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Enantioselective Characteristics and Montmorillonite (MMT)-mediated Removal Effects Of #-Hexachlorocyclohexane (#-HCH) in Laying Hens Xueke Liu, Zhigang Shen, Peng Wang, Chang Liu, Guojun Yao, Zhiqiang Zhou, and Donghui Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01111 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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Environmental Science & Technology

1

Enantioselective

Characteristics

and

Montmorillonite

(MMT)-mediated

2

Removal Effects Of α-Hexachlorocyclohexane (α-HCH) in Laying Hens

3

Xueke Liu, Zhigang Shen, Peng Wang, Chang Liu, Guojun Yao, Zhiqiang Zhou and Donghui Liu*

4

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of

5

Applied Chemistry, China Agricultural University, Beijing, 100193, P. R. China.

6

*Corresponding author:

7

Donghui Liu, Department of Applied Chemistry, China Agricultural University, Beijing,

8 9

100193, P. R. China; Tel.: +86 010-62731294; Fax: +86 010-62731294; E-mail: [email protected]

10 11 12

1

ACS Paragon Plus Environment


13

Abstract

14

α-Hexachlorocyclohexane (α-HCH) is a chiral organochlorine pesticide that is

15

often ubiquitously detected in various environmental matrices and may be absorbed

16

by the human body via food consumption with serious detriments to human health. In

17

this study, enantioselective degradation kinetics and residues of α-HCH in laying hens

18

was investigated after a single dose of exposure to the pesticide, whereas

19

enantioselectivity and residues of α-HCH in eggs, droppings, and various tissues were

20

investigated after long-term exposure. Meanwhile, montmorillonite (MMT), a feed

21

additive with high capacity of adsorption, was investigated for its ability to remove

22

α-HCH from laying hens. Most non-brain tissues enantioselectively accumulated

23

(-)-α-HCH, while (+)-α-HCH was preferentially accumulated in the brain. The

24

enantiomer fractions (EFs) in most tissues gradually decreased, implying continuous

25

depletion of (+)-α-HCH in laying hens. After 30d exposure and 31d elimination, the

26

concentration of α-HCH in eggs and tissues of laying hens with MMT-containing feed

27

was lower than that with MMT-free feed, indicating the removal effects of MMT for

28

α-HCH in laying hens. The findings presented herein suggest that modified MMT

29

may potentially be useful in reducing the enrichment of α-HCH in laying hens and

30

eggs, thus lower the risk of human intake of α-HCH.

31 32

2


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33

Introduction

34

Hexachlorocyclohexane (HCH) is an broad-spectrum ingested and contact

35

insecticide that has been extensively used during the last century1. Due to its

36

persistency, bioaccumulation, easy transportation and potential toxic effect, HCH has

37

been banned in most countries before 1993 and added to the list of persistent organic

38

pollutants (POPs) in 20092. However, relatively high concentrations of HCH can still

39

be detected in the environment currently.3-6 HCH can be transported into the human

40

body through food consumption7. As is well known, chicken and eggs are essential

41

sources of protein and comprise a large portion of the human diet. However, in the

42

process of breeding of laying hens, chicken and eggs could be compromised by

43

various contaminants including HCH in feed. The concentrations of HCH in eggs and

44

chicken from Beijing were reported as 0.824 and 0.082 ng/g. respectively5, values that

45

pose serious risk to food safety.

46

Generally, HCH was used in two formulations: technical HCH (α-HCH 60-70%,

47

β-HCH 5-12%, γ-HCH 10-12%, δ-HCH 6-10% and ε-HCH 3-4%), and lindane (pure

48

γ-HCH)8. Compared with other isomers, α-HCH caused the highest incidence of

49

hepatocellular carcinomas and hepatic nodules in orally exposed mice, and could

50

cross the blood-brain barrier more easily 8. In addition, the Environmental Protection

51

Agency (EPA) has classified α-HCH as probable human carcinogens9. Due to its

52

lipophilicity, α-HCH is easily accumulated in tissues of high fat content10. Of all

53

isomers, only α-HCH has a pair of enantiomers. Though (+)-α-HCH and (-)-α-HCH

54

have the same physicochemical properties, they can behave differently in the

55

biochemical process11; for example, enantioselective (+)-α-HCH had a significantly 3



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56

stronger toxicity to primary rat hepatocytes12. Additionally, it was reported that

57

enantioselective behaviors of α-HCH occurred in various environmental matrices13-15;

58

for example, (+)-α-HCH was degraded faster than (-)-α-HCH in sewage sludge16.

59

Meanwhile, several studies have reported enantioselective bioaccumulation and

60

elimination of α-HCH in various animals. (+)-α-HCH was more abundant in rabbits11,

61

loach fish17, quails18 and eider ducks19, while (-)-α-HCH dominated in sheep20, pigs21

62

and non-brain tissues of mice18.

63

Montmorillonite (MMT) is a mineral substance that is ubiquitously found in the

64

environment. MMT has a mesoporous nature and large surface area (240 m2/g), which

65

gives it a high adsorption capacity

66

suggested as additives that can effectively reduce mycotoxins in feed23, 24. Previous

67

studies demonstrated that 5 g MMT/kg complete feed did not show toxic effects in

68

biochemical, hematological and immunological examinations25,

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evaluation by the European Food Safety Authority (EFSA) considers that MMT as a

70

feed additive does not adversely affect the environment because of the mineral

71

components occurring in MMT that are ubiquitous in the environment27. Recently,

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some reports provided evidence of the removal effects of mycotoxins via addition of

73

MMT in contaminated feed. However, to date, no research investigated the removal

74

effects of MMT for persistent organic pollutants.

