1
Supporting Information
2
Compound specific isotope analysis of hexachlorocyclohexane
3
isomers: a method for source fingerprinting and field
4
investigation of in situ biodegradation
5
6
Michelle Chartrand1, Elodie Passeport2,3*, Carla Rose1, Georges Lacrampe Couloume1, Terry F.
7
Bidleman4, Liisa M. Jantunen5, Barbara Sherwood Lollar1
8
9
1
Department of Earth Sciences, University of Toronto, 22 Russell Street, Toronto, ON M5S 3B1,
10
11
Canada
2
Department of Civil Engineering, University of Toronto, 35 Russell Street, Toronto, ON M5S
12
13
1A4, Canada
3
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200
14
College Street, Toronto, ON M5S 3E5, Canada
4
15
16
17
5
Dept. of Chemistry, Umeå University, Linnaeus väg 6, SE-901 87 Umeå, Sweden
Air Quality Processes Research Section, Environment Canada, 6248 Eighth Line, Egbert, ON
L0L1N0, Canada
18
19
*Corresponding author e-mail: elodie.passeport@utoronto.ca
20
21
Short title: CSIA of HCH isomers: source fingerprint and in situ biodegradation
1
22
23
2
24
S1. Details on TG-HCH samples
Technical
Grade HCH
Manufacturer Details
Pesticide 666
α-HCH
β-HCH
γ-HCH
% in
Concentration
% in
Concentration
% in
Concentration
-1
-1
Mixture
(mg L )
Mixture
(mg L )
Mixture
(mg L-1)
57
245
14
60
19
80
25
China
Obtained from Midwest Research Institute,
Kansas City, MO, USA; manufacturer lot #
BHC-K500
80-238-3
75
241
17
55
6
20
country of origin unknown
US EPA Pesticide Repository lot K500
Ittehad Pesticide, Lahore, Pakistan
BHC-K503
52
181
8
27
30
103
US EPA Pesticide Repository lot K503
Hooker Chemical, Niagara Falls, NY, USA
BHC-K501
Fortified BHC
26
123
6
27
43
202
US EPA Pesticide Repository lot K501
Table S1. Manufacturer details and relative amounts of α-, β-, and γ-HCH in the four TG-HCH samples. Analysis was performed by capillary gas
26
chromatography-electron capture negative ion mass spectrometry using an Agilent 6890 GC-5973 Mass Selective Detector with a 30-m HP-5MS
27
capillary column (0.25 mm i.d., 0.25 μm film thickness, J&W, Agilent Technologies via Chromatographic Specialties Inc., Brockville, ON,
28
Canada). Samples were injected splitless (2 μL, split opened after 0.5 min), with the following temperature program: initial temperature 90°C,
29
15°C min-1 to 160°C, 1°C min-1 to 180°C, hold for 2 min; 20°C min-1 to 270°C, hold for 10 min. Quantification was performed using 5 standards
30
that spanned the concentration range of the TG-HCH mixtures.
3
31
32
33
S2. Analytical method
S2.1 Comparison of offline vs online (i.e. continuous flow) isotope analysis
Two laboratory working standards from Sigma Aldrich (lot no. 19328 and 5227X), referred
34
to as α-HCHSA and -HCHSA, respectively, different from those used in the pre-concentration
35
protocol tests (that consisted of a separate “α-HCH”, referred to without the subscript “SA”), were
36
isotopically characterized to compare the 13C values obtained by IRMS analysis using traditional
37
offline combustion tube preparation (and dual inlet isotope analysis) and gas chromatography – inline
38
combustion (“online”, or continuous flow isotope analysis). [1] In the offline preparation method, free
39
product samples (3 mg) were placed in sealed quartz tubes in the presence of CuO, Cu, and Zn and
40
connected to a vacuum line. Samples were combusted to CO2 at 850°C in a furnace. The CO2-
41
converted HCH isomers were introduced into the Finnigan MAT 252 isotope ratio mass spectrometer
42
by a dual inlet port. For the online method, saturated solutions of α-HCHSA and -HCHSA were
43
separately prepared in pentane. Liquid injections (2 µL) were made on a Varian 3400 gas
44
chromatograph interfaced with a combustion oven (Thermo Fisher / Finnigan, Bremen, Germany)
45
held at 980 ºC in line with a Finnigan MAT 252 isotope ratio mass spectrometer for isotope ratio
46
analysis (GC/C-IRMS). A RESTEK Stx®-CL Pesticide column was used (30 m × 0.32 mm, i.d.
47
0.5 µm; flow 1.2 mL min-1, Restek, Bellefonte, PA, USA, via Chromatographic Specialties Inc.,
48
Brockville, Canada). The injector temperature was 250°C, and the GC column temperature
49
started at 140°C and was held for 5 min, increased to 175°C at 5°C min-1 and held for 9 min, then
50
increased to 300°C at 20°C min-1.
51
4
Injection #
1
2
3
4
5
6
Average
1 SD
-HCHSA
Offline-IRMS GC/C-IRMS
-31.5
-32.0
-32.2
-32.0
-31.9
-31.9
-31.9
-31.9
-32.0
-31.8
NA
-31.8
-31.9
-31.9
0.2
0.1
-HCHSA
Offline-IRMS GC/C-IRMS
-26.9
-26.7
-27.1
-27.0
-27.0
-26.5
-26.8
-26.6
-26.9
-26.4
NA
-26.4
-26.9
-26.6
0.1
0.2
52
Table S2: Stable carbon isotope values for α-HCHSA and -HCHSA laboratory working standards,
53
measured by offline combustion IRMS and GC/C-IRMS (“online” or continuous flow isotope analysis).
54
S2.2 Characterization of the α-HCH standard used for the first three pre-concentration steps
55
Several liquid injections (10 µL) of a solution of an α-HCH pure standard solution (100
56
mg L-1 in acetone) were made via GC/C-IRMS where the gas chromatograph was equipped with
57
either a DB-1 column (60 m × 0.25 mm i.d.) or HP-5 column (30 m × 0.32 mm i.d.) coupled with
58
DB-5MS column (60 m × 0.25 mm i.d.). All columns were purchased from Agilent, Santa Clara,
59
CA, USA, through Chromatographic Specialties Inc., Brockville, ON, Canada. Details on
60
temperature programs and isomer peak resolution are provided below. The mean, one standard
61
deviation (SD), and number of determinations (n) of the δ13C values of α-HCH pure standard
62
were –27.0‰, 0.3‰, and 28, respectively. The signal size for these 28 injections ranged from
63
2.1 to 5.5 V. Changes in analytical conditions did not significantly affect isotopic measurements.
64
65
5
66
67
68
S2.3 Pre-concentration steps for HCH isotope analysis
Figure S1 provides a schematic of the first three pre-concentration steps. Each pre-
69
concentration step was performed with a fresh 200 mL aliquot of the 50 or 500 μg L-1 aqueous stock
70
α-HCH solution. The 13C value of the α-HCH standard was −27.0 ± 0.5‰. Isotopic analysis was
71
performed on each extract after each pre-concentration step to confirm that gthere was no significant
72
isotopic fractionation (< 0.5‰) associated with any step. Each pre-concentration step sample was
73
stored in the freezer until analyzed.
74
Step 1. The first pre-concentration step, DCM extraction, involved shaking a 200 mL aliquot
75
of the 50 or 500 μg L-1 aqueous α-HCH stock solution with 200 mL of DCM in a 500-mL separatory
76
funnel. The DCM extract was drained into a funnel containing sodium sulfate and glass wool to
77
remove the excess water, which subsequently drained into a 500-mL round bottom flask. The
78
extraction was then repeated with an additional 100 mL of DCM. Boiling chips were added to the
79
combined DCM extracts (300 mL) in the round bottom flask, and the DCM was concentrated to
80
approximately 3 mL using rotary evaporation. The DCM was further reduced to 1 mL under a stream
81
of N2, and the remaining 1-mL sample was transferred to a 2-mL screw cap vial.
82
Step 2. The second pre-concentration step, solvent exchange into iso-octane, was performed
83
in an identical manner to the DCM extraction step, except that 3 mL of iso-octane was added to the
84
300 mL of DCM extract, which was subsequently rotary evaporated to remove all the DCM, with 3
85
mL of iso-octane remaining. The 3 mL of iso-octane was reduced to 1 mL under a stream of nitrogen,
86
and stored in a 2-mL screw cap vial.
87
Step 3. The third pre-concentration step, H2SO4 clean-up, was performed identically to the
88
second pre-concentration step with the following additional steps: 1 mL of 18 M H2SO4 was added to
89
the 1-mL iso-octane extract, the sample was shaken on a Vortex shaker for 2 minutes, then
6
90
centrifuged at 1,000 rpm for 5 minutes to re-separate the H2SO4 and iso-octane. The iso-octane layer
91
was then sub-sampled and transferred to a 2-mL screw cap vial.
92
93
94
Figure S1. Schematic of the preparation method for the α-HCH pre-concentration step samples. Arrows
95
indicate the points in the process where samples were taken for δ13C analysis to test for isotopic effects in
96
the pre-concentration process (e.g. Protocol test).
7
97
98
Figure S2: 13C values of α-HCH following DCM extraction (circles), solvent exchange into iso-octane
99
(squares), and H2SO4 clean-up (triangles) versus the amplitude of the signal from GC/C-IRMS analysis, as
100
in Sherwood Lollar et al. [2] The solid line represents the characterized value of the α-HCH working
101
standard (−27.0‰, see Section S2.2 above), and the dashed lines represent ± 0.5‰ around the
102
characterized value. None of the pre-concentration steps create significant isotopic fractionation from the
103
isotopically characterized α-HCH standard.
104
105
106
S2.4 Step 4. Additional pre-concentration step
For some field groundwater samples, the volumes of some of the extracts were further
107
reduced to approximately 100 μL under a stream of nitrogen to improve the detection of the HCH
108
isomers. It was already shown that liquid-liquid extraction (e.g., extraction of an aqueous sample
109
with pentane) does not involve significant isotopic fractionation even when the water to solvent
8
110
ratio was as high as 1000:1. [3] Additional protocol tests were conducted to verify the absence of
111
significant isotopic fractionation when further reducing the sample volume by evaporating the
112
solvent under a gentle stream of N2. For that, the -HCHSA, -HCH, and δ-HCHSA standards were
113
first combined in pentane, then evaporated to dryness under a stream of N2, and finally
114
reconstituted in acetone. The characterized values for -HCHSA and δ-HCHSA are provided in
115
Table S2. These data demonstrate that no significant isotope effects (± 0.5‰) occur due to this
116
final step. The 13C values of the HCH isomers in the initial pentane solution were -31.8 ± 0.5 ‰
117
(-HCHSA), -27.9 ± 0.5 ‰ (-HCH), and -26.6 ± 0.5 ‰ (δ-HCHSA). The corresponding values in
118
acetone after evaporation to dryness and reconstitution in acetone were the same: -31.9 ± 0.5 ‰
119
(-HCHSA), -27.9 ± 0.5 ‰ (-HCH), and -26.3 ± 0.5 ‰ (δ-HCHSA).
120
121
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122
123
S 2.5 GC conditions
Initially, a DB-1 column from Agilent, Santa Clara, CA, USA (60 m × 0.25 mm i.d.; flow rate
124
1.2 mL min-1) purchased via Chromatographic Specialties Inc., Brockville, ON, Canada was used,
125
with a temperature program beginning at 90 ºC, increased at 15 ºC min-1 to 150 ºC, and increased to
126
250 ºC at 3 ºC min-1. The injector temperature was 200 ºC. This GC column and temperature program
127
was successful for measuring the α-HCH pure standard, the α-HCH pre-concentration step samples,
128
as well as α-HCH in the 4 TG-HCH mixtures and field samples, as the α-HCH isomer was
129
sufficiently resolved from the next eluting isomer, β-HCH. However, due to the large amount of
130
acetone or iso-octane from the direct liquid injections, solvent tailing was observed. In addition, β-
131
HCH partially co-eluted with the next eluting isomer, γ-HCH, in the four TG-HCH mixtures and in
132
the field samples.
