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Supplementary Material
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Selective Bioaccumulation and Elimination of Hexachlorocyclohexane isomers in Tubifex
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tubifex (Oligochaeta, Tubificidae)
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Shanshan Di1,2, Ledan Huang2, Jinling Diao2, Zhiqiang Zhou1,2*
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1 Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural
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University, Yuanmingyuan west road 2, Beijing 100193, China
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2
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Beijing 100193, China
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*Corresponding author:
Department of Applied Chemistry, China Agricultural University, Yuanmingyuan west road 2,
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Zhiqiang
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Department of Applied Chemistry, China Agricultural University, Yuanmingyuan west road 2,
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Beijing 100193, P.R. China; Tel: +8610-62733547; Fax: +8610-62733547;
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E-mail: zqzhou@cau.edu.cn
Zhou,
Beijing Advanced Innovation Center for Food Nutrition and Human Health,
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Number of Pages: 5
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Number of Tables: 3
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Samples extraction and Chemicals Analysis.
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The overlying water samples were thawed at room temperature and extracted by petroleum
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ether (20 mL) in a 50 mL polypropylene centrifuge tube. After vortex-mixing for 3 min, the
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solvent phase was filtered through 5 g of anhydrous sodium sulfate for dehydration, transferred to
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a pear-shaped flask, and the extraction was repeated twice using fresh solvent. Then the combined
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extraction evaporated to dryness at 35 °C by a vacuum rotary evaporator (Shanghai Yarong
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Biochemistry Instrument Factory, Shanghai, China). The extractive was redissolved with 500 µL
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n-hexane after passing through a filter membrane (pore size, 0.45 μm).
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Microwave-assisted solvent extraction (MAE, Mars 6 Xpress, CEM, Matthews, NC, USA) was
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applied to analyze the T. tubifex and sediment samples. T. tubifex samples (3 g) were homogenized
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with an Ultra-Turrax T18 homogenizer for 30 s, and transferred into the microwave extraction
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vessels (TCmX was added as recovery surrogate) with 20 mL extraction solution (acetone:
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petroleum ether=1:1, V /V). The vessels were covered with pressure-resistant holders and placed
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into the rotary base. The extraction temperature was 100 ºC and programmed as follows: ramped
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to 100 ºC in 5 min, held for 10 min and cooled to room temperature. The extraction solvent was
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dehydrated by filtering through anhydrous sodium sulfate to a pear-shaped flask, and concentrated
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to dryness by a vacuum rotary evaporator. Florisil-SPE cartridge (1000 mg, 6 mL, Agilent
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SampliQ Products) was used to clean up interfering substances. The cartridge was preconditioned
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by eluting with 10 mL of acetone, following 5 mL of n-hexane, and equilibrating with 5 mL
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elution solvents (acetone: n-hexane=1:15, v/v). The elution was done with 6 ml elution solvents.
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The eluate was concentrated to dryness under a gentle nitrogen flow, and diluted to 500 µL with
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n-hexane. The sediment samples (10 g) were extracted with the same method above.
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HCH isomers were analyzed with an Agilent 7890A gas chromatograph equipped with electron
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capture detector (μECD) and a HP-5 column (30 m, 0.32 mm i.d., 0.25 μm film; Agilent
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Technologies Inc.). One microliter of sample solution was injected in splitless mode with the split
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outlet opened after 0.75 min. The injector and detector temperatures were 270 ºC and 290 ºC,
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respectively. The oven temperature program was as follows: initial temperature was 100 ºC (hold
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2 min), increased to 180 ºC at 15 ºC/min (hold 5 min), to 185 ºC at 5 ºC/min (hold 2 min), to 270
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ºC at 20 ºC/min, and finally increased to 290 ºC at 20 ºC/min (hold 10 min). Nitrogen was used as
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the carrier gas at a flow rate of 1 mL/min.