75

In this study, the enantioselectivity of α-HCH and removal effects of MMT for

76

α-HCH in laying hens were investigated. Kinetics or residues of α-HCH and their

77

enantiomers in blood and various tissues were characterized by single oral exposure

22

. MMT and its modified products have been

4


26

. Meanwhile, an

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experiment. In long-term exposure experiments, dynamic residues in blood, eggs,

79

droppings, and terminal residues in other various tissues were investigated to evaluate

80

the removal effects of MMT for α-HCH in laying hens. The results from this study

81

suggest that MMT may be used to reduce enrichment of α-HCH in laying hens and

82

eggs, and reduce the risk of human intake of α-HCH.

83

Materials and Methods

84

Animals and Reagents

85

Laying hens (about 2 kg in weight and 35 weeks in age) were purchased from Fujia

86

Poultry Center, Beijing, China. Rearing conditions were room temperature at 25±2 °C,

87

humidity at 50±10% and light/dark (17/7 h one day) cycle. The laying hens were

88

acclimated for 1 week before experiment, and caged individually with free access to

89

water and feed.

90

α-HCH (≥98% purity) and hexachlorobenzene (HCB) standard solution were

91

purchased

from

Aladdin

Industrial

Corporation

(Shanghai,

China).

92

Na-montmorillonite PGV clay was purchased from Nanocor Inc. (Arlington Heights,

93

Illinois, USA). Trimethyloctadecyl ammonium bromide (STAB, 99%) was purchased

94

from Sigma-Aldrich. Chromatographic grade isooctane was purchased from Fisher

95

Chemicals (Fair Lawn, NJ). All other reagents were of analytical reagent and

96

purchased from Beijing Chemical Reagent Factory, China.

97

Synthesis of organic modified montmorillonite

98

Exactly 5.66 g of STAB was dissolved in 100 mL of ethanol and water mixture

99

solution (volume ratio of 1:1). Then 10.0 g of MMT was added to the solution and 5



100

stirred for uniform dispersal. Next, the reaction of device was transferred into a 60 °C

101

water bath and stirred for 2 hours with ultrasonic assistance to achieve ion exchange.

102

Then the modified MMT was separated by filter and washed with mixture solution

103

(ethanol/water, v/v, 1:1). After drying at 60 °C in vacuum, the modified MMT was

104

activated at 105 °C for 20 min. The chemical structure of modified MMT and the

105

infrared spectroscopy of MMT, STAB and STAB-MMT are shown in Figure S1 and

106

S2.

107

Single oral exposure experiment

108

Before commencing the experiment, 12 laying hens were fasted for 12 h, and

109

three laying hens were selected as the control. Next, α-HCH (0.1 mg/kg b.w.) was

110

dissolved in corn oil, which was placed into capsules and deposited in the stomach.

111

After capsule deposit, 1 mL blood sample was drawn from wing vein of each animal

112

at 0.083, 0.166, 0.25, 0.5, 0.75, 1, 1.5, 2, 6, 12, 24, 48, 72 h. The blood samples were

113

placed in glass tubes (with 56 ng heparin sodium). The plasmas were separated from

114

blood samples by centrifugation at 3000 rpm for 10 min, and stored at -20 °C until

115

analysis. Three laying hens were sacrificed at 24, 72, and 168 h after exposure,

116

respectively. Brain, kidney, heart, liver, lung, muscular stomach, thigh muscle, breast

117

muscle, skin, and fat were sampled and stored at -20 °C until analysis.

118

Long-term exposure experiment and removal effect of modified MMT

119

According to China Hygienical Standard for Feeds, the maximum limit

120

concentration of HCH was set at 0.3 mg/kg. Thus, fortified feed contained 0.1 mg/kg

121

α-HCH in this exposure experiment. The α-HCH was dissolved in n-hexane and 6


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added to feed. After n-hexane was removed, the fortified feed was ready. According

123

to European Food Safety Authority (EFSA), 5g bentonite (dioctahedral MTT)/kg

124

complete feed29 and 20 g Friedland clay/kg complete feed are both safe for chicken,

125

and Friedland clay contains more than 35% of MMT27. Thus, 5 g the modified

126

MMT/kg complete feed was used in the experimental group.

127

Hens in the experimental group were fed feed I (supplemented with modified

128

MMT and α-HCH), and control group was fed the feed II (supplemented with only

129

α-HCH). Each group contained 9 laying hens, and all animals were caged individually

130

with free access to water and feed. After 30 days of exposure, the feed of

131

experimental group was replaced with the feed III (supplemented with only modified

132

MMT), and the feed of control group was replaced with feed IV (untreated feed).

133

Three laying hens of experimental and control group were sacrificed at 30 d after

134

exposure, respectively. The remaining laying hens were sacrificed at 61 d. During the

135

experiment, 1 mL blood sample was drawn from wing vein at days 1, 3, 7, 10, 15, 20,

136

30, 31, 33, 35, 37, 40, 45, 50, 56, 61d. Blood samples were handled in a manner

137

similar to the single oral exposure experiment. Eggs and droppings were collected

138

daily and stored at -20 °C, while egg yolks and egg whites were separated before

139

storage. After euthanization, brain, kidney, heart, liver, lung, muscular stomach, thigh

140

muscle, breast muscle, skin, and fat were sampled and stored at -20 °C until analysis.

