1
Fate of Ag-NPs in sewage sludge after application on agricultural soils
2
Supporting Information
3
Ana.E Pradas del Real1, Hiram Castillo-Michel2, Ralf Kaegi3, Brian Sinnet3, Valérie Magnin1, Nathaniel
4
Findling1, Julie Villanova4, Marie Carrière5,6, Catherine Santaella7, Alejandro Fernández-Martínez1,
5
Clément Levard8, Géraldine Sarret1
1
6
ISTerre (Institut des Sciences de la Terre), Université Grenoble Alpes and CNRS, Grenoble, France.
2
7
ID21, ESRF-The European Synchrotron, CS40220, 38043 Grenoble Cedex 9, France
3
8
4
9
Eawag, Particle Laboratory, Dübendorf, Switzerland.
ID16b, ESRF-The European Synchrotron, CS40220, 38043 Grenoble Cedex 9, France
5
10
Univ. Grenoble-Alpes, INAC-SCIB, F-38000 Grenoble, France
6
11
12
7
CEA, INAC-SCIB, F-38000 Grenoble, France
Lab Ecol Microb Rhizosphere & Environ Extrem, UMR 7265 CEA-CNRS-Aix Marseille Université, CEA Cadarache,
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14
Saint Paul Les Durance, France
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Aix-Marseille Université, CNRS, IRD, CEREGE UM34, 13545, Aix en Provence, France
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Number of pages: 23
17
Number of figures: 12
18
Number of tables: 8
19
20
2. MATERIALS AND METHODS
21
2.1 Nanoparticle characterization
22
PVP-coated Silver Nanoparticles (PVP-Ag-NPs) were provided by NanoAmor, Nanostructured and
23
Amorphous Materials Inc. (USA). These Ag-NPs were chosen based on their broad size distribution
24
which probably closely reflects real NPs inputs to WWTPs. Ag-NPs were thoroughly characterized in
S1
25
powder and in suspension. Shape and nominal diameter were determined by Transmission Electron
26
Microscopy (TEM-TECNAI OSIRIS). Specific Surface Area (BET) was determined by the Volumetric Gas
27
(N2) Adsorption Method at 77K (BELSORP-max, BelJapan.Inc). Coherent domain size was determined
28
by X-Ray diffraction (XRD). XRD patterns were recorded with a Bruker D5000 powder diffractometer
29
equipped with a SolX Si(Li) solid state detector from Baltic Scientific Instruments using CuKα1+2
30
radiation. Intensities were recorded at 0.04°, 2-theta step intervals from 15 to 70° (5 s counting time
31
per step). Rietveld refinement of the powder diffraction patterns were carried out using the FullProf
32
package1. In suspensions of 100 mgPVP-Ag-NPs·L-1 in ultrapure water, the hydrodynamic diameter
33
was measured by Dynamic Light Scattering (DLS), Z potential was also measured over a range of pH
34
from 2 to 10 (Zetasizer Nano ZS Malvern Instruments). In the same suspensions, ionic Ag+
35
concentration was measured with an ion specific silver/sulfide Ion selective electrode (Thermo
36
Scientific) calibrated with a series of AgNO3 standard solutions.
37
38
-1
39
*Hydrodynamic diameter zeta potential and ionic Ag+ were measured in suspensions of 100 mg PVP-Ag-NPs ·L at pH 7
40
Figure S1: PVP-Ag-NPS characterization: a) Summary of the main characteristics (shape and nominal diameter were
41
investigated in 120 particles), b) TEM image of the PVP-Ag-NPs, c) Experimental and Rietveld refined X-ray diffraction
42
patterns, d) Determination of the point of zero charge of Ag-NPs according to the pH.
S2
43
PVP-Ag-NPs suspensions for spiking into the plant were prepared in 4mM citric acid. They were
44
subjected to ultrasonication for 30 min in a Bandelin Sonoplus sonicator at 50W with pulses of 5s in
45
5min intervals, and pH was adjusted to 7 with NaOH (1M).