133
To improve the resolution of the β- and γ-HCH peaks and to decrease solvent tailing, a 30 m
134
× 0.32 mm i.d. HP-5 column (Agilent, Santa Clara, CA, USA, via Chromatographic Specialties Inc.,
135
Brockville, ON, Canada) coupled with a 60 m × 0.25 mm i.d. DB-5MS column (Agilent, Santa
136
Clara, CA, USA, via Chromatographic Specialties Inc., Brockville, ON, Canada; HP-5 + DB-5MS)
137
was used. Both the HP-5 and DB-5MS columns had similar stationary phases ((5%-phenyl)-
138
methylpolysiloxane and phenyl arylene polymer, which is virtually equivalent to (5%-phenyl)-
139
methylpolysiloxane, for the HP-5 and DB-5MS columns, respectively). The GC temperature program
140
started at 120 ºC and was held for 10 min, increased to 150 ºC at 15 ºC min-1 and held for 10 min,
141
increased to 200 ºC at 3 ºC min-1 and held for 10 min, and increased to 250 ºC at 3 ºC min-1. The
142
injector temperature was 100 ºC, held for 10 min, then increased at 25 ºC min-1 to 200 ºC, and held at
143
this temperature for the duration of the sample run. This method successfully minimized solvent
144
tailing and co-elution of β- and γ-HCH. The HP-5 + DB-5MS column was used to re-analyze the α-
145
HCH pure standard to ensure that changing the column and GC parameters did not affect the
10
146
measured δ13C value. Thereafter, the β- and γ-HCH pure standards, the lindane sample, and all HCH
147
isomers in the 4 TG-HCH mixtures and the field samples were all analyzed using this column setup.
148
S2.6 Field sample from well 28-S
149
In groundwater samples from well 28-S, the HCH isomer concentrations were reported to
150
be below detection limit by Law et al. [4] However, based on retention time comparisons with
151
other samples run under identical GC conditions, γ- and δ-HCH were identified in this well,
152
because of the greater sensitivity of the mass spectrometer. The retention time of the γ-HCH
153
standard ranged between 2448 and 2457 s, which corresponded to a peak in the chromatogram
154
for well 28-S (2443 – 2446 s). Furthermore, the elution order for the HCH isomers on the DB5-
155
MS column is α-HCH, followed by β-HCH, γ-HCH and finally δ-HCH (US EPA method
156
8081A). [5] The retention time of the next major peak in the chromatogram ranged from 2608 to
157
2638 s for all well samples, and was therefore identified as δ-HCH. This was further supported by
158
the fact that the δ13C values of this peak for wells 28-S, 19-D and 7-S were isotopically identical
159
within uncertainty to the δ13C value of α- and γ-HCH, suggesting that this peak was indeed δ-
160
HCH. The sample from well 28-S was pre-concentrated according to the four steps outlined in
161
Sections S2.3 and S2.4, which were shown not to cause measurable isotopic fractionation.
162
Duplicate analyses of sample 28-S had peak sizes greater than 0.4 V for all HCH isomers.
163
S2.7 Estimation of the limit of detection
164
For the pre-concentration steps, 10 μL of the 1-mL extract were injected onto the gas
165
chromatograph for isotopic analysis by GC/C-IRMS. Assuming 100% efficiency in the extraction
166
process (between 75 and 125 % recovery was reported by Law et al.
[4]
for the same extraction
11
167
process), the total mass of α-HCH injected onto the gas chromatograph for GC/C-IRMS analysis
168
from the pre-concentration step samples can be calculated using the following equation:
169
minj = C0 × Vsol × Vinj / Vext
(Eq. S1)
170
where minj is the total mass of α-HCH injected onto the gas chromatograph for GC/C-IRMS
171
analysis (μg), C0 is the initial concentration of α-HCH in the aqueous solution (μg L-1), Vsol is the
172
volume of the α-HCH solution used for extraction (L), Vinj is the volume of the extract injected
173
onto the gas chromatograph for GC/C-IRMS analysis (10 μL for all injections), and Vext is the
174
final volume of the extract (1,000 µL for all samples). For the 50 and 500 μg L-1 extracts, 100 and
175
1,000 ng of α-HCH were injected in a 10 μL sample injection onto the gas chromatograph for
176
GC/C-IRMS analysis, respectively (Table S3). The total mass of the α-HCH injected onto the gas
177
chromatograph for GC/C-IRMS analysis from the α-HCH pure standard (10-μL injection from
178
100-mg L-1 solution) is also 1,000 ng.