141

Sample extraction and cleanup

142

Tissues, egg yolks, egg whites, and droppings were subjected to homogenization; and

143

0.5 g of the samples (plasma, tissues, droppings or egg white) was treated with HCB 7



144

as internal standard before extraction. The samples were extracted with 5 mL ethyl

145

acetate for 5 min on a vortex mixer and then centrifuged at 3000 rpm for 5 min. The

146

ethyl acetate was transferred to 12 mL glass tube. The same extraction operation was

147

repeated again with 5 mL ethyl acetate. The second ethyl acetate was merged in the 12

148

mL glass tube. The ethyl acetate was evaporated to dryness under a gentle stream of

149

nitrogen at 30 °C. The remaining extracts were dissolved by 1 mL of n-hexane and

150

purified by a column (6 mL). The column was prepared by adding 1mL concentrated

151

sulphuric acid on 1.5 g silica gel, and successively pre-washed with 5mL of

152

trichloromethane and 5 mL of n-hexane. After transferring to the top of the column,

153

remaining extracts were eluted with 10 mL mixture of trichloromethane and n-hexane

154

(v/v, 7/3). The eluents were evaporated to dryness under a gentle stream of nitrogen at

155

30 °C. The residue was reconstituted in 200 µL of n-hexane for analysis. Egg yolk

156

was extracted by a mixture of n-hexane and ethyl acetate (v/v, 3/1), and purified by

157

the above method.

158

Sample analysis

159

Gas chromatography tandem triple quadrupole mass spectrometer (Thermo

160

Scientific TSQ Quantum XLS) was used for the achiral analysis of α-HCH with a

161

HP-5 MS column (30 m × 0.25 mm × 0.25 µm). Ultrapure grade helium was used as

162

the carrier gas at 1.0 mL min−1 flow. The samples were injected in splitless mode. The

163

injector temperature was 250 °C. The GC oven temperature was programmed from an

164

initial temperature of 90 °C (1 min hold), ramped at 20°C min−1 up to 250 °C. Then,

165

the oven was programmed to 290 °C at 25 °C min−1 (10 min hold). Mass spectrometry

166

was carried out on a triple quadrupole mass spectrometer coupled with an electron 8


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167

impact ionization source. The parameters of mass spectrometry were set as follows:

168

ionization voltage 70 eV, ion source temperature 290 °C. Argon (99.999%) was used

169

as collision gas and the pressure was set at 1.5 mTorr. The chromatograms were

170

acquired in selected reaction monitoring (SRM) scan mode. Collision energy voltages

171

and tube lens are listed in Table S1. Chiral analysis was performed with an Agilent

172

7890A GC-ECD with a BGB-172 column (30 m × 0.25 mm × 0.25 µm; BGB

173

Analytik. AG). The nitrogen flow rate was 1.0 mL min-1 and the injection mode was

174

splitless. The injector and detector temperatures were 250 and 280 °C, respectively.

175

The initial oven temperature was 60 °C, increased to 150 °C at 15 °C min−1, then

176

increased to 176 °C at 0.8 °C min−1(10 min hold), to 180 °C at 2 °C min−1(4 min hold),

177

and finally to 220 °C at 2 °C min−1(10 min hold). EF was used to evaluate

178

enantioselective behaviors of α-HCH in the tissues of laying hens, and defined as the

179

ratio of the peak area of (+)-α-HCH (A(+)-α-HCH) to the sum of the peak areas of

180

(+)-α-HCH and (-)-α-HCH (A(-)-α-HCH).

181

was first eluted out, which has been reported in a previous study28.

182

183

EF =

A

EF was calculated by equation 1. (-)-α-HCH

(+)-α - HCH

(A(+)-α-HCH + A(-)-α-HCH )

(1)

Analytical quality control

184

HCB was used as an internal standard for quantification of α-HCH. Recovery

185

and precision were valued by spiking the samples with the analytes at three different

186

concentrations in triplicate. The recoveries were in 76-105% in the all samples. The

187

detection limits, based on signal-to-noise ratio (S/N) of 3, were 0.005 ng/g for α-HCH.

188

Detailed information on quantification method of α-HCH is outlined in Table 1–2. 9



189

Statistical analysis

190

The pharmacokinetic parameters were obtained by DAS (Dug and statistic,

191

version 2.0). In single oral exposure experiment, a two-compartment model was

192

adopted to fit the dynamic changes of α-HCH in blood. In long-term exposure

193

experiment, the eliminations of α-HCH in blood were evaluated using the first-order

194

kinetics model. The statistical analysis was conducted using OriginPro 8.0, and charts

195

were obtained by OriginPro 8.0.

196

Results and Discussion

197

Dynamics of α-HCH in blood after single oral exposure

198

The dynamics of α-HCH in blood after single oral exposure are presented in Figure

199

1(a). The concentration of α-HCH rapidly increased to its peak value at 0.75 h, and

200

then declined gradually to a stable state. The dynamics trend of α-HCH in chicken

201

blood was very similar to what was previously reported for quail blood18, which was

202

reasonably well fitted for a two-compartment model with R2 0.978. The blood

203

pharmacokinetic parameters of α-HCH are shown in Table S2. The calculated

204

absorption half-lives (t1/2α) and elimination half-lives (t1/2β) were 0.92 and 36.02 h,

205

respectively. The calculated apparent volume of distribution was 768.50 L/kg, which

206

suggested that α-HCH had a high preferential conjunction with other tissues.