46
2.2. Description of WWTP and spiking process
47
The sludge was produced in a pilot treatment plant at EAWAG (Dübendorf, Switzerland). The
48
background of Ag in the sludge was 14 mgAg·kg-1, this sludge was used as control. Then the objective
49
was to obtain a final digested sludge polluted with 18 mgAg·kg-1 (low dose) and 400mgAg·kg-1 (high
50
dose). A schematic layout of the the WWTP is given in Figure S3. Municipal wastewater was first
51
settled in a larger sedimentation tank (primary clarifier, 9m3) and then fed into the pilot WWTP (16
52
L·h-1). The pilot WWTP consisted of a non-aerated tank (denitrification, 91 L), an aerated tnak
53
(nitrification, 120 L), a secondary clarifier (120 L), a thickener (32 L) and an anaerobic digester (300 L).
54
The hydraulic retention time was 21 hours and the average sludge age was 14 days. In the biological
55
process, the secondary sludge was continuously recirculated to the denitrification tank to reduce the
56
nitrate produced in the nitrification stage. The treated wastewater from the top of the clarifier left
57
the pilot WWTP (16 L·h-1). In the thickener, 16 L of secondary sludge from the nitrification tank were
58
mixed once a day with 16 L of primary sludge. After 45 min of mixing, the sludge was settled for 3 h
59
and then transferred to the anaerobic reactor (8 L·day-1). The remaining 24 L in the thickener were
60
discharged into the sewer system. 8 L of digested sludge leaves the anaerobic reactor per day. The
61
hydraulic residence time in the anaerobic reactor was 37.5 days. On average, the total suspended
62
solids (TSS) were ~5.4 g·L-1 in the primary sludge, ~1.7 g·L-1 in the secondary sludge and ~10 g·L-1 in
63
the anaerobic digester (Apollo 1).
64
Nitrification tests were performed before the spiking of PVP-Ag-NPs into the pilot WWTP to evaluate,
65
if at planned working doses, Ag is toxic to the nitrifying bacteria of the biological treatment.
66
Standards batch nitrification test were conducted with 0, 100 and 1000 mg·kg-1 of Ag spiked to the
67
mixed liquor (7 L) collected from the nitrifying stage of the main WWTP at EAWAG. A control assay
S3
68
was carried out in parallel to each Ag assay. NH4HCO3 (85mg·L-1, WWR International, LLC) was added
69
to each reactor and after 30 min, selected concentrations of Ag were spiked to the reactor. Dissolved
70
oxygen (mg·L-1), pH, EC (µS·cm-1) and T(°C) were monitored with a WTW 3430 multiparameter sensor.
71
Samples were collected from both reactors each 15min for 2h, filtered through 0.7 µm and 0.45 µm
72
filters (Nanocolor 50 chromafil GF/PET) and stored at 4°C until analysis. Samples were analyzed for
73
total NH4+, NO2- and NO3- by Ion chromatography. Dissolved oxygen (mg·L-1), pH, EC and T (°C) were
74
stable during experiments and nitrification was still operating at all doses (10 to 1000 mg·Kg-1), and
75
did not jeopardize the water treatment process (Figure S2).
S4
76
77
+
Figure S2: Decrease of NH4 over time for control and Ag-spiked sludge.
S5
78
In order to load the digested sludge with the desired concentration of Ag, suspensions (described
79
above) of PVP-Ag-NPs were spiked into two different stages of the WWTP: the denitrification tank
80
and the thickener. In the denitrification tank the Ag-NPs suspension was continuously spiked. In the
81
thickener PVP-Ag-NPs suspensions were spiked once a day for 30min, after 5 min of mixing both
82
primary and secondary sludge. After the spiking, the sludge was mixed for another 10 min and
83
settled for 3h before being transferred to the anaerobic reactor. PVP-Ag-NPs were spiked into the
84
plant for 5 weeks until the desired concentration in the digested sludge was reached.
