Supporting Information for:
BIODEGRADATION OF THE UV FILTER BENZOPHENONE-3 UNDER
DIFFERENT REDOX CONDITIONS
YOU-SHENG LIU †, ‡, GUANG-GUO YING †, ‡, *, ALI SHAREEF ‡ AND RAI S.
KOOKANA ‡
†
State Key Laboratory of Organic Geochemistry, Guangzhou Institute of
Geochemistry, Chinese Academy of Sciences, Guangzhou, P R China
‡
CSIRO Land and Water, Water for a Healthy Country Flagship, PMB No.2, Glen
Osmond SA, Australia
The Supporting information section contains 1 table and 3 figures.
1
GC-MS and LC-MS/MS analysis of degradation products for BP-3
The biodegradation products of BP-3 under different conditions (oxic, nitrate
reducing, sulfate reducing, Fe (III) reducing and anoxic unamended) after 42 days
incubation were extracted (80 ml of slurry sample each) by using SPE as described in
the section 2.3.1 and analyzed using an Agilent 6890 gas chromatography coupled to
a 5973 mass spectrometric detector (GC-MS) and liquid chromatography-tandem
mass spectrometry (LC-MS/MS).
GC-MS was applied to analyzed biotransformation products in the extracts (2 μl
each injection) in the pulsed splitless mode. The compounds were separated on a
HP-5MS column (30m × 0.22mm, 0.25μm thickness) with helium as carrier gas at a
linear flow rate of 1mL/min. The GC oven temperature was programmed from 70 oC
(hold 2 min) to 280 oC (hold 10 min) at a rate of 8 oC /min. The injection port
temperature was set at 280 oC. The transfer line temperature was 280 oC. The MS was
operated in scan mode with its ion source temperature at 230 oC. Ionization was
carried out in electron impact (EI) mode at 70 eV. All of the major peaks in the total
ion chromatograms (TIC) obtained for samples from the BP-3 biodegradation
experiments were analysed by using Agilent Chemistation ver. D02, NIST 05 mass
spectral library and NIST Mass spectral Search Program 2.0. The mass spectrum of
each peak in the TIC was deconvoluted, and peaks were assigned identities using
automated mass spectral deconvolution and identification system (AMDIS) (National
Institute of Standards and Technology, Gaithersburg, MD, USA), which is able to
identify chemical structures, estimate molecular weight, and generate chemical
2
formulas for compounds corresponding to the respective peaks [1,2].
LC-MS/MS, a Finnigan TSQ Quantum Discovery Max (Thermo Electron, San Jose,
CA, USA), was also applied in the determination of biodegradation products of BP-3
in sample extracts (10 μl each, reconstituted in methanol). LC experiments were
conducted using a Finnigan SurveyorTM HPLC system (Thermo Electron, San Jose,
CA, USA). Separation was performed on a BDS Hypersil C18 150×2.1 mm (3 μm
particle size) column with a mobile phase flow rate of 0.2 ml/min. The mobile phase
composition was ultrapure water and methanol using the following gradient
parameters: 15% methanol (0-3 min), 90% methanol (8-12 min) and 15% methanol
(12-20 min). Mass spectrometry analysis was undertaken using atmospheric pressure
electrospray ionization (ESI) in full scan mode in the mass range between 50-500 amu
in positive ionization mode. Data acquisition and process were performed with
Xcalibur 2.0.7 software (Thermo Finnigan, USA).
Microbiological analysis
Oxic treatments
Total numbers of culturable bacteria in each BP-3 treatment were monitored on
each sampling occasion using the most probable number (MPN) technique.
Sub-samples (100 μl) were collected from each treatment bottle immediately upon
removal from the incubator. Five 10 μl replicates of the collected sub-sample were
then used to inoculate 90 μl of nutrient broth (Oxoid) in five wells of the first row of
wells in a 96-well microtitre tray. These inoculated wells were then diluted in 10-fold
3
increments in the following wells of the microtitre plates. Inoculated microtitre plates
were then incubated at 25 oC for 72 h. Growth was confirmed by visual examination
of the microtitre tray for evidence of microbial growth in each of the wells, and where
necessary through microscopic examination. The number of culturable
microorganisms in each of the original samples was then calculated by the using the
most probable number tables [3,4].
