ESI-tqMS MRM (277→79, 279→79/81, 281→81) chromatograms of 3,5-dibromo-4-hydroxybenzaldehyde, the representative chlorinated cooking water sample, the cooking water sample spiked with 3,5-dibromo-4-hydroxybenzaldehyde, respectively; (d–f) UPLC/ESI-tqMS product ion scan spectra of m/z 277, 279 and 281 of the cooking water sample, respectively; (g) High-resolution MS spectrum and proposed structure of ion cluster m/z 533/535/537/539 of the cooking water sample. The y-axes of charts (b) and (c) are on the same scale.

Fig. 4. Proposed transformation pathways of polar Br-DBPs with chain structures in chloraminated cooking water.

171.0

x2

100

265.3 321.2 337.0

%

(a)

395.3 465.1

0

0 171.1

x2 265.4

440.5 469.3 525.6

321.4

209.3 265.2

265.4 321.3

200

300

100 375.3 440.5

400

457.0

500

%

(c) 171.0

523.7

m/z 600

Cl2 + SRNOM + Rice flour x2

170.9 214.9 485.5 551.7 277.1 337.1 395.4 455.5

%

0 100

0 216.9 Cl2 + Starch

x2 448.5 296.0375.4 409.4 489.6 545.5

170.9

0 Cl2 + Maltose

171.0 215.0

285.4

0 Cl2 + Glucose

% 0 100

216.9

170.9

200

300

171.1

400

x5 NH2Cl + Starch

(l) 171.0

0

285.0

302.9

554.9 553.1

m/z 600

500

553.4 457.5 551.4 555.4

217.0 275.2 303.0 346.9 455.3

100 (m) 413.4

457.3 489.7

509.5 594.5

x2

237.1 321.4

300

553.1

509.5 443.4 473.5 512.5

x5 NH2Cl + Maltose

x2

100 (i)

100 (j)

100 %

%

100 (h)

200

100 (k)

0

512.5

x4 473.1 536.1 407.4 425.3

x5 NH2Cl + SRNOM + Rice flour

100 (g)

x4

x2 NH2Cl + SRNOM + Rice flour x4 209.3 (f) 471.2 455.2 265.3 302.9 409.3

%

%

x2 Cl2 +209.3 SRNOM + Rice flour

0 100

%

(e)

0

0 100

x2 NH2Cl + SRNOM 209.3 302.9 346.8 265.3 171.0

(d)

x2 NH2Cl + Rice flour

100 %

Cl2 +209.3 Rice flour

(b)

%

%

100

100 369.5

400

457.5 513.7 545.5

500

m/z 600

171.1

221.0

301.0 347.0

0 %

%

209.3 100 Cl2 + SRNOM

x5 NH2Cl + Glucose 303.0 346.9 171.0 237.3

456.5 512.8

(n)

0 100

200

300

456.6 512.6

400

500

m/z 600

Fig. 5. (a–f) ESI-tqMS PIS spectra of m/z 79 of the simulated chlorinated and chloraminated cooking water samples prepared with only SRNOM, only rice flour, and both SRNOM and rice flour, respectively; (g–n) ESI-tqMS PIS spectra of m/z 79 of the simulated chlorinated and chloraminated cooking water samples prepared with both SRNOM and rice flour, only starch, only maltose, and only glucose, respectively. The y-axes of charts (a–c), (d–f), (g–j) and (k–n) are on the same scales with maximum intensities of 1.21×108, 6.06×108, 3.06×106 and 2.36×107, respectively. “×2” and “×5” mean that the y-axes of the specific m/z regions are enlarged by 2 and 5 times, respectively.