85
At the end of each experiment the digested sludges was collected from the anaerobic digester and
86
dewatered by two consecutives centrifugation cycles of 5 and 15 min at 4000 rpm. The centrifuged
87
sludge was air dried and grounded by using an agate mortar. In the high dose batch, samples were
88
collected from the anaerobic digester and from the denitrification and the nitrification tanks
89
(secondary sludges) as well. They were centrifuged at 4000 rpm for 10min and stored at -80°C until
90
analysis.
91
S6
92
93
Figure S3: Schematic layout of the pilot WWTP.
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95
2.3. Pot experiment
96
Sludge and soil characteristics were determined by ISO certified methods (Total Organic Matter an
97
total Organic Carbone: ISO 14235, Total N and C/N ratio: ISO 13878, SO4-2: ISO 11263, S total: ISO
98
11885, O2O5: ISO 11263, NO3- and NH4: ISO 14256-2). Exangables K2O, MgO, CaO and Na2O were
99
determind by the AFNOR method NFX 31-108 in order to calculate the Cation Echange Capicty (CEC).
100
pH was measured by mixing the samples with MQ water in 1:2.5 ratio. Results are given in table S1.
101
Taken into account their % humidity, the soil and the corresponding sludges were mixed in 1/10 ratio
102
(sludge/soil). The homogenization was performed by intercalating a layer of soil (~1000 g) with a
103
layer of sludge (~100 g). 4 layer of each were supoerposed and thoroughly mixed. 200g of each
104
mixture were placed in the corresponding pots. Blank pots were filled just with soil. Seven replicates
S7
105
were used for each treatment (3 plant experiment+ 3 microbiology + 1 pot to record data before
106
plant culture, T0). Afterwards, the weight of each pot was determined and noted. 50 mL of tap water
107
were dded to each pot to reach saturation (100% moisture content) and weights were noted again.
108
Until planting time pots were keep at 65-70% water content (considered as no water stress
109
conditions) in a chamber 16/8h photoperiod, 21/15°C, 70% humidity. Soils were rewatered as
110
necessary every two days to maintain constant moisture content, water to be added was calculated
111
by weight. One week after incubation in that way, soil samples were recorded (T0) and pots were
112
planted with the corresponding specie.
Soil
113
-2
P2O5
-
+
C total
N total
(g·Kg )
-1
(g·Kg )
5.07
87.9
50.8
6.27
8.1
18
0.42
0.16
181
17
14.29
6.54
6.59
6.84
296
372
390
171
251
225
42.4
53.4
45.9
4
4
4.9
308
501
451
9.93
12.3
10.8
0.88
0.77
0.73
31
8.1
30
2064
3273
558
97.29
110.71
115.86
pH
38% sand 43% silt 20% clay
Silty
Sludge control
Sludge LD
Sludge HD
OM total
(g·Kg )
Texture
-1
-1
C/N
SO4
S total
(mg·Kg ) (g·Kg-1)
-1
-1
)
(g·Kg
N-NO3
N-NH4
CEC
-1
(mg·Kg ) (mg·Kg ) (meq·100g)
-1
114
Table S1: Sludge and soil characteristics.
115
The pore water of soils was studied by placing one Rhizon soil moisture sampler (Rhizon SMS 19.21,
116
length: 5 cm, diameter: 2.5 mm) in each pot to extract about 4.5 mL solution from the rhizosphere.
117
The pore water was collected 30 min after saturation of soils. pH was measured inmediatly after
118
collection.Samples were frozen (-20°C) untill analisys for Total Organic Carbon (TOC-Vcsnn,
119
Shimadzu).
120
2.4. Silver concentrations in soils and pore water
121
Silver concentrations were determined by ICP-MS in soil samples, plant tissues and in pore water of
122
all treatments after plant culture. In the high dose treatment, Ag was also measured in the CaCl2 and
123
DTPA exchangeable fractions. Concentrations in other metals (Zn, Fe, Cu) were determined by ICP-
124
AES on the same samples.