207

Residues of α-HCH in tissues after single oral exposure

208

Results of α-HCH residues in tissues are shown in Figure 1(b). The data showed

209

that the concentrations of α-HCH were highest in fat, followed by brain and skin. Due

210

to its lipophilicity, α-HCH easily accumulates in tissues of high lipid content10., and a 10


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211

similar order of accumulation has been reported for α-HCH in mice18 and rabbits11,

212

and for cis/trans-chlordane in cockerels7. The highest concentration of α-HCH

213

occurred at 24 h in muscular stomach. With increase in time, the concentrations of

214

α-HCH decreased in most tissues, but significantly increased in fat, likely due to its

215

lipophilic nature.

216

Enantioenrichment of α-HCH in blood and other tissues after single oral

217

exposure

218

EF values were obtained by measured concentrations of (+)- and (-)-α-HCH. The

219

EFs in blood are shown in Figure 1(c), and EFs in other tissues are presented in

220

Figure 1(d). Because the limit of detection for chiral analysis of α-HCH was rather

221

high, low concentrations of enantiomers of α-HCH in some samples could not be

222

detected. Thus, the trend of EF values started at 0.5 h in blood; and 0.5 h and 48 h

223

after exposure, EFs of α-HCH were 0.46 and 0.34 in blood, respectively. With the

224

increase in exposure time, EFs gradually decreased in blood, showing a continuously

225

slight enantioselective depletion of (+)-α-HCH in blood. Similar EF changes were

226

also previously observed in mice18, but opposite enantioselective effect was observed

227

in quails18 and rabbits11.

228

Most of non-brain tissues had a slight enantioenrichment of (-)-α-HCH at 24, 72,

229

and 168 h after exposure, and enantioenrichment of (-)-α-HCH increased over time.

230

Similar enantioenrichment of (-)-α-HCH was also previously seen in relevant tissues

231

of mice18 and sheep20. Interestingly, the enantioenrichment of (-)-α-HCH of non-brain

232

tissues in laying hens was contrary to that in other birds such as quail18, 11



233

double-crested cormorant30 and eider duck19. This phenomenon was similar to the

234

relationship between non-brain tissues of mice18 and rabbit11. Though mice and rabbit

235

are all mammals, they likely have different enantioselective enzymatic systems, which

236

resulted in differing enantioselectivity. Moreover, the enantioenrichment of

237

(+)-α-HCH in brain of laying hens was contrary to that in non-brain tissues. EFs in

238

brain were greater than 0.93 at 24, 72, and 168 h after exposure. Although (-)-α-HCH

239

dominated in blood and non-brain tissues, (+)-α-HCH in brain still was completely

240

dominant. The results likely depended on the strong enantioenrichment of (+)-α-HCH,

241

rather than elimination of (-)-α-HCH. Again, a similar phenomenon was reported in

242

most animals such as mice18, rabbit11, quail18 and eider duck19. Likely, the strong

243

enantioenrichment of (+)-α-HCH resulted from the enantioselective transport of

244

(+)-α-HCH across the blood-brain barriers31-33.

245

Dynamics of α-HCH in blood during long-term exposure

246

Concentrations of α-HCH in blood were measured at various time intervals after

247

the initial exposure. As shown in Figure 2(a), the concentration of α-HCH in blood

248

was plotted over time for both treatment groups. During the experimental period, the

249

concentrations of α-HCH in blood of two groups showed the similar trend. During the

250

30 days of exposure, the concentrations of α-HCH gradually increased. After stopping

251

exposure, the eliminations of α-HCH in blood of two groups were both fitted to

252

first-order kinetics well (R2=0.9574-0.9860). The elimination half-life (t1/2) was

253

obtained by fitting data from the elimination period to first-order kinetics equation.

254

The t1/2 of experimental group (2.01 d) was lower than the control group (6.71 d). This 12


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255

was likely because α-HCH in various tissues and blood was consistent in the

256

experimental group, while higher α-HCH in tissues of control group was constantly

257

transferred to blood. As illustrated in Figure 2(a), the concentration of α-HCH in

258

experimental group was lower than the control group. In addition, the concentrations

259

of α-HCH in experimental group was approximately half that in the control group at

260

30 d after exposure. Those results demonstrated that the modified MMT could

261

effectively reduce the absorption of α-HCH in laying hens.

262

Dynamics of α-HCH in egg yolks and egg whites during long-term exposure

263

Concentrations of α-HCH in egg yolks and egg whites were also measured at

264

various time intervals after the initial exposure. The concentration of α-HCH in egg

265

yolks was plotted over time for both the experimental and control groups. As shown

266

in Figure 2(b), the change trends of α-HCH in egg yolks of the two groups were

267

similar. However, the concentration of α-HCH in experimental group was

268

significantly lower than the control group over the course of the experiment, and

269

about half of the control group at 30 d after exposure. Those also demonstrated that

270

the modified MMT could effectively reduce the concentration of α-HCH in egg yolks.