375.6 395.6 455.8

127.3 Cl2: Together 209.6

x2

%

215.4 5 min

100 (c)

Salt

%

277.2

127.1

200

Cl2: 0209.6 min

%

100 (g)

215.3 277.3

400

471.6 489.8

553.6

m/z 600

500

x3 NH2Cl: Together 209.4

375.4 455.3

171.1

0

265.3 302.9

409.5

5 min

Salt x3 NH2Cl: Rice flour 375.5 455.4 209.4 409.3 302.9 171.2

0 100

200

300

400

x2 NH 209.3 2 Cl: 0 min

487.6 525.9

189.1

x2 471.3 553.1 557.3 x2 473.2 553.3 557.3 x2 551.3 473.4 509.2 557.3

455.2 x2 471.2

265.3

321.4

m/z 600

500

409.4

551.1

0

%

215.2 277.3

127.3

x2 NH 209.3 2 Cl: 5 min

(l)

x2

265.4 189.1

321.4

375.4

x2 NH 209.3 2 Cl: 10 min

100 (m)

265.6 375.6 393.6 451.6

0 min Cl : 20 209.5 100 (j)2

509.6 579.9

321.6

300

375.7

400

171.2

455.7 489.7

500

m/z 600

0 100

171.0

200

455.2

471.1 x2

265.4

321.4

455.2 471.1 551.1 409.4

265.3 321.4

455.3 471.2 551.0 409.4

0 Cl: 20 min x2 NH 209.3 100 (n) 2

x2 265.7

%

375.6 393.6485.6 509.6 x2

171.2 215.2

200

100 0

0 Cl : 10 min 209.6 100 (i)2

171.4

x2

%

Cl : 5209.5 min

100 (h)2

%

375.3 265.3 303.1 409.4

100 (k)

127.1

0 100

Rice flour

100 (f)

x2 393.6

5 min

(e)

%

0

300

572.0

x2

375.6 395.7

171.1

%

0 100

457.6 553.7

(d)

0 100

265.7 375.7 393.6

127.3 Cl2: Rice flour 209.6

509.4 565.8

Salt x3 NH2Cl: 209.4

%

100 (b) 0

100

%

% 0

x2

%

(a)

Rice flour

215.3 277.3

%

5 min

Cl2: Salt 209.6

100

x2

300

400

500

m/z 600

Fig. 6. (a–f) ESI-tqMS PIS spectra of m/z 79 of the simulated chlorinated and chloraminated cooking water samples prepared with addition of edible salt followed by addition of rice flour after heating for 5 min, addition of edible salt and rice flour simultaneously, and addition of rice flour followed by addition of edible salt after heating for 5 min; (g–j) ESI-tqMS PIS spectra of m/z 79 of the simulated chlorinated cooking water samples prepared with addition intervals between rice flour and edible salt of 0, 5, 10, and 20 min, respectively; (k–n) ESI-tqMS PIS spectra of m/z 79 of the simulated chloraminated cooking water samples prepared with addition interval between edible salt and rice flour of 0, 5, 10, and 20 min, respectively. The y-axes of charts (a–c), (d–f), (g–j), and (k–n) are on the same scales with maximum intensities of 7.57×106, 4.52×107, 9.89×106 and 2.00×108, respectively. “×2” and “×3” mean that the y-axes of the specific m/z regions are enlarged by 2 and 3 times, respectively.

Before optimization (chlorination) Before optimization (chloramination) After optimization (chlorination) After optimization (chloramination)

Cell viability (%)

100 80 60

sample before optimization (chlorination) after optimization (chlorination) before optimization (chloramination) after optimization (chloramination)

40 20 0

concentration range

EC50

r2 a

ANOVA test statistic b

7.8–1000×

125×

0.996

F9,10=954.5; P≤0.001

7.8–1000×

288×

0.980

F9,10=1126.8; P≤0.001

7.8–1000×

220×

0.998

F8,9=946.6; P≤0.001

7.8–1000×

282×

0.992

F8,9=2749.4; P≤0.001

a

0.0

0.2

0.4

0.6

0.8 3

Concentration factor (10 )

1.0

r2 is the coefficient of determination for the regression analysis upon which the EC50 value was calculated. b the degrees of freedom for the between groups and residual associated with the calculated F–test result and the resulting probability value.

Fig. 7. Comparative Hep G2 cell cytotoxicity of the simulated chlor(am)inated cooking water samples prepared before and after cooking condition optimization.

•

Bromide contents in 20 edible salts from nine countries were determined.

•

Structures/formulae of 25 polar Br-DBPs were identified in cooking water samples.

•

Formation of polar Br-DBPs under various cooking conditions were explored.

•

Cooking condition optimization reduced cytotoxicity of cooking water samples.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: