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Effects of temperature on UV-B-induced DNA damage and photorepair in Arabidopsis
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thaliana
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Shaohua Wang1,2, Ping Wang2,*
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1. Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
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E-mail: jesc@263.net (first author’s email address)
2. School of Life Sciences and Technology, Jinan University, Guangzhou 510632, China
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Abstract: DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and
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photoproducts (6-4 PPs) induced by UV-B radiation in Arabidopsis thaliana at different
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temperatures was investigated using the technologies with specific monoclonal antibodies.
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CPDs and 6-4 PPs increased during 3 hr UV-B exposure, but further exposure led to
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decreases. Contrary to the commonly accepted view that DNA damage induced by UV-B
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radiation is temperature-independent because of its photochemical nature, we found UV-B-
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induction of CPDs and 6-4 PPs in Arabidopsis to be slower at a low than at a high
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temperature. Photorepair of CPDs at 24℃ was much faster than that at 0 and 12℃, with 50%
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CPDs removal during 1 hr exposure to white light. Photorepair of 6-4 PPs at 12℃ was very
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slow as compared with that at 24℃, and almost no removal of 6-4 PPs was detected after 4 hr
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exposure to white light at 0℃. There was evidence to suggest that temperature-dependent
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DNA damage and photorepair could have important ecological implications.
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Key words: cyclobutane pyrimidine dimers; Arabidopsis thaliana; DNA repair; uvrA; UvrA
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*
Corresponding author. E-mail: jesc@263.net
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Introduction
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There is growing awareness of the potential biological effects of stratospheric ozone
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depletion as part of global climatic change (Leun et al., 1995 (three or more authors)). The
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most important consequence of the ozone depletion is an increase in the amount of UV-B
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(280-315 nm) radiation reaching the surface of earth. Even modest increase in UV-B radiation
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is likely to cause significant biological damage (Björn et al., 1999a). Investigations during the
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past two decades have demonstrated that UV-B radiation has many direct and indirect effects
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on plants, including damage to DNA. The most common DNA lesion caused by exposure to
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UV-B is the formation of dimers between adjacent pyrimidines in the same strand, i.e., the
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cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidinone adducts (6-4 PPs)
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(Britt, 1996 (one author)). Unrepaired dimers are leathal to cells because they deform the
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DNA helix, interfering with both replication and transcription. Both types of DNA damage
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can be reversed by subsequent exposure to radiation ranged from 360-420 nm (UV-A to blue
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light). This phenomenon is termed photoreactivation or photorepair and is due to the actions
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of one or more proteins tremed “photolysis”. These enzymes specifically recognize and bind
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to pyrimidine dimers (Britt and Mori, 1999 (two authors)).
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DNA damage and repair has been investigated in several plant species (Mitchell et al.,
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1993a, 1993b; Pang et al., 1991; Takeuchi, 1996; Taylor, 1996; Björn et al., 1996, 1999), but
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information on the effects of environmental factors such as temperature is limited. Interaction
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of increased solar UV-B radiation with other climatic change factors such as global warming
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is scarce.
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In the present study, we examined the temperature effects on the formation and photo
repair of DNA damage induced by UV-B radiation in Arabidopsis thaliana.
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1 Materials and methods
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1.1 Soil samples
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Soil samples were collected from surface layer (0-20 cm in depth) of a paddy field
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located in Sumen Village, Binjiang Country, Guixi City, Jiangxi Province, central subtropical
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China (28°20.307′N and 117°14.133′E). This about 20,600 m2 paddy fields have been
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contaminated with Cu and Cd by sewerage from an adjacent smelting factory for more than
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20 years (Hu et al., 2004). The paddy soil was developed from red sandstone with 13.0% clay,
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40.5% silt and 46.5% sand, and the main properties of soil are shown in Table 1. The total
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concentrations of Cu and Cd significantly exceed the environmental quality standard for
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agricultural soils (Cu 50 mg/kg and Cd 0.3 mg/kg in GB 15618-1995) issued by State
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Environmental Protection Administration of China. Unfortunately, rice is still planted on the
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contaminated soils by local farmers due to drive of compensation mechanism and poverty.
