supporting information for occurrences and behaviors of...
TRANSCRIPT
1
Supporting Information
for
Occurrences and Behaviors of Naphthenic Acids in a Petroleum Refinery Wastewater
Treatment Plant
Beili Wang1, Yi Wan
1*, Yingxin Gao
2, Guomao Zheng
1, Min Yang
2, Song Wu
3, Jianying Hu
1
1Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences,
Peking University, Beijing 100871, China
2Chinese Acad. Sci., State Key Lab Environ Aquat Chem, Ecoenviron Sci Res Ctr, Beijing
100085, Peoples R China
3Petrochina Ji Dong Oilfield Company, Tangshang 063200, China
(Received )
*Address for Correspondence:
Address for Correspondence
Dr. Yi WAN
College of Urban and Environmental Sciences
Peking University
Beijing 100871, China
TEL & FAX: 86-10-62759126
Email: [email protected]
2
This file includes: (1) chemicals and reagents and UPLC-QTOF-MS analysis; (2) analysis of
aromatic NAs; (3) assay of agonist and antagonist; (4) calculations of mass flow, mass
proportions and solid-water partition coefficients; (5) treatment parameters in the petroleum
refinery wastewater treatment plant during the sampling period; (6) characteristics of
collected wastewater, suspended solids and sludge samples; (7) concentrations of NAs (mg/kg)
in suspended solids and sludge samples; (8) mass flux (g/d) of NAs in the petroleum refinery
wastewater treatment plant; (9) sample locations at the petroleum refinery wastewater
treatment plant; (10) profiles of NAs in suspended solids from different treatment units in the
petroleum refinery wastewater treatment plant; (11) profiles of NAs in activated sludge and
dewatered sludge; (12) mass flows (g/d) of individual aromatic NAs (C11H16O2) in the
petroleum refinery wastewater treatment plant; (13) dose-response curves of anti-estrogen
activities in wastewater samples collected from different treatment units in the petroleum
refinery wastewater treatment plant.
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Chemicals and reagents.
Fourteen NAs and three oxy-NAs were used as model compounds:
4-n-propylcyclohexanecarboxylic acid (cis- and trans-) (C10H18O2),
trans-4-pentylcyclohexane carboxylic acid (C12H22O2), 12-oxochenodeoxycholic acid
(C24H38O5), cyclohexanecarboxylic acid (C7H12O2), 1-pyrenebutyric acid (C20H16O2), abietic
acid (C20H30O2), 1-adamantaneacetic acid (C12H18O2), 2-hexyldecanoic acid (C16H32O2) and
12-hydroxysteric acid (C18H36O3) were purchased from TCI (Tokyo Chemical Industry Co.,
Tokyo, Japan); 1,2,3,4-tetrahydro-2-naphthoic acid (C11H12O2), dicyclohexylacetic acid
(C14H24O2), 5-beta-cholanic acid (C24H40O2), cyclohexane pentanoic acid (C11H20O2) and
12-hydroxydodecanoic acid (C12H24O3) were purchased from Sigma Aldrich (Oakville, ON,
Canada); 1-methyl-1-cyclohexane carboxylic acid (C8H14O2) was purchased from Alfa Aesar
(Ward Hill, MA); trans-4-tert-butylcyclohexanecarboxylic acid (C11H20O2) was purchased
from Acros Organics (Morris Plains, NJ); 1-adamantane carboxylic acid (C11H16O2) was
purchased from J&K Chemical (Beijing, China). The commercial mixture of NAs was from
Acros Organics. Methanol, hexane, ethyl acetate (EA) and methyl tert-butyl ether (MTBE)
were obtained from Fisher Chemicals (Fair Lawn, NJ). HPLC grade ammonium acetate was
purchased from Dima-Tech Inc. (Richmond Hill, ON, Canada). Hydrochloric acid and
ammonia were purchased from Beijing chemicals. Distilled water was prepared by a Milli-Q
Synthesis water purification system (Millipore, Bedford, MA).
UPLC-QTOF-MS analysis. An ACQUITY UPLC system (Waters, Milford, MA)
coupled to a Xevo QTOF-MS (G2, Waters) equipped with an electrospray ionization (ESI)
source was used in the analysis of naphthenic acids. Instrument control was performed using
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MassLynx Software (Waters, software version V4.1). All the model compounds were
separated on a Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1×100 mm). The
column was maintained at 40°C at a flow rate of 0.2 mL min-1
and the injection volume was 3
µL. The mobile phases consisting of ultrapure water containing 10 mM ammonium acetate
(A) and methanol (B) were used with gradient elution. The initial conditions were 10% B for
2 min, ramped to 60% by 3 min, ramped to 70% by 7 min, then ramped to 100% by 13 min,
and finally held for 1 min before returning to initial conditions, which were equilibrated for 5
min before injection of the next sample.
The mass spectra of NAs and oxy-NAs were acquired in the negative ion mode. The
analysis was performed in full scan mode in the mass range of 80-700 Da with a 1s scan time.
According to our preliminary experiments, the optimized parameters were as follows: source
capillary voltage of 2.0 kV, sampling cone voltage of 45 V, extraction cone voltage of 4.0 V,
source temperature 100 °C, desolvation temperature 250 °C, cone gas flow rate 50 L/h and
desolvation gas flow rate 600 L/h. The [M-H]- ion of leucine-enkephalin (200 pg/µL infused
at 5 µL/min) was used as a reference lock mass (m/z 554.2615). The QTOF detector was
calibrated with a sodium formate solution to achieve mass accuracy lower than 3 ppm by
using leucine-enkephalin as the lock mass in negative mode. The accuracy of mass
measurement in combination with the retention times in UPLC were used to calculate
empirical formulae NAs and oxy-NAs.
Analysis of aromatic NAs. Isolation of the aromatic NAs were conducted followed a
method reported previously.1 Aliquot of elute was evaporated to dryness, heated with BF3–
methanol (70 °C, 4 h) and extracted into hexane. The extract was then loaded onto solid phase
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extraction (SPE) cartridge (6 mL Discovery® Ag-Ion SPE cartridge, 750 mg sorbent; Sigma–
Aldrich, Dorset, UK). Cartridges were subsequently eluted using hexane (3×5 mL), 95%
hexane 5% diethyl ether (4×5 mL), 90% hexane 10% diethyl ether (3×5 mL) and 100%
diethyl ether (5 mL). The second fractions were redissolved in hexane for GC-MS analysis.
Derivatized aromatic NAs was analyzed using a gas chromatography−mass spectrometer
(GC-MS) (Agilent 6890N) equipped with 5975C mass spectrometer (Agilent Technologies) in
EI (electron impact) mode. Chromatographic separation was achieved on a HP-5MS capillary
column (30 cm × 0.25 mm × 0.25 µm film thickness; J&W Scientific). A splitless injector
was used, and the injector was held at 250 °C. The temperature program increased from 40 (1
min) to 250 °C at 5 °C/min, then to 300 °C (10 min) at 20 °C/min. The ion source temperature
was maintained at 300 °C. The carrier gas was helium at a constant flow rate of 1 mL/min.
Assay of agonist. In this study, the yeast two-hybrid assay with the human estrogen
receptor ERa and the coactivator TIF2 was used to test the estrogen agonist activity of
extracts of wastewater samples as previously described.2 Briefly, two expression plasmids,
pGBT9-ERLBD and GAAD424-TIF-2, were introduced into yeast cells (Saccharomyces
cerevisiae Y190) with a b-galactosidase reporter gene. The yeast cells were preincubated
overnight at 30 °C (about 14–16 h) in 5 ml medium (6.7 g/L Difco yeast nitrogen base
without amino acids, 2% glucose, 300 mg/L L-isoleucine, 1,500 mg/L L-valine, 200 mg/L
L-adenine hemisulfate salt, 200 mg/L L-arginine HCl, 200 mg/L L-histidine HCl
monohydrate, 300 mg/L L-lysine HCl, 200 mg/L L-methionine, 500 mg/L L-phenylalanine,
200 mg/L L-threonine, 300 mg/L L-tyrosine, 200 mg/L L-uracil (Sigma, St. Louis, MO, USA).
The 50-µl overnight culture and 2.5-µl DMSO solution diluted to the desired concentrations
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were then added to 200 µl fresh medium, respectively. After yeasts were cultured for 4 h at 30
°C, 150 µl of the previously described culture were fractionated, and its absorbance at 595 nm
was detected. The residual culture (100 µl) was centrifuged at 4 °C (12,000 rpm) for 5 min,
and the collected cells were resuspended in 200 µl of Z buffer (0.1 M sodium phosphate [pH=
7.0], 10 mM KCl, 1 mM MgSO4) containing 1 mg/ml Zymolyase 20T (Seikagaku, Tokyo,
Japan) and incubated for 20 min at 30°C. The enzymatic reaction was started by addition of
40 µl of 4 mg/ml 2-nitrophenyl-b-D-galactoside (ONPG, Tokyo Kasei, Tokyo, Japan) and
incubated for 20 min at 30 °C. Then the enzymatic reaction was stopped by adding 1 M
Na2CO3 (100 µl). After centrifuging the previously described solution, 150-µl aliquots were
taken into each of 96 wells of a microplate. Absorbances at 414 and 570 nm were read on a
microplate reader (Bio RAD 550, Richmond, CA, USA) to estimate estrogenic activity, and
β-galactosidase activity was calculated by
U= 1,000× ([OD414] - [1.75×OD570])/([v] ×t × [OD595]) (1)
where U = β-galactosidase activity, t = time of reaction (min), v = volume of culture used in
assay (ml), observed density (OD)595 = cell density at the start of the assay, OD414 =
absorbance by o-nitrophenol at the end of reaction, and OD570 = light scattering at the end of
reaction. In this assay, the 17β-estradiol (Sigma, 98%) was used as positive controls.
Assay of antagonist. A similar assay was used to test estrogen antagonist activity by
measuring the ability of the sample extracts to inhibit β-galactosidase induction by
17β-estradiol as described previously.3 The difference between two assays of agonist and
antagonist is that besides 50 µl overnight culture and 2.5 µl DMSO solution of sample
extracts, 2.5 µl DMSO solution of 17β-estradiol (200 nM) were also added to the fresh
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medium, and the final culture volume was 255 ml. After incubation for 4 h at 30 °C, the
β-galactosidase activity in each tube was determined following the same method as described
in the assay of agonist. The β-galactosidase activity was converted to percentage inhibition of
test chemical to the β-galactosidase induction by 17β-estradiol according to Equation 2 and
plotted versus concentration of test chemical in 255 µl culture.
Inhibition (%) = (Unitmax - Unitx)/Unitmax × 100 (2)
where Unitmax = the β-galactosidase activity of 2,000 pM 17β-estradiol and Unitx = the
observed β-galactosidase activity. In this assay, tamoxifen (Sigma) was used as positive
controls. The yeast toxicity of sample extracts was determined by measuring the decreased
β-galactosidase activity when the yeast cell was exposed to this sample as previously
described.4
Data analysis. Mass balances were performed by multiplying concentrations of target
analyte by average daily flow rates (eq 3)
W = Cwater-phase × Q + Csorbed × Q ×CTSS (3)
where “W” is the total mass of individual compound in aqueous and sorbed phase;
“Cwater-phase ”and “Csorbed” represent the water-phase and sorbed concentrations, respectively; Q
is the water flow; and “CTSS” represents the concentration of total suspended solids.
The sorption behavior of NAs was assessed using the apparent solid-water partition
coefficient (Kd, L/kg) as calculated by Csorbed/Cwater-phase, where “Csorbed” and “Cwater-phase”
represent the chemical concentrations in sorbed and water-phase from the activated sludge
treatment units, respectively.