179
The peak amplitudes from the isotopic analysis ranged from 0.2 to 0.3 V (mean 0.24 V)
180
for the 50 μg L-1 extracts, from 2.0 to 3.9 V (mean 2.5 V) for the 500 μg L-1 extracts, and 2.1 to
181
5.5 V (mean 3.2 V) for the α-HCH standard. The instrument linearity in this study was 0.2 to 5.5
182
V. Using the minimum peak amplitude of 0.2 V, the calculated theoretical minimum mass
183
(TMM) required for α-HCH carbon isotopic analysis can be calculated using:
184
TMM = minj × minp / meanp
(Eq. S2)
185
where minj is obtained from Eq. S1, minp is the minimum peak amplitude for carbon isotopic
186
analysis (0.2 V) and meanp is the mean peak amplitude of the extracts. The TMM was 83 and 80
187
ng of α-HCH (20 ng of carbon delivered on column) for the 50 and 500 μg L−1 extracts,
188
respectively, and 63 ng for the α-HCH pure standard (17 ng of carbon). The three calculated
189
TMMs are in close agreement, suggesting that this method of evaluating the TMM required for
190
carbon isotopic analysis is robust and can be applied to a wide range of α-HCH concentrations.
12
191
Furthermore, the calculated TMM agrees closely with the on-column detection limit for carbon
192
CSIA documented in other studies using a similar analytical system (12 ng of carbon). [6]
193
194
195
196
197
198
199
200
201
From the pre-concentration step samples, the theoretical detection limit (TDL) in μg L-1
for carbon isotopic analysis can be calculated using:
TDL = TMM × Vext / (Vinj × Vsol)
(Eq. S3)
where TMM is obtained from Eq. S2.
Table S3. Total mass of α-HCH injected onto the gas chromatograph for GC/C-IRMS analysis and the
theoretical minimum mass required for α-HCH carbon isotopic analysis for the α-HCH standard and the
pre-concentration samples, and the theoretical detection limit for carbon isotopic analysis calculated based
on results from the pre-concentration samples.
Total mass of
Theoretical
Theoretical
Volume of
Theoretical
Sample
α-HCH (ng)
minimum
minimum
water
detection limit
mass of HCH
mass of
extracted (L)
(μg L-1)
(ng)
carbon (ng)
-1
50 μg L
100
83
20
0.2
42
aqueous stock
500 μg L-1
1000
80
20
0.2
40
aqueous stock
100 ng μL-1
1000
63
17
NA
NA
α-HCH in
acetone
202
13
203
S3. Source differentiation: summary of the δ13C values of the four TG-HCH and lindane
204
samples, and three HCH standards.
205
206
207
208
209
Table S4: δ13C values for each isomer in each standard or sample analyzed.
a
Analytical uncertainty for all δ13C HCH isomer measurements is ± 0.5‰, which includes both
accuracy and reproducibility after the method of Sherwood Lollar et al. [2] b SD = standard deviation
on replicates (precision) is provided for all standards and samples for which replicates were run.
Sample
HCH isomer
α-HCH standard
β-HCH standard
γ-HCH standard
Lindane
Pesticide 666
α-HCH
β-HCH
γ-HCH
γ-HCH
α-HCH
β-HCH
γ-HCH
α-HCH
γ-HCH
α-HCH
γ-HCH
α-HCH
β-HCH
BHC-K501
BHC-K503
BHC-K500
Average
δ13C
(‰)a
-27.0
-26.6
-26.4
-28.0
-26.1
-25.6
-25.4
-27.7
-28.2
-28.4
-27.8
-32.9
-31.8
Number of
analyses (n)
Mean ± SDb of all δ13C for all
isomers in sample (‰)
28
9
3
8
6
1
2
2
2
2
1
4
2
-27.0 ± 0.3
-26.6 ± 0.3
-26.4 ± 0.1
-28.0 ± 0.2
-25.9 ± 0.4
-27.9 ± 0.3
-28.2 ± 0.4
-32.5 ± 0.7
14
210
211
212
213
214
215
216
217
S4. Summary of δ13C values, concentration, and pH values for the groundwater well samples
Table S5: pH, EF values, concentration (Conc), individual δ13C measurements of two replicate analyses (referred to as Rep 1 and Rep 2),
mean of the duplicate δ13C measurements, and one standard deviation (SD) for α-, γ- and δ-HCH isomers in groundwater from wells at the
HCH-contaminated field site. Although previous study by Law et al. reported concentrations of γ- and δ-HCH below detection limits in well
28-S, [4] sample pre-concentration following the method outline in Sections S2.3, S2.4, and S2.6 led to significant peaks (> 0.4V) from
GC/C-IRMS analysis. These peaks were clearly identified as γ- and δ-HCH based on retention times and allowed for 13C measurement.