271

The highest concentration of α-HCH in egg whites was 0.17 µg/kg. As illustrated

272

in Figure 2(c), the concentrations of α-HCH in egg whites of two groups were not

273

significantly different. As previously mentioned, α-HCH easily accumulates in tissues

274

of high fat content due to lipophilicity 10. Previous studies have shown that egg yolks

275

enrich fat-soluble contaminants more readily. The highest concentration of α-HCH in

276

the egg yolks of control group hens was 6.71 µg/kg, which was much less than the 13



277

extraneous maximum residue limits (0.1 mg/kg). That also showed that the

278

concentrations of α-HCH in feed II was not excessive. Meanwhile, the

279

supplementation of modified MMT effectively reduced the α-HCH in eggs.

280

Dynamics of α-HCH in droppings during long-term exposure

281

Because of the strong adsorption of the modified MMT, it was difficult to

282

extract and analyze α-HCH in droppings of experimental group. Thus, the dynamics

283

of α-HCH in droppings of only the control group was investigated. As shown in

284

Figure 2(d), the concentrations of α-HCH gradually increased during the period of

285

exposure, and then rapidly reduced to a stable level during the period of elimination.

286

Because α-HCH in droppings might cause secondary pollution of the

287

environment, the droppings were collected. Previously, it was reported that α-HCH in

288

droppings may be degraded by addition of microorganisms or other methods of

289

accelerating the degradation of α-HCH34, which we did not pursue in the current study

290

but should be investigated in future studies.

291

Residues of α-HCH in tissues after long-term exposure

292

Residues of α-HCH in tissues of two groups at 30 d after exposure are shown in

293

Figure 2(e). The concentrations of α-HCH in both groups were highest in fat,

294

followed by brain and skin, and the contents of α-HCH in these three tissues were

295

significantly higher than other tissues. This was the same order of expression

296

observed in the single oral exposure experiment, and similarly observed in mice18 and

297

rabbits11. The highest concentration of α-HCH in these tissues of control group was

298

76.2 µg/kg in fat, which was less than the extraneous maximum residue limit (0.1 14


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299

mg/kg). Commonly edible chicken tissues, such as thigh muscle, breast muscle, skin,

300

and fat, are a factor of concern for food safety. The concentrations of α-HCH in

301

these tissues of experimental group hens were lower than in the control group. The

302

results demonstrated that modified MMT reduced the concentration of α-HCH in

303

these commonly edible tissues.

304

Residues of α-HCH in tissues of two groups at 61 d are demonstrated in Figure

305

2(f). Compared with those at 30 d, the concentrations of α-HCH at 61 d were

306

significantly decreased. Moreover, the concentration of α-HCH in fat was higher;

307

while that in other tissues were low, though not significantly different. This may be

308

due to the fact that the brain mainly expresses phospholipids and cholesterol, while fat

309

mainly is comprised of triglycerides. Moreover, triglycerides, as neutral fats, can

310

effectively enrich lipophilic organic pollutants21. On the other hand, the

311

concentrations of α-HCH in all tissues of experimental group were lower than

312

control group. The results implied that the modified MMT contributed to the removal

313

of α-HCH from laying hens.

314

Enantioselectivity of α-HCH in long-term exposure experiment

the

315

EFs in the blood, egg yolk, and droppings were plotted over time in Figure 3.

316

Due to higher the limit of detection for chiral analysis of α-HCH, chiral data of some

317

samples could not be obtained. Additionally, because of the low concentration of

318

α-HCH in egg whites, their EFs were not obtained. As illustrated in Figure 3(a), EFs

319

in blood of two groups were consistently less than 0.5 and decreased with increasing

320

time, which also implied that (-)-α-HCH was dominant in the blood of laying hens.

321

This enantioselectivity was contrary to what was previously observed in quail18. 15



322

They might have different enzymatic system, which resulted in opposite degradation

323

of the two α-HCH enantiomers11, 18, 32.

324

group were higher than the control group. After adding the adsorbent, the absorptive

325

dose of α-HCH was reduced in the experimental group. This implied that the

326

concentration difference between (-)-α-HCH and (+)-α-HCH in blood was related to

327

the absorptive dose of α-HCH. After 10 days of elimination, EFs of experimental

328

group and control group were both approximately 0.4, this was similar with EFs in the

329

single oral exposure after the elimination period.

Moreover, EFs in the blood of experimental

330

As shown in Figure 3(b), during the 30 days of exposure and initial stage of

331

elimination, EFs in egg yolks of two groups were less than 0.5 and decreased with

332

increasing time. However, during last stage of elimination, EFs in egg yolks of two

333

groups rapidly increased with time and both were greater than 0.5 at 60 d. Initially,

334

(-)-α-HCH dominated in the egg yolks, while in the last stage of the experiment,

335

(+)-α-HCH was dominant in egg yolks. Increase of EFs may be related to the

336

reduction in concentration of α-HCH in laying hens. There is likely a big difference

337

between sources and transport mechanisms of various materials in formation process

338

of egg yolk. On the other hand, EFs in the egg yolks of environmental group were

339

also higher than control group. That showed that enantioselectivity of α-HCH was

340

weakened after adding the adsorbent.