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1.2 Plant material and growth conditions
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Arabidopsis thaliana ecotype Columbia-0 was used in all experiments. Seeds were
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surface-sterilized with 75% ethanol, rinsed with water, and incubated for 2 days at 4℃, then
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distributed in commercial mixture medium and covered with glass for 48 hr to ensure high
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humidity for an even germination. After growing for 10 days, young plants were transplanted
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to 6 cm × 6 cm plastic pots (5 plants in each pot) and grown in a greenhouse under 800
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µmol/(m2·sec) photosynthetically active radiation (PAR, 400-700 nm), supplied by 400 W
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dysprosium lamps (Osram Powerstar, Germany). Spctrum of this type of dysprosium was
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shown in our former paper (Li, 2002).
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Fully expanded leaves were used as plant materials. Detached leaves with abaxial
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surface up floating on distilled water in Petri dish, were exposed to UV-B radiation at a
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distance of 20 cm from the lamps, and without any other illumination.
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1.3 UV-B and white light irradiation
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UV-B radiation (also containing UV-A) was obtained from 6 UVB-313 lamps (Q-
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PANEL, USA) and filtered through 0.13 mm cellulose diacetate. All radiation below 280 nm
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was filtered out. Measurement of spectral irradiance was same as our privious report (Li,
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2002b). Irradiance of the UV-B region (280--315 nm) was 2.95 W/m2. White light, 150 W/m2
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in the interval 400-700 nm, used for photorepair experiments, was supplied by a 400 W lamp
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(Osram Powerstar, Germany) and filtered through a 10 cm depth of water in a transparent
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polystyrene container to remove excess infrared radiation. Radiation measurements were
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carried out with a model 754-6S spectroradiometer (Optronic Laboratories, USA). Spectral
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irradiances of UV-B and white light for photorepair experiments were shown in our privious
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report (Li, 2002).
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In the isotherm experiments, 4 g of samples were mixed with 100 mL Pb 2+ solution (80,
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400, 800, 2000 and 4000 mg/L) at 200 r/min and 25℃ for the equilibrium time. To gain a
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comprehensive knowledge of the Pb2+ adsorption process, the adsorbents included S, P, SP1
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(0, 2, 4, 6, 9 and 10 days), SP2 (10 days) and SP3 (10 days). The Pb 2+ solution after
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adsorption was collected and measured using the method mentioned above, with each
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treatment being in triplicates. The total number of treatments in the experiment was 150
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(5×10×3). The Pb2+ adsorbed amount (Q) was calculated by Eq. (1):
Q  (C0  C ) V / m
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(1)
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where, C0 (mg/L) and C (mg/L) are the initial and final Pb2+ concentrations, respectively; V (L)
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is the solution volume in the flask, m (g) is the dry mass of the absorbent, including S, P, and
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SP.
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The foregoing steps were repeated four times to obtain the irreversible parameter of
adsorption-desorption (H), which was calculated by Eq. (2) (Yang and Zhang, 2007):
H
bdes
bads
(2)
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where, H is the irreversible parameter of adsorption-desorption, and bdes and bads are
adsorption constant and desorption constant, respectively.
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2 Results and discussion (results and discussion can be written separately)
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2.1 UV-B induced DNA damage in Arabidopsis thaliana
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Arabidopsis thaliana plants grown in greenhouse were exposed to UV-B radiation in
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dark room at 24℃. Both types of dimeric pyrimidine photoproducts were induced in plant
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leaves. The CPDs content of leaves increased during 3 hr UV-B exposure and a smaller
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increase of 6-4 PPs was observed (Fig. 1). Further exposure of UV-B radiation led to decrease
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of both types of DNA damage. The decrease is probably due to photorepair activity driven by
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the UV-A radiation supplied together with the UV-B. It was deduced from our result that A.
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thaliana, as quatitified by dimer formation in DNA, was very sensitive to UV-B radiation.
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2.2 Effect of temperature on DNA damage
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Figure 2 shows that UV-B-induced DNA damage in A. thaliana depends on
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temperature. When detached leaves were exposed to UV-B radiation for 2 hr at 12 and 24℃,
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more CPDs and 6-4 PPs accumulated than at 0℃ (t-test, P < 0.01) (Fig. 2a), but the
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difference of 6-4 PPs formation between at 12 and 24℃ was not significant. Both CPDs and
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6-4 PPs were induced by UV-B radiation even at 0℃ (Fig. 2b).