125
For the extraction of the fine fraction, 15 g of soils were diluted in 500mL of MQ water and placed in
126
centrifuge. After 2 days of agitation, they were centriguged 40 min at 4000 rpm. Based on equation
S8
127
1, the top 7cm were collected. Remaining suspension was diluted agin to 500 mL. The procredeure
128
was repeated several times. CaCl2 1M was added to the collected supernatant suspensions to
129
flucolate the particles. They were decanted overnight and centrifugued (4300 rpm, 30min) to remove
130
the supernatant. The remainning pellet corresponded to the fine fraction (<2 µm) and was
131
lyophyliced to remove the water.
132
To obtain the CaCl2 and DTPA exangeable fractions, 1 g of soil were shaken for 7 h in 10 mL of either
133
0.1 M CaCl2 or DTPA solution (0.1 M triethanolamine TEA, 0.01 M CaCl2 and 0.005 M DTPA buffered
134
at pH 7.3). The suspension was then centrifuged for 10 min at 3073 g. The supernatant was filtered
135
on 0.22 μm cellulose acetate filters and acidified with HNO3 (ultrapure grade reagent) and kept at 4°C
136
until analysis.
137
The bioavailability of Ag in the amended soils was investigated by measuring Ag concentration in the
138
extracted pore water.
139
Solid soil dried samples and lyophilized plant tissues were mineralized before Ag determination by
140
adding HNO3 and HF. Then samples were heated at 120°C for 4h. After acid evaporation, samples
141
were diluted in HNO3 5% untill analysis. Ag was determined by ICP-MS and Fe, Cu, and Zn by ICP-OES,
142
all samples were filtered to 20 µm before analysis. NIST 2782 reference matherial (Industrial Sludge)
143
was used to test the efficency of the process. The recovery of Ag was 105%, taken into account that
144
Ag2S is the main specie found in sludges and that Ag2S is one the Ag species with the lowest
145
solubility, we can assume a high efficiency in the extraction of all Ag species by the applied methods.
146
The recovery percentages were 111 % for Cu, 125 % for Zn and 91 % for Fe.
147
Metal concentration data were analysed by General Lineal Model (GLM) using the statistical package
148
SPSS version 19.0. GLM was followed by a post hoc Duncan test to assess the significance of
149
differences among sludge amendments and plant treatments for each variable. Values given in tables
150
and figures indicate mean values (n=3) ± standard error (S.E.).
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2.5. Silver speciation and distribution by synchrotron techniques
S9
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Ag reference compounds
153
The following solid state Ag references were diluted in boron nitride and pressed into 5 mm diameter
154
pellets: Ag diethyldithiocarbamate (C5H10AgNS2, Ag-DEDTC), AgPO4, AgCl, AgNO3, Ag2O, Ag2CO3, PVP-
155
Ag-NPs, natural macrocrystalline Ag2S (acanthite) and nano-Ag2S obtained from the sulfidation of the
156
PVP-Ag-NPs. To get this last reference PVP-Ag-NPs were sulfidized as described in Levard et al.,
157
(2011)2. Briefly, a suspension of 1mM PVP-Ag-NPs (pH=7) was mixed with a 1mM Na2S in a 0.01M
158
NaNO3 electrolyte. After 3 days of reaction, solutions were centrifigued, washed three times with
159
MQ water and dried. The total transformation of Ag0 from the PVP-Ag-NPs to Ag2S was tested by X-
160
ray Difraction (XRD). The obtained nanoAg2S was characterized as described for the PVP-Ag-NPS
161
Ag-GSH aqueous reference compounds at different Ag/GSH ratios and pH (1/10 pH7, 1/1 pH 7.5, 1/2
162
pH 7.5, 1/1 pH5, 1/1 pH 4) were prepared in anoxic conditions and mixed with 20% glycerol to
163
prevent ice crystal formation during cooling.
164
165
Figure S4: Ag2S reference compound characteristics: a) summary of main characteristics, b)TEM image of synthesized
166
nanoAg2S, c) Experimental and Rietveld refined X-ray diffraction patterns of synthesized nanoAg2S c) and mineral Ag2S
167
(Acanthite) d).