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To assess the contribution of sorption and degradation to the removal of NAs in the
wastewater treatment plant, the raw sewage loading (including water-phase and suspended
solid phases) was taken as the system input (100%), while the system output consisted of (i)
secondary effluent, and (ii) dewatered sludge. The third part was expressed as (iii) lost, due to
the total effect of degradation or transformation mechanism in each treatment unit, and was
calculated as
Wlost = Winfluent – Weffluent – Wsludge (4)
where the “W” is the mass of total NAs within the treatment plant. To assess the mass
variations of NAs under different treatment processes, the mass change percentage (%) in
each treatment unit was calculated using (Winflow-Woutflow)/Winflow×100%, where Winflow and
Woutflow represent the total mass flow, respectively. The calculations of mass flow and
solid-water partition coefficient (Kd) in the treatment units were provided in the SI Table S4
and Table 2.
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Table S1. Characteristics of collected wastewater, suspended solid and sludge samples.
Samples Time
Sampling Frequency
Petroleum
wastewater
influent
Gravity
settling
effluent
Coagulation
effluent
Walnut
shell
effluent
Flotation
effluent
A/O
process
effluent
Secondary
effluent
Wastewater samplesa
May 15-16, 2013
(summer) 2 2 2 2 2 2 2
May 15-16, 2014
(summer) 2 2 2 2 2 2 2
October 30-31, 2013
(winter) 2 2 2 2 2 2 2
Suspended solid samplesb
May 15-16, 2013
(summer) 2 2 2 2 2 2 2
May 15-16, 2014
(summer) 2 2 2 2 2 2 2
Sludgec
May 15-16, 2013
(summer) 1
1
May 15-16, 2014
(summer) 1 1
a: each wastewater sample was a 12-h composite sample with a sampling interval of 3h.
b: suspended solid samples were collected by filtering the water sample, and the glass fiber filters used for filtering four composite water
samples collected in each treatment unit in summer were extracted as one sample for analysis.
c: the sludge samples collected in summers were mixed and extracted as one sample for analysis.
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Table S2. Treatment parameters in the petroleum refinery wastewater treatment plant during the sampling period.
Petroleum
wastewater
Gravity
settling
unit
Coagulation
unit
Walnut shell
unit
Flotation
unit
A/O process
unit
Secondary
unit
water flow (m3/d) 13000 13000 13000 9000 9000 9000 9000
TSS (mg/L) 235 ± 68 68 ± 20 97 ± 31 151 ± 85 56 ± 3 34 ± 16 32 ± 13
HRT (h) 3.5 3.5 3.5 3.5 16 4
CODCr(mg/L) 140 88 32
NO2N (mg/L) 0.03 ± 0.01 0.02 ± 0.02
NO3N (mg/L) 0.005 ± 0.001 0.009 ± 0.003
H2SO4 (mg/L) 6.3 ± 3.8 33 ± 27
Sulfur Compounds
(SCs) (mg/L) 10 0.2
Suspend Solids (SS)
(mg/L) 9.0 ± 3.5 5.0 ± 1.0
Petroleum Content
(mg/L) 36 3.9 2.2 1.0 9.4 4.2 1.5
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Table S3. Concentrations of NAs (mg/kg) in suspended solids and sludge samples collected from different treatment units in a petroleum
refinery wastewater treatment plant, north China.
NAs Petroleum
wastewater
Gravity settling
effluent
Coagulation
effluent
Walnut shell
effluent
Flotation
effluent
A/O process
effluent
Excess
sludge
Dewater
sludge
Total
Z=0
Z=-2
Z=-4
Z=-6
Z=-8
Z=-10
Z=-12
Z=-14
860
31
86
189
184
139
91
79
61
1720
96
193
399
371
259
175
130
98
1680
94
182
361
350
275
180
134
105
1289
101
171
277
244
197
140
96
64
2772
261
597
594
454
360
237
159
110
1564
111
146
236
299
356
222
108
86
1594
70
155
288
319
359
194
114
95
2489
194
419
605
485
326
198
149
113
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Table S4. Mass flux (g/d) of NAs in the petroleum refinery wastewater treatment plant.