341

EFs in droppings of control group were consistently greater than 0.5 and

342

increased with time (Figure 3(c)). (+)-α-HCH was dominant in droppings of laying

343

hens, which was contrary to the dominant (-)-α-HCH in blood of laying hens. 16


Page 16 of 26

Page 17 of 26


344

Previous studies have suggested that chiral organochlorine pesticides

exhibit

345

enantioselectivity, which is not from absorption or excretion based on passive

346

diffusion processes, but from enantioselective metabolism35; a phenomenon that was

347

demonstrated in the enantioselectivity of cis/trans-chlordane in cockerels as being due

348

to enantioselective metabolism rather than absorption7. However, renal excretion and

349

biliary excretion are the main excretion process of xenobiotic pollutants, and active

350

transport processes may occur by carrier proteins. Urine and feces are excreted at the

351

same time in birds, thus may be one reason why the (+)-α-HCH was dominant in

352

droppings. (+)-α-HCH was dominant in droppings of laying hens and superiority of

353

(+)-α-HCH was more obvious after stopping exposure, which further demonstrated

354

that excretion process was a factor in the enantioselectivity of α-HCH.

355

EFs in tissues of the two groups at 30 d and 61d are shown in Figure 3(d) and

356

Figure 3(e), respectively. Similar to the single oral exposure experiment, (+)-α-HCH

357

in brain was still completely dominant, while (-)-α-HCH was dominant in non-brain

358

tissues. After stopping exposure, the enantioenrichment of (+)-α-HCH in brain was

359

decreased, and the dominant position of (-)-α-HCH in non-brain tissues was increased.

360

These results showed that the ratios of (+)-α-HCH in all tissues were reduced, which

361

might be due to (+)-α-HCH being preferentially eliminated in the excretion process.

362

Supporting Information

363

The parameters of mass spectrometry (Table S1), the blood pharmacokinetic

364

parameters of α-HCH (Table S2), chemical structure (Figure S1) and the infrared

365

spectroscopy (Figure S2) of modified MMT. 17



366

Page 18 of 26

Acknowledgments This work was supported by National Natural Science Foundation of China

367 368

(Contract Grant Number: 21307155)

369

References

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405

(1) Li, Y. F., Global technical hexachlorocyclohexane usage and its contamination consequences in the environment: from 1948 to 1997. Sci. Total Environ. 1999, 232, (3), 121-158. (2) Nadal, M.; Marques, M.; Mari, M.; Domingo, J. L., Climate change and environmental concentrations of POPs: A review. Environ. Res. 2015, 143, (Pt A), 177-85. (3) Yu, Y.; Hung, H.; Alexandrou, N.; Roach, P.; Nordin, K., Multiyear Measurements of Flame Retardants and Organochlorine Pesticides in Air in Canada's Western Sub-Arctic. Environ. Sci. Technol. 2015, 49, (14), 8623-8630. (4) Romanic, S. H.; Klincic, D.; Kljakovic-Gaspic, Z.; Kusak, J.; Reljic, S.; Huber, D., Organochlorine pesticides and polychlorinated biphenyl congeners in wild terrestrial mammals from Croatia: Interspecies comparison of residue levels and compositions. Chemosphere 2015, 137, 52-58. (5) Yu, Y.; Tao, S.; Liu, W.; Lu, X.; Wang, X.; Wong, M., Dietary Intake and Human Milk Residues of Hexachlorocyclohexane Isomers in Two Chinese Cities. Environ. Sci. Technol. 2009, 43, (13), 4830-4835. (6)

Pucko,

M.;

Stern,

G.

A.;

Macdonald,

R.

W.;

Barber,

D.

G.,

alpha-

and

gamma-Hexachlorocyclohexane Measurements in the Brine Fraction of Sea Ice in the Canadian High Arctic Using a Sump-Hole Technique. Environ. Sci. Technol. 2010, 44, (24), 9258-9264. (7) Lu, Z.; Xue, M.; Shen, G.; Li, K.; Li, X.; Wang, X.; Tao, S., Accumulation Dynamics of Chlordanes and Their Enantiomers in Cockerels (Gallus gallus) after Oral Exposure. Environ. Sci. Technol. 2011, 45, (18), 7928-7935. (8) Willett, K. L.; Ulrich, E. M.; Hites, R. A., Differential toxicity and environmental fates of hexachlorocyclohexane isomers. Environ. Sci. Technol. 1998, 32, (15), 2197-2207. (9) Dorsey, A., Toxicological Profile for Alpha-, Beta-, Gamma-, and Delta-Hexachlorocyclohexane. US Department of Health & Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry: 2005. (10) Ruus, A.; Skaare, J. U.; Ingebrigtsen, K., Disposition and depuration of lindane (gamma-HCH) and polychlorinated biphenyl-110 (2,3,3 ',4 ',6-pentachlorobiphenyl) in cod (Gadus morhua) and bullrout (Myoxocephalus scorpius) after single oral exposures. Environ. Toxicol. Chem. 2001, 20, (10), 2377-2382. (11) Xue, M.; Shen, G.; Yu, J.; Zhang, D.; Lu, Z.; Wang, B.; Lu, Y.; Cao, J.; Tao, S., Dynamic changes of alpha-hexachlorocyclohexane and its enantiomers in various tissues of Japanese Rabbits (Oyctolagus cuniculus) after oral or dermal exposure. Chemosphere 2010, 81, (11), 1486-1491. (12) Moller, K.; Huhnerfuss, H.; Wolfle, D., Differential effects of the enantiomers of α-hexachlorocyclohexane (α-HCH) on cytotoxicity and growth stimulation in primary rat hepatocytes. Organohalogen Compd. 1996, 29, 357-360. (13) Falconer, R. L.; Bidleman, T. F.; Gregor, D. J.; Semkin, R.; Teixeira, C., ENANTIOSELECTIVE BREAKDOWN OF ALPHA-HEXACHLOROCYCLOHEXANE IN A SMALL ARCTIC LAKE AND 18