168
S10
169
170
Ag-K edge X-ray absorption spectroscopy
171
Ag K-edge (25.5 keV) bulk XANES and EXAFS measurements were performed in the beamline FAME
172
(BM30B) at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Pellets of samples
173
(frozen hydrated in the case of sludge from the various reactors) and reference compounds were
174
transferred into a helium cryostat operating at 10K for analysis. Spectra were recorded using a Si
175
(220) double crystal monochromator and in fluorescence mode with a 30-element solid-state Ge
176
detector (Canberra, France). To limit beam damage, spectra were collected on several points of the
177
pellets, 10 to 17 scans of 40 min were averaged. XANES and EXAFS data were treated both by
178
principal components Analysis (PCA) and linear combination fitting (LCF) and by shell fitting.
179
By using ATHENA software 3, backgrounds were subtracted and spectra were normalized. Principal
180
components Analysis (PCA) of both the XANES and the EXAFS spectra was performed as stated in
181
Ressler et al. (2000)4 by the use of the PCA software developed in beamline 10.3.2 at the Advanced
182
Light
183
(https://sites.google.com/a/lbl.gov/microxas-lbl-gov/software). Briefly, the number of components
184
needed to describe the system was evaluated statistically with the IND indicator. Then, the Ag model
185
compounds that constitute the most probable components of the original XANES and EXAFS spectra
186
were identified by Target Transformation.
187
Once main Ag species were identified, their proportion in the samples was determined by Least-
188
Squares Combination Fitting (LCF) of the experimental spectra with the spectra of selected reference
189
compounds in the 25550-25600 eV range. The quality of the fits was estimated using the NSS
190
parameter as defined for Ag k-edge. In general, it is consided that the accuracy of the LCF method is
191
10% so an species is not included in the LCF unles it accounts for more than a 10%5-6. Ag K-edge
192
EXAFS spectra were collected when the Ag concentrations were high enough. The extraction of
193
EXAFS signal was performed according to standard methods. The k3-weighted EXAFS spectra were
Source,
Lawrence
Berkeley
National
Lab
(ALS-LBNL,
Berkeley,
CA)
S11
194
least-squares fitted over a k range of 3.0 –11 Å-1, using natural Ag2S and Ag-DEDTC as components.
195
The quality of the fit was estimated by the NSS parameter where NSS = Ʃ[k3 χexp − k3 χ fit]2 / Ʃ [k3 χ exp]2
196
x 100).
197
In parallel, the structural parameters of Ag reference compounds and samples were determined by
198
shell simulations using ARTEMIS (Ravel and Newville, 2005). Phase and amplitude functions of Ag
199
standards were calculated by FEFF 6.0 from Ag-structures found in the literature. EXAFS spectra were
200
Fourier transformed over a k range between 2.4 and 11.7Å-1 depending on the sample. The
201
contribution of the first, second and third shells was simulated in k and R space.
202
Transmision Electron Microscopy Measurements
203
Samples of the final dried sludge were prepared for Transmission Electron Microscopy as described
204
in Kaegi et al. (2011)7. The dried sludge was diluted 1:100 in MQ water and centrifuged for 2h at
205
3000g directly on TEM grids. Samples were examined in a TEM (TECNAI-OSIRIS) in scanning mode
206
with a DF4 detector and an acceleration voltage of 200 keV. Elemental analysis was performed with a
207
Energy Dispersive X-ray system (EDX)
208
209
210
211
212
213
214
215
S12
216
217
3. RESULTS AND DISCUSSION
218
3.1. pH and Total Organic Carbon in the pore water
219
Table S2: pH and Total Organic Carbon (TOC) in the porer water collected before (T0) after 4 weeks of plant growth (TF).