Anoxic treatments
Total numbers of bacteria were monitored for the anoxic unamended,
nitrate-reducing, sulfate reducing and Fe(III) reducing samples as described for the
oxic samples except that minimal salts, nitrate-reducing, sulfate reducing and Fe(III)
reducing mediums (minimal salts medium described in section 2.2 and amended with
either NaNO3 (20 mM), Na2SO4 (20 mM), or Fe (III) citrate (20 mM)), respectively,
were used as the growth medium and all manipulations and incubations were
undertaken in the anaerobic chamber.
Biodegradation kinetic model
The following first-order kinetic model was applied to fit the biodegradation data
of BP-3:
dC
  k 1  C  Ct  C 0  e  k 1  t
dt
Where, C0 is the initial concentration of BP-3; Ct is the concentration at time t; and k1
is the first-order rate constant. Using this equation, half-lives, t1/2 can be calculated as
4
(ln 2) / (k1).
References:
1. Ausloos P, Clifton C, Lias S, Mikaya A, Stein S, Tchekhovskoi D, Sparkman O,
Zaikin V, Zhu D. 1999. The critical evaluation of a comprehensive mass spectral
library.
J Am Soc Mass Spectrom 10: 287-299.
2. Pongsuwan W, Fukusaki E, Bamba T, Yonetani T, Yamahara T, Kobayashi A.
2007. Prediction of Japanese green tea ranking by gas chromatography /mass
spectrometry-based hydrophilic metabolite fingerprinting. J Agric Food Chem 55:
231-236.
3. Woomer PL. Most probable number. In: Weaver RW, Angle S, Bottomley P,
Bezdicek D, Smith S, Tabatabai A, Wollum A. (Eds.), 1994. Methods in Soil
Analysis. Part 2-Microbiological and Biochemical Properties. Soil Science
Society of America Inc., Wisconsin, pp.59-80.
4. Ying GG. Toze S, Hanna J, Yu XY, Dillon PJ, Kookana RS. 2008. Decay of
endocrine disrupting chemicals in oxic and anoxic groundwater. Water Res 42:
1133-1141.
5
Table S1 - The optimum GC-MS/MS parameters including retention time, collision
energy, dwell and precursor and product ions
Compound
benzophenone-3
(BP-3)
Benzylcinnamate
IS C
Retention
Time (min)
12.67
Precursor
Ion
Product Ion a
Dwell
(ms)
CE b
(V)
227.2
184.1
23.7
25
227.2
212.1
10
20
250
105
14.9
20
131
103.1
10
10
131
77
10
30
13.03
a
Bold letter indicates m/z used for quantization
CE, collision energy.
C
IS, internal standard.
b
6
NO3- concnetration (mM)
25
20
15
10
5
0
0
6
12
18
24
30
36
42
Incubation Time (days)
Figure S1 - Time course of nitrate depletion in nitrate reducing treatment of
benphenone-3. Nitrate was re-amended on day 4 to maintain the nitrate reducing
condition.
7
SO42- concentration (mM)
25
20
15
10
5
0
0
6
12
18
24
30
36
42
Incubation Time (days)
Figure S2 - Time course of sulfate depletion in sulfate reducing treatment of
benphenone-3.
8
4e+9
Abundance
[M+1]= 215
3e+9
Oxic
2e+9
1e+9
0
0
5
10
15
20
4e+9
Abundance
[M+1]= 215
3e+9
Anoxic unamended
2e+9
1e+9
0
0
5
10
15
20
Abundance
4e+9
3e+9
[M+1]= 215
2e+9
Nitrate reducing
1e+9
0
0
5
10
15
20
4e+9
Abundance
[M+1]= 215
3e+9
2e+9
Sulfate reducing
1e+9
0
0
5
10
15
20
4e+9
Abundance
[M+1]= 215
3e+9
2e+9
Fe(III) reducing
1e+9
0
0
5
10
15
20
Retention time
Figure S3 - Total ion chromatograms of LC-MS/MS of benzophenone-3 (BP-3) under
oxic, anoxic unamended, nitrate reducing, sulfate reducing and Fe (III) reducing
conditions after incubation 42 days.
9