Page 19 of 26


406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449

ITS WATERSHED. Environ. Sci. Technol. 1995, 29, (5), 1297-1302. (14) Ridal, J. J.; Bidleman, T. F.; Kerman, B. R.; Fox, M. E.; Strachan, W. M. J., Enantiomers of alpha-hexachlorocyclohexane as tracers of air-water gas exchange in Lake Ontario. Environ. Sci. Technol. 1997, 31, (7), 1940-1945. (15) Law, S. A.; Bidleman, T. F.; Martin, M. J.; Ruby, M. V., Evidence of enantioselective degradation of alpha-hexachlorocyclohexane in groundwater. Environ. Sci. Technol. 2004, 38, (6), 1633-1638. (16) Buser, H. R.; Muller, M. D., ISOMER AND ENANTIOSELECTIVE DEGRADATION OF HEXACHLOROCYCLOHEXANE ISOMERS IN SEWAGE-SLUDGE UNDER ANAEROBIC CONDITIONS. Environ. Sci. Technol.1995, 29, (3), 664-672. (17) Ma, R.; Liu, D.; Qu, H.; Zhou, G..; Zhou, Z.; Wang, P., Enantioselective toxicokinetics study of the bioaccumulation

and

elimination

of

alpha-hexachlorocyclohexane

in

loaches

(Misgurnus

anguillicaudatus) and its environmental implications. Chemosphere 2013, 90, (7), 2181-2186. (18) Yang, D.; Li, X.; Tao, S.; Wang, Y.; Cheng, Y.; Zhang, D.; Yu, L., Enantioselective Behavior of alpha-HCH in Mouse and Quail Tissues. Environ. Sci. Technol. 2010, 44, (5), 1854-1859. (19) Kallenborn, R.; Huhnerfuss, H.; Konig, W. A., GAS-CHROMATOGRAPHIC SEPARATION OF THE ENANTIOMERS OF ORGANIC MARINE POLLUTANTS .2. ENANTIOSELECTIVE METABOLISM OF (+/-)-ALPHA-1,2,3,4,5,6-HEXACHLOROCYCLOHEXANE IN ORGANS OF THE EIDER DUCK. Angewandte Chemie-International Edition in English 1991, 30, (3), 320-321. (20) Moeller, K.; Huehnerfuss, H.; Rimkus, G., On the diversity of enzymatic degradation pathways of alpha-hexachlorocyclohexane as determined by chiral gas chromatography. HRC (J. High Res. Chrom.) 1993, 16, (11), 672-673. (21) Covaci, A.; Gheorghe, A.; Schepens, P., Distribution of organochlorine pesticides, polychlorinated biphenyls and alpha-HCH enantiomers in pork tissues. Chemosphere 2004, 56, (8), 757-766. (22) Ahmed, O. S.; Dutta, D. K., Generation of metal nanoparticles on montmorillonite K 10 and their characterization. Langmuir 2003, 19, (13), 5540-5541. (23) Desheng, Q.; Fan, L.; Yanhu, Y.; Niya, Z., Adsorption of aflatoxin B-1 on montmorillonite. Poultry Sci. 2005, 84, (6), 959-961. (24) Lemke, S. L.; Grant, P. G.; Phillips, T. D., Adsorption of zearalenone by organophilic montmorillonite clay. J. Agric. Food Chem. 1998, 46, (9), 3789-3796. (25) Abbes, S.; Ouanes, Z.; ben Salah-Abbes, J.; Houas, Z.; Oueslati, R.; Bacha, H.; Othman, O., The protective effect of hydrated sodium calcium aluminosilicate against haematological, biochemical and pathological changes induced by Zearalenone in mice. Toxicon 2006, 47, (5), 567-574. (26) Abbes, S.; Ben Salah-Abbes, J.; Hetta, M. M.; Ibrahim, M.; Abdel-Wahhab, M. A.; Bacha, H.; Oueslati, R., Efficacy of Tunisian montmorillonite for in vitro aflatoxin binding and in vivo amelioration of physiological alterations. Appl. Clay Sci. 2008, 42, (1-2), 151-157. (27) EFSA Panel on Additives and Products or Substances used in Animal Feed, Scientific Opinion on the safety and efficacy of Friedland clay (montmorillonite-illite mixed layer clay) when used as technological additive for all animal species. EFSA Journal 2014, 12, (11), 3904. (28) Falconer, R. L.; Bidleman, T. F.; Szeto, S. Y., Chiral pesticides in soils of the Fraser Valley, British Columbia. J. Agric. Food Chem. 1997, 45, (5), 1946-1951. (29) EFSA Panel on Additives and Products or Substances used in Animal Feed, Scientific opinion on the safety and efficacy of bentonite (dioctahedral montmorillonite) as feed additive for all species. EFSA Journal 2011, 9, (2), 2007. (30) Iwata, H.; Tanabe, S.; Iida, T.; Baba, N.; Ludwig, J. P.; Tatsukawa, R., Enantioselective 19