-1
TOC (mg·L )
T0
TF
pH
T0
Wheat
Soil
6.5
19.9 ± 1.5
7.2
a
68.4 ± 1.9
c
6.4
20.8 ± 2.3
Wheat
7.0
108.9 ± 6.4
7.8
non planted
7.0
b
390.4 ± 15.1
85.34 ± 0.8
b
7.0
8.0
Rape
7.6
a
162.7 ± 7.3
c
6.5
Wheat
180.9 ± 3.8
643.55 ± 47.6
a
246.8 ± 5.0
c
non planted
6.6
107.1 ± 8.6
Wheat
7.3
138.2 ± 7.1
8.0
Rape
non planted
220
39.8 ± 0.8
non planted
Soil+Sludge control Rape
Soil+ Sludge HD
b
6.8
Rape
Soil+Sludge LD
TF
7.1
b
644.03 ± 33.7
a
178.9 ± 4.6
b
7.6
143.9 ± 4.8
221
Results are the values measured in the pull of 6 samples. Data of TOC are the average ± SE of three analytical
222
measurements in the pull of 6 samples. Lowercase letters indicate differences between plant species for the same
223
treatment
224
3.2. Total and bioavailable Silver in soil
225
Table S3: Ag, Cu, Fe and Zn concentrations in the amended soils at the end of the experiment (TF).
total Ag
(mg· Kg-1)
226
227
DTPA
CaCl2
Extractabl extractable
e Ag (%)
Ag (%)
Total Cu
(mg· Kg-1)
DTPA
extractable
Cu (%)
Total Fe
(mg· Kg-1)
DTPA
extractable
Fe (%)
total Zn
(mg· Kg-1)
DTPA
CaCl2
extractable extractable
Zn (%)
Zn (%)
soil
TF, Rape
TF, Wheat
TF,unplanted
udl
udl
udl
12.67 ± 0.88
12.67 ± 2.60
10.00 ± 0.57
TF, Rape
0.80 ± 0.16
Soil+Sludge
control
TF, Wheat
udl
TF,unplanted
Soil+Sludge LD
TF, Rape
TF, Wheat
TF,unplanted
Soil+Sludge HD
TF, Rape
60.78 ± 27.09 0.04 ± 0.02 0.05 ± 0.03 40.33 ± 12.55 30.93 ± 8.95 14691.3 ± 1341.898 0.433 ± 0.059 125.00 ± 21.55 21.90 ± 1.90
udl
TF, Wheat 50.42 ± 7.98
udl
0.06 ± 0.04 37.33 ± 4.25 21.78 ± 6.71 11418.3 ± 507.562 0.348 ± 0.049 114.00 ± 4.58 22.09 ± 1.18 1.16 ± 0.14
TF,unplanted 35.16 ± 15.20 0.03 ±0.01 0.02 ± 0.01 28.00 ± 6.66 21.70 ± 3.10 12746.3 ± 1689.739 0.518 ± 0.025 93.33 ± 15.24 20.17 ± 0.15 2.16 ± 0.48
17060.3 ± 826.822
14532.7 ± 1894.123
15274.3 ± 255.248
72.67 ± 19.06
121.00 ± 21.38
74.00 ± 4.36
67.00 ± 11.01
15596.7 ± 646.153
127.33 ± 38.34
56.33± 18.37
14683.3 ± 2824.743
147.00 ± 8.50
0.59 ± 0.07
48.33 ± 10.83
13787.7 ± 431.275
152.00 ± 24.19
1.15 ± 0.32
0.78 ± 0.01
1.07 ± 0.11
49.33 ± 6.01
48.33 ± 3.18
54.66 ± 3.38
16695.3 ± 796.859
20358.3 ± 3643.213
15899.7 ± 638.101
151.67 ± 24.37
93.33 ± 15.25
133.33 ± 9.84
Results are means ± SE (n = 3)
S13
-1
228
229
230
231
232
233
*Detection limits in soils (mg·Kg ), 0.4-0.5 (Ag), 40-50 (Cu, Fe, Zn); in extractable fractions (%), 0.000001(Ag), 0.0001 (Cu,
Fe, Zn).