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3 Conclusions
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Temperature is one of the major environmental factors controling survial, growth,
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reproduction, and thus geographic distribution of plants. The study of combined temperature
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and UV-B radiation could be of importance with respect to possible effects of climatic change,
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especially global warming and increasing levels of UV-B radiation caused by the depeletion
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of stratospheric ozone layer. The present investigation provided molecular evidence for
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temperature-dependence of UV-B-induced DNA damage and photorepair.
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Acknowledgments
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This work was supported by the National Natural Science Foundation of China (No.
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30200030) and the Natural Science Foundation of Educational Department of Guangdong
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government (No. 20070506).
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References
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Aleksander-Kwaterczak, U., Helios-Rybicka, E., 2009. Contaminated sediments as a potential
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source of Zn, Pb, and Cd for a river system in the historical metalliferous ore mining and
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smelting industry area of South Poland. J. Soils Sediments 9(4), 13-22.
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Anderson, C.G., 2012. The metallurgy of antimony. Chemie der Erde. 72(S4), 3-8.
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Douay, F., Roussel, H., Fourrier, H., Heyman, C., Château, G., 2007. Investigation of heavy
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metal concentrations on urban soils, dust and vegetables nearby a former smelter site in
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Mortagne du Nord, Northern France. J. Soils Sediments 7(3), 143-146.
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Duan H., Huang Q., Wang Q., Zhou B., Li J., 2008. Hazardous waste generation and
management in China: A review. J. Hazard. Mater. 158(2-3), 221-227.
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Levinsky, N.G., 2008. Fluid and electrolytes. In: Thorn, G.W., Adams, R.D., Braunwald E. et
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al., (Eds.), Harrison’s Principles of Internal Medicine (3rd ed.). McGraw-Hill, New York,
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pp. 364-375.
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148
149
150
151
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155
156
Gebel, T., 1997. Arsenic and antimony: comparative approach on mechanistic toxicology.
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List of tables
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Table 1 Heavy metal contents in the slag and dust samples
Element
DS
WQS
AAR
BFD
As (mg/kg)
700
5700
86000
1280
Sb (mg/kg)
6930
11100
316000
234000
Cd (mg/kg)
0.44
0.07
1.04
5.60
Co (mg/kg)
2.29
32.6
0.23
10.7
Cr (mg/kg)
28.3
213
12.0
116
5
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DS: desulfurized slag; WQS: water-quenched slag; AAR: arsenic-alkali residue; BFD: blast furnace dust.
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List of figures
- 4 .5
ln k c a p p ( ( m o l/L ) s e c - 1 )
0
- 5 .0
y = - 7 .3 9 + 0 .9 8 x
2
-1
ln ( C A /C A 0 )
-1
0  m o l/L
-2
2 .5  m o l/L
5 .0  m o l/L
-3
7 .5  m o l/L
R = 0 .9 9 5
- 5 .5
- 6 .0
- 6 .5
1 0  m o l/L
a
1 5  m o l/L
-4
0
10
20
30
b
- 7 .0
40
1 .0
1 .5
2 .0
2 .5
ln C R u 0 (  m o l/L )
T im e ( m in )
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Fig. 1 (a) Influence of Ru(III) concentrations on the performance of Ru(III)-catalyzed permanganate
164
oxidation and (b) the plot of lnkcapp versus lnCRu0. Reaction conditions: CMn0 = 50 µmol/L, CA0 = 5 µmol/L,
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pH = 7.0 ± 0.1 and temperature 25 ℃.
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1.0
-1
0.5W/mL, k=0.0307 min
-1
1.0W/mL, k=0.0687 min
-1
2.0W/mL, k=0.191 min
0.8
c/c0
0.6
0.4
0.2
0.0
0
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168
169
170
20
40
60
80
100
120
Time (min)
Fig. 2 BPA degradation as function of ultrasonic power densities. Experimental conditions: initial BPA
concentration of 1mg/L, ultrasonic frequency of 800 kHz, and power intensity of 3 W/cm2.
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6