(a)
A third replicate analysis of sample from well 22-S led to δ13C values of: −26.2 (α-HCH), −25.2 (γ-HCH), and −26.1 (δ-HCH).
Well
pH
γ-HCH
-HCH
-HCH
EF
Conc Rep 1 Rep 2 Mean
Conc Rep 1 Rep 2 Mean
Conc Rep 1 Rep 2 Mean
µg
±1
µg L−1
±1
µg L−1
±1
L−1
SD
SD
SD
22-S(a)
<4
0.508
34
−25.8 −26.3 −26.1
43
−25.9 −26.3 −25.8
30
−24.7 −25.7 −25.5
± 0.3
± 0.5
± 1.0
<4
0.503
420
−27.0 −26.6
−26.8
± 0.3
360
−27.0 −26.4
−26.7
± 0.4
290
−26.8
−26.0
−26.4
± 0.6
7-S
neutral
0.504
4.9
−25.3 −26.3
−25.8
± 0.7
5.9
−26.1 −25.8
−26.0
± 0.2
5.9
−25.7
−26.1
−25.9
± 0.3
28-S
neutral
0.613
1.0
−24.2
−24.4
−24.3
± 0.1
<0.02a
−26.9
−26.0
−26.5
± 0.6
<0.02a
−24.0
−23.8
−23.9
± 0.1
19-D
neutral
0.507
9.9
−24.6 −24.6
−24.6
± 0.0
2.7
−20.9 −22.2
−21.6
± 0.9
5.4
−23.4
−22.4
−22.9
± 0.7
34-D
218
219
15
220
References
221
222
223
224
225
226
227
[1] D. Hunkeler, R. U. Meckenstock, B. Sherwood Lollar, T. C. Schmidt, J. T. Wilson. A guide
for assessing biodegradation and source identification of organic ground water contaminants
using Compound Specific Isotope Analysis (CSIA) 2008, EPA 600/R-08/148, 67 pp. Available
from:
http://cfpub.epa.gov/si/si_public_record_Report.cfm?dirEntryId=202171&CFID=18587818&CF
TOKEN=16703234&jsessionid=cc30446f8848a49f57876e26556164371e69 (accessed on Dec.
18, 2014)
228
229
230
[2] B. Sherwood Lollar, S. K. Hirschorn, M. M. G. Chartrand, G. Lacrampe-Couloume. An
approach for assessing total instrumental uncertainty in compound-specific carbon isotope
analysis: Implications for environmental remediation studies Anal. Chem. 2007, 79, 3469.
231
232
233
[3] H. S. Dempster, B. Sherwood Lollar, S. Feenstra. Tracing organic contaminants in
groundwater: A new methodology using compound-specific isotopic analysis Environ. Sci.
Technol. 1997, 31, 3193.
234
235
236
[4] S. A. Law, T. F. Bidleman, M. J. Martin, M. V. Ruby. Evidence of enantioselective
degradation of alpha-hexachlorocyclohexane in groundwater Environ. Sci. Technol. 2004, 38,
1633.
237
238
239
[5] USEPA. Method 8081B Organochlorine pesticides by gas chromatography 2007, 57.
Available from: http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8081b.pdf (accessed on
Dec. 18, 2014)
240
241
242
243
[6] T. C. Schmidt, L. Zwank, M. Elsner, M. Berg, R. U. Meckenstock, S. B. Haderlein.
Compound-specific stable isotope analysis of organic contaminants in natural environments: a
critical review of the state of the art, prospects, and future challenges Anal. Bioanal. Chem. 2004,
378, 283.
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