*udl: under the detection limit
* CaCl2 extractable Cu and Fe were not detectable
*Ag was not detectable in the pore water
234
Table S4: Biomass of plants harvested at the end of the experiment, total silver concentrations in plant tissues (shoots and
235
roots) and % of total Ag extracted buy roots from the soils.
total Ag
Fresh weight
( mg·plant-1)
shoots
soil
Soil+Sludge
control
Soil+Sludge LD
Soil+Sludge HD
236
237
238
239
240
241
242
243
Rape
Wheat
Rape
Wheat
Rape
Wheat
Rape
Wheat
B
490 ± 20
NS
805 ± 50
A
1300 ± 220
NS
700 ± 100
AB
790 ± 130
NS
680 ± 100
1390 ± 180A
NS
700 ± 35
(mg· Kg-1 D.M)
roots
A
278 ± 70
AB
136 ± 20
B
39 ± 7
A
172 ± 23
B
19 ± 5
B
91 ± 8
12 ± 5B
B
88 ± 14
% Ag
extracted
shoots
roots
by roots
udl
udl
udl
udl
udl
udl
udl
udl
udl
udl
udl
udl
udl
udl
0
0
0
0
0
0
0.02 ± 0.015b
a
0.21 ± 0.044
16.33 ± 4.09ns
ns
32 ± 14.17
Results are means ± SE (n = 3). Capital letters indicate differences in the biomass among treatments for the same plant
specie (p<0.05, Duncan test). Lowercase letter indicate differences between plant species for the same treatment.
*udl: under the detection limit
3.3. Ag speciation
244
S14
3
245
Figure S5: Ag K-edge extended X-ray absorption fine structure (EXAFS) of selected Ag-S reference compounds: a) k χ(k)-
246
spectra and b), Fourier transforms; and of PVP-Ag-NPs c) k χ(k)-spectra and d), Fourier transform. Solid lines, experimental
247
data; dashed lines, simulations
3
248
249
3
250
Figure S6: k χ(k)-spectra of Ag K-edge extended X-ray absorption fine structure (EXAFS) of Ag reference compounds: a)
251
AgNO3 (blue), Ag-malate (red), AgO (green) and Ag2CO3 (purple); b) AgPO4 (blue) and AgCl (red).
252
Table S5: EXAFS structural parameters for selected Ag-S reference compounds and sludge and soil samples.
First shell (Ag-S)
Atom CN R (Å)
standards
synthetic nano Ag2S
253
254
natural Ag2S (acanthite)
Ag-DEDTC
Ag-GSH 1/10 pH 7
Ag-GSH 1/1 pH 7.4
Ag-GSH 1/2 pH 7.5
Ag-GSH 1/1 pH 5
Ag-GSH 1/1 pH 4
Samples
TF, Wheat planted soil
TF, Rape planted soil
TF, unplanted soil
T0, before plant culture
final dried sludge
σ
Second Shell (Ag-Ag)
2
Atom CN R (Å)
σ
2
Third Shell (Ag-Ag)
Atom CN R (Å)
σ
2
e0
R-factor
S
2.1
2.50
0.0071
Ag
6.0
3.09
0.0077
Ag
2.9
4.2
0.0083
3.48
0.0177
S
S
S
S
S
S
S
2.0
3.1
2.5
2.1
3.1
1.9
1.9
2.50
2.53
2.46
2.39
2.48
2.45
2.48
0.0060
0.0074
0.0074
0.0057
0.0086
0.0079
0.0085
Ag
Ag
Ag
Ag
Ag
Ag
Ag
6.0
1.2
0.8
0.4
1.2
0.3
0.6
3.08
2.91
2.98
2.95
2.89
2.93
2.96
0.0080
0.0040
0.0048
0.0050
0.0070
0.0035
0.0052
Ag
….
….
….
….
….
….
2.2
….
….
….
….
….
….
4.20
….
….
….
….
….
….
0.0070
….
….
….
….
….
….