NAs Petroleum
wastewater
Gravity
settling
effluent
Coagulation
effluent
Walnut shell
effluent
Flotation
effluent
A/O process
effluent
Secondary
effluent
Excess
sludge
Dewatered
sludge
water-phase
Z=0 455 ± 91 266 ± 16 274 ± 64 250 ± 59 290 ± 124 46 ± 31 56 ± 47 - -
Z=-2 981 ± 187 821 ± 161 778 ± 52 756 ± 90 998 ± 163 176 ± 215 53 ± 18 - -
Z=-4 977 ± 277 815 ± 92 753 ± 118 735 ± 68 981 ± 52 222 ± 206 133 ± 64 - -
Z=-6 469 ± 283 319 ± 116 295 ± 97 295 ± 74 451 ± 43 201 ± 63 212 ± 173 - -
Z=-8 280 ± 204 168 ± 52 145 ± 46 143 ± 29 187 ± 32 155 ± 72 180 ± 155 - -
Z=-10 167 ± 110 102 ± 33 90 ± 28 92 ± 21 118 ± 27 107 ± 48 125 ± 100 - -
Z=-12 119 ± 95 68 ± 29 59 ± 25 59 ± 18 73 ± 24 106 ± 67 141 ± 121 - -
Z=-14 80 ± 72 42 ± 20 37 ± 18 36 ± 13 42 ± 19 90 ± 62 117 ± 114 - -
sorbed
Z=0 65 ± 29 59 ± 33 82 ± 33 137 ± 43 132 ± 50 34 ± 14 - 0.4 ± 0.1 27 ± 13
Z=-2 183 ± 80 118 ± 67 159 ± 64 233 ± 74 301 ± 115 45 ± 19 - 0.9 ± 0.3 58 ± 29
Z=-4 400 ± 175 244 ± 139 315 ± 128 376 ± 120 299 ± 115 72 ± 30 - 1.7 ± 0.6 84 ± 42
Z=-6 389 ± 171 227 ± 129 305 ± 124 331 ± 105 229 ± 88 92 ± 38 - 1.9 ± 0.6 67 ± 34
Z=-8 294 ± 129 159 ± 90 240 ± 97 268 ± 85 181 ± 69 109 ± 46 - 2.2 ± 0.7 45 ± 23
Z=-10 193 ± 84 107 ± 61 157 ± 64 190 ± 60 119 ± 46 68 ± 28 - 1.2 ± 0.4 27 ± 14
Z=-12 167 ± 73 79 ± 45 117 ± 48 130 ± 41 80 ± 31 33 ± 14 - 0.7 ± 0.2 21 ± 10
Z=-14 129 ± 57 60 ± 34 92 ± 37 87 ± 28 56 ± 21 26 ± 11 - 0.6 ± 0.2 16 ± 7.8
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Figure S1. Sample locations at the petroleum refinery wastewater treatment plant, north China.
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Figure S2. Profiles of NAs in suspended solids from different treatment units in the petroleum refinery wastewater treatment plant, (a)
petroleum wastewater, (b) gravity setting, (c) coagulation, (d) walnut shell, (e) flotation and (f) A/O process.
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Figure S3. Profiles of NAs in activated sludge (a) and dewatered sludge (b).
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Figure S4. Mass flows (g/d) of individual aromatic NAs (C11H16O2, selected ions: 133, 105, 77 ref by 1) in a petroleum refinery wastewater
treatment plant, north China.
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Figure S5. Dose-response curves of anti-estrogen activities in wastewater samples collected from different treatment units in a petroleum
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refinery wastewater treatment plant, north China. a) petroleum wastewater, b) gravity settling effluent, c) coagulation effluent, d) walnut shell
effluent, e) flotation effluent, f) A/O process effluent, g) secondary effluent and h) tamoxifen as an anti-estrogenic positive control.
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M. Development of the antagonist assay system for estrogen receptor using yeast and its
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4. Hu, J. Y.; Xie, G. H.; Aizawa, T. Products of aqueous chlorination of 4-nonylphenol and
their estrogenic activity. Environ. Toxicol. Chem. 2002, 21, 2034-2039.