450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467

accumulation of alpha-hexachlorocyclohexane in northern fur seals and double crested cormorants: Effects of biological and ecological factors in the higher trophic levels. Environ. Sci. Technol. 1998, 32, (15), 2244-2249. (31) Luurtsema, G.; de Lange, E. C. M.; Lammertsma, A. A.; Franssen, E. J. F., Transport across the blood-brain barrier: Stereoselectivity and PET-tracers. Mol. Imaging Biol. 2004, 6, (5), 306-318. (32) Ulrich, E. M.; Willett, K. L.; Caperell-Grant, A.; Bigsby, R. M.; Hites, R. A., Understanding enantioselective processes: A laboratory rat model for alpha-hexachlorocyclohexane accumulation. Environ. Sci. Technol. 2001, 35, (8), 1604-1609. (33) Dewailly, E.; Mulvad, G.; Pedersen, H. S.; Ayotte, P.; Demers, A.; Weber, J. P.; Hansen, J. C., Concentration of organochlorines in human brain, liver, and adipose tissue autopsy samples from Greenland. Environ. Health Perspct. 1999, 107, (10), 823-828. (34) Qu, J.; Xu, Y.; Ai, G.; Liu, Y.; Liu, Z.., Novel Chryseobacterium sp PYR2 degrades various organochlorine pesticides (OCPs) and achieves enhancing removal and complete degradation of DDT in highly contaminated soil. J. Environ. Manage. 2015, 161, 350-357. (35) Wong, C. S.; Lau, F.; Clark, M.; Mabury, S. A.; Muir, D. C. G., Rainbow trout (Oncorhynchus mykiss) can eliminate chiral organochlorine compounds enantioselectively. Environ. Sci. Technol. 2002, 36, (6), 1257-1262.

468 469

20


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Page 21 of 26


470

471

Table 1 The calibration curves and the LOD of α-HCH in samples Sample Correlation coefficient( r ) Linearity (ng/g)

LOD（ng/g）

Plasma

0.9985

0.1-5.0

0.005

Egg yolk

0.9994

0.1-5.0

0.005

Egg white

0.9996

0.01-1.00

0.005

Droppings

0.9998

0.1-5.0

0.005

Muscle

0.9998

0.1-5.0

0.005

Fat

0.9992

1.0-100.0

0.005

Heart

0.9999

0.5-50.0

0.005

Lung

0.9998

0.5-50.0

0.005

Kidney

0.9999

0.5-50.0

0.005

Liver

0.9998

0.5-50.0

0.005

Muscular stomach

0.9997

0.5-50.0

0.005

Brain

0.9989

1.0-100.0

0.005

Skin

0.9999

1.0-100.0

0.005

LOD, limit of detection.

472

21



473

Table 2 The recovery and precision of α-HCH in samples Sample

Plasma

Egg yolk

Egg white

Droppings

Muscle

Fat

Heart

Lung

Kidney

Liver

Muscular stomach

Brain

Concentration（ng/g）

Recovery(%)

RSD(%)

5 1 0.5 5 1 0.1 1 0.1 0.01 5 1 0.1 5 1 0.1 50 5 1 50 5 1 50 5 1 50 5 1 50 5 1 50 5 1 50 5 1 50

94 91 97 90 91 97 91 95 103 86 88 89 87 88 93 88 96 97 84 76 93 83 78 82 105 87 95 78 76 82 94 99 96 105 103 105 96

1.5 4.4 6.9 2.3 2.3 8.1 4.4 8.3 11.4 2.9 6.8 6.2 4.4 8.0 10.7 5.7 7.3 6.8 5.4 1.6 7.8 10.0 10.2 14.5 7.6 2.8 2.3 3.9 9.1 9.6 7.9 2.3 5.0 4.3 2.9 4.8 2.1

5

100

5.8

1

95

9.0

Skin 474

Page 22 of 26

RSD, Relative standard deviation

22


Page 23 of 26


TOC 84x47mm (300 x 300 DPI)



Figure 1 The dynamic curves (a) and EF curves (c) of α-HCH in blood and residues (b) and EF (d) of α-HCH in tissues in single oral exposure experiment, (A=Brain, B=Kidney, C=Heart, D=Liver, E=Lung, F=Muscular Stomach, G=Thigh Muscle, H=Breast Muscle, I=Skin, J=Fat; bars are standard error). 81x63mm (300 x 300 DPI)


Page 24 of 26

Page 25 of 26


Figure 2 The dynamic curves of α-HCH in blood (a), egg yolk (b), egg white (c), droppings (d) and residues of α-HCH in tissues at 30d (e) and 61d (f) in long-term exposure experiment (A=Brain, B=Kidney, C=Heart, D=Liver, E=Lung, F=Muscular Stomach, G=Thigh Muscle, H=Breast Muscle, I=Skin, J=Fat; bars are standard error). 59x28mm (300 x 300 DPI)



Figure 3 EF curves of α-HCH in blood (a), egg yolk (b), droppings (c) and EF of α-HCH in tissues at 30d (d) and 61d (e) in long-term exposure experiment (A=Brain, B=Kidney, C=Heart, D=Liver, E=Lung, F=Muscular Stomach, G=Thigh Muscle, H=Breast Muscle, I=Skin, J=Fat; bars are standard error). 59x28mm (300 x 300 DPI)


Page 26 of 26

Enantioselective Characteristics and ... - ACS Publications

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