Supplementary online material
FORMATION OF BOUND RESIDUES BY SULFADIMETHOXINE IN SOIL
MEDIATED BY EXTRACELLULAR OXIDOREDUCTASES
Rashmi Singh, Sudeep S Sidhu, Hao Zhang and Qingguo Huang*
Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223
The supplementary material contains two tables (Table S1, S2) and two figures (Figure S1, S2)
Archives of Environmental Sciences and Pollution Research
Jan 21, 2015
Five Pages
Two Tables
Two Figures
1
I. Physico-chemical properties of soil used in the study
TABLE S1. Physico-chemical characteristics of soil
Parameter
Value
Sand content (%)
66.00
Silt content (%)
23.60
Clay content (%)
10.40
pH
5.15
Procedure used
Reference
Hydrometer method
Bouyoucos,1 Day,2
0.01 M CaCl2 (1:1)
soil/solution ratio
Cuniff 3
Organic matter content (%)
2.90
Loss on ignition
Cation exchange capacity
3.70
Mehlich 1 extraction- Isaac and Johnson,4 Mehlich,5
(meq/100 g)
dilute HCl and H2SO4
Base saturation (%)
65.93
Soluble salts (mmhos/cm)
0.04
Conductivity method
Bower and Wilcox,6
II. Residual activity of laccase, horseradish peroxidase (HRP), and lignin peroxidase (LiP)
in soil
To assess enzyme activity in soil, 1 g soil samples were incubated individually with 1.5
mL solution of laccase, HRP, and LiP with and without sulfadimethoxine (SDM) in 30 mL glass
tubes. The total enzyme activity used in the study was 10 U for each enzyme, and samples were
prepared in triplicates. The laccase solution was prepared in citrate-phosphate buffer (pH 3.8)
while HRP and LiP solutions were prepared in phosphate (pH 6.0), and tartarate buffer (pH 3.0)
respectively. At pre-determined time intervals the soil samples were extracted and analyzed for
remaining enzyme activity. All the soil samples were sequentially extracted three times using 1.5
mL of their respective buffer solution. For extraction, soil samples were shaken at room
2
temperature for 30 min on an orbital shaker. The tubes were then centrifuged at 250 g for 20 min.
The enzyme activity in the supernatant was measured at the end of each extraction, and the total
activity was determined by addition of the activity in each extract. The detail procedure used for
measuring enzyme activity is described in Materials & Methods section 2.2.
In the procedure described above, the enzyme extraction was performed three sequential
times because our preliminary tests have shown that after the third extraction the supernatant did
not contain laccase activity anymore. However, it should be noted that this does not necessarily
preclude that some active enzymes may still be sorbed on soil and thus unrecovered by the
extraction, or some enzyme activity may become lost during the extraction. As such, our
measurement may provide a conservative estimate of the remaining active enzyme.
Enzyme activity (U/g)
10
Lac
HRP
LiP
Lac + SDM
HRP + SDM
LiP + SDM
8
6
4
2
0
0
24
72
120
Duration (Hours)
192
264
Figure S1. Laccase, HRP, and LiP activity in soil over 264 h. Open circle, triangle, and diamond
represent the systems without sulfadimethoxine (SDM), while solid circle, triangle, and diamond
represent the systems with SDM. Data points represent means of three replicates and error bars
represent standard deviations.
3
Extractable SDM (%)
III. Effects of enzyme activities on SDM transformation
100
90
80
70
60
50
40
30
20
10
0
Average SDM after 72h
a
b
c
d
Control
Laccase
10
d
Laccase HRP 10
25
Treatments
HRP 25
Figure S2. SDM recovery after 72 h of incubation in systems applied with laccase and HRP at 10
and 25 U g-1 soil are represented as Laccase 10, Laccase 25, HRP 10, and HRP 25, respectively.
The initial SDM concentration was 2 µg g-1 soil. Values are the means of three replicates and
error bars are standard deviations. The same lower case letters are not considered to be
statistically different according to LSD at α = 0.05.
IV. First order kinetic study of the effect of enzyme treatments on extractable SDM
A kinetic analysis was conducted on extractable SDM (the time-course data in Figure 1 in the
paper) by data fitting to the pseudo-first-order rate equation in the form ln (Ct/C0) = kt, where
C0 and Ct are extractable SDM concentrations at time 0 and t, respectively, and k is the pseudofirst-order rate constant, and the results are shown as following.
Table S2. First order reaction kinetics parameters of extractable SDM with different enzyme
treatments and different treatment methods.
Treatment
Control
LiP SAM
Lac SAM
Lac MAM
HRP SAM
HRP MAM
Degradation rate
constant k (h-1)
-0.0006
-0.0011
-0.0018
-0.0020
-0.0023
-0.0027
Half life (h)
1155.24
630.13
385.08
346.57
301.37
256.72
4
Regression
coefficient
0.7255
0.5526
0.9417
0.9640
0.6036
0.8164
V. References
1.Bouyoucos, G.J. 1936. Directions for making mechanical analysis of soils by the hydrometer
method. Soil Sci. 42:3-12.
2. Day, P.R. 1965. Particle fractionation and particle-size analysis. In: Methods of soil analysis.
Part I. C.A. Black, editor Soil Sci. Soc. Amer.
3. Cuniff, P.A. 1995. Official methods of analysis of AOAC International, 16th edition, method
2.7.08. Chapter 2. P 37
4. Isaac, R.A., and W.C. Johnson. 1983. High speed analysis of agricultural samples using
iductively coupled plasma-atomic emission spectroscopy. Spectrochemica Acta. 38 B: 277-282.
5. Mehlich, A. 1953. Determination of P, Ca, Mg, K, Na, and NH4. North Carolina Soil Test
Division (Mimeo, 1953). North Carolina Dep. Of Agric. Raleigh, NC.
6. Bower, C.A., and L.V. Wilcox. 1965. Soluble salts. In C.A. Black, editor, Methods of soil
analysis. Soil. Sci. Soc. Amer. P 933-951.
7. Swift, R. S. Organic matter characterization. In Methods of soil analysis: part 3, chemical
methods; Sparks, D. L., Page, A. L., Helmke, P. A., Loeppert, R. H., Soltanpour, P. N.,
Tabatabai, M. A., Johnston, C. T., Sumner, M. E., Eds.; Soil Science Society of America:
Madison, WI, 1996; pp 1018.
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