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Agilent 7890A GC-μECD equipped with a BGB-172 (30 m, 0.25mm i.d., 0.25mm film; BGB
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Analytik AG, Switzerland) chiral column was used to analyze the enantiomers of α-HCH. The
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injector and detector temperatures were 250 ºC and 280 ºC, respectively. The oven temperature
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program was as follows: initial temperature was 60 ºC, increased to 150 ºC at 15 ºC/min, to 176 ºC
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at 0.8 ºC/min (hold 10 min), increased to 180 ºC at 2 ºC/min (hold 4 min), and finally increased to
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220 ºC at 10 ºC/min (hold 1.5 min). Nitrogen was applied as carrier gas at a flow rate of
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0.7ml/min. (+)-α-HCH and (-)-α-HCH were identified on the basis of retention time.
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The average recoveries for HCHs at levels between 0.01 and 0.1 mg/kg ranged between 71.6%
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and 110.0% in T. tubifex tissue, between 70.2% and 109.9% in sediment, between 73.7% and
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108.8% in overlying water (0.001-0.01 mg/kg), and SD below 20% (n=3 for each sample type).
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The limit of detection (LOD) defined as the concentration with a signal-to-noise ratio of 3, was
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0.35, 0.82, and 0.45 µg/kg for α-HCH, β-HCH and γ-HCH respectively.
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Table S1. The EF values of α-HCH enantiomers in T. tubifex, overlying water and sediment for the
+TSE, -TSE and EE treatments.
Exposure time (days)
Enantiomer
factor EF
worms-(+TSE)
sediment-(+TSE)
sediment-(-TSE)
1
2
3
5
7
10
0.489±0.001
0.496±0.001
0.500±0.001
0.492±0.001
0.501±0.001
0.502±0.002
0.497±0.001
0.505±0.001
0.499±0.001
0.504±0.002
0.504±0.003
0.509±0.002
0.493±0.001
0.501±0.003
0.506±0.002
0.490±0.001
0.495±0.001
0.505±0.001
Exposure time (days)
Enantiomer
factor EF
worms-(EE)
overlying water-(EE)
sediment-(EE)
0.5
1
3
5
0.501±0.001
0.494±0.007
0.494±0.007
0.504±0.001
0.495±0.003
0.495±0.003
0.505±0.001
0.490±0.009
0.501±0.008
0.505±0.002
0.490±0.008
0.505±0.002
Table S2. The kinetic parameters of first-order kinetics equation for elimination processes in
sediment.
C(t)=C(t=0)×exp(-kt)
α-HCH-(+TSE)
β-HCH-(+TSE)
γ-HCH-(+TSE)
C0
k
R2
t1/2
0.0280±0.0007
0.0299±0.0008
0.0707±0.0016
0.0826±0.0063
0.0290±0.0063
0.2239±0.0083
0.977
0.807
0.996
8.39
23.90
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Table S3. The water quality parameters of overlying water in +TSE, -TSE, TSE and EE
treatments.
Treatments
days
pH
units
mV
COND
TDS
SALL
RES
mV
mS/cm
g/L
ng/L(ppt)
MΩ·cm
+TSE
1
2
3
5
7
10
7.91
7.73
7.72
7.69
8.20
8.43
-66
-56
-55
-58
-78
-96
2.07
3.03
3.56
4.59
5.50
5.47
1.52
2.27
2.72
3.49
4.07
4.19
1.05
1.56
1.85
2.41
2.80
2.88
504
332
282
218
189
184
-TSE
1
2
3
5
7
10
8.57
8.65
8.55
8.88
8.74
8.80
-105
-109
-103
-122
-114
-117
2.02
1.95
2.05
2.52
2.53
2.70
1.47
1.42
1.56
1.87
1.88
2.01
1.02
0.98
1.08
1.29
1.30
1.38
495
514
470
398
394
372
TSE
1
8.08
-75
2.95
2.22
1.52
338
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EE
2
3
5
7
10
7.88
7.58
7.64
8.19
8.42
-64
-46
-50
-82
-95
3.62
4.46
6.25
6.52
6.40
2.77
3.39
4.92
5.18
5.09
1.88
2.33
3.33
3.46
3.41
276
224
159
154
155
0.5
1
3
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8.45
8.41
7.85
8.04
-91
-89
-57
-68
3.74
3.52
4.50
5.80
2.87
2.68
3.43
4.50
1.94
1.83
2.36
3.07
267
283
222
172
mV:Ionic potential
COND:Conductivity
TDS:Total dissolved solids
SALL:Salinity
RES:Resistivity
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