1.40
5.81
1.77
0.85
2.72
0.17
-0.09
0.0097
0.0073
0.0144
0.0193
0.0210
0.0284
0.0145
S
S
S
S
S
1.8
1.8
1.9
1.9
1.8
2.51
2.52
2.51
2.51
2.5
0.0060
0.0060
0.0060
0.0060
0.0060
Ag
Ag
Ag
Ag
Ag
4.1
3.5
4.7
3.5
4.1
3.05
3.04
3.06
3.06
3.05
0.0100
0.0100
0.0100
0.0100
0.0100
Ag
Ag
Ag
Ag
Ag
1.5
1.9
6.7
1.4
1.6
4.22
4.18
4.88
4.18
4.19
0.0100
0.0100
0.0100
0.0100
0.0100
0.73
0.64
1.27
1.62
0.47
0.0160
0.0350
0.0111
0.0199
0.0242
CN: coordination number; R: inter-atomic distance (Å), σ2: Debye Waller factor, e0: energy shift , R-factor: fit residual
255
256
257
S15
258
259
260
261
262
Table S6: Structural parameters obtained by shell fitting for the other Ag- reference compounds.
263
264
CN: coordination number; R: inter-atomic distance (Å), σ2: Debye Waller factor, e0: energy shift , R-factor: fit residual.For
265
AgPO4 a fourth shell was fitted as well with the following parameters: CN=6.5, R=3.67 Å, σ2=0.0051. For Ag2CO3 a fourth
266
shell was fitted with the following parameters: CN=2.0, R=3.38 Å, σ2=0.002; a fith shell was fitted as well: CN=6.2, R=3.53 Å,
267
σ2=0.013
268
269
Table S7 gives the IND values, (indicator value of Malinowski), a measure of the usefulness of adding
270
another component, and the eigen values, that represent the contribution to the data made by that
271
particular component and obtained by the Principal Component Analysis (PCA) of the set of spectra.
272
Based on the IND values, two components have been used to describe the system. Figure S7 shows
273
the experimental spectra of references compounds together with their linear least-squares fit to the
274
weighted sum of components (target transformations). The spoil values give a measure of how much
275
the target transformation disagrees with the input. Based on these results mineral Ag2S (accanthite),
276
Ag2S from sulfidation and Ag-DEDTC were chosen as better compounds to describe the system. Note
277
that some Ag-GSH gets better results in the analysis of the XANES but worse in the EXAFS. Whereas
278
XANES gives information about the oxidation state and the coordination number, EXAFS is also
279
sentitive to the bond distance of the neighbouring atoms. Ag-GSH references shows shorter Ag-S and
S16
280
Ag-Ag bond distances (table S5 below) than the Ag-DEDTC and the samples, explaining why these
281
compounds gets higher Spoil factors than the AgDEDTC, specially when looking to the EXAFS.
282
Similiarlly, though both Ag2S references showed similar features, synthesized Ag2S showed higher
283
amplitude at higher k values which is translated in slightly higher coordination number for the third
284
shell (Table S5). The spoil of mineral Ag2S was lower in the PCA analyses of the EXAFS because all the
285
experimental spectra shows low coordinations numers for the thrid shell. So, mineral Ag2S was finally
286
selected to do the linear Combination Fitting (LCF).
287
288
Table S7: indicator (IND) value of Malinowski (1991) and Eigen values of each component obtained by Principal Colmponent
289
Analysis (PCA).
EXAFS
XANES
Component
Eigen value
IND value
Eigen value
IND value
1
4.06E+01
2.52E-01
2.41E+01
2.08E-03
2
5.04E+00
4.04E-01
2.42E-01
1.36E-03
3
4.54E+00
7.73E-01
8.52E-02
1.52E-03
4
3.67E+00
2.38E+00
6.42E-02
1.73E-03
5
*
*
4.74E-02
1.85E-03
6
*
*
1.84E-02
3.92E-03
7
*
*
1.74E-02
1.37E-02
290
291
292
293
S17
294
295
Figure S7: Experimental spectra (solid lines) and Target Transformations (dotted lines) for two componets in the XANES
296
region a) and in the EXAFS b) for all tested Ag reference compounds
297
298
Table S8: Ag species distribution (%) in sludge samples obtained by Linear Least squares Combination Fitting of Ag k-edge
299
XANES spectra.
% of Ag species
Ag-Thiol and/or
Ag2S
amorphous Ag2S
Sample
66
78
62
81
Final sludge dried
Final sludge frozen
Denitrification frozen
Nitrification frozen
24
23
39
20
SUM
NSS(%)
90
101
101
101
10.14
1.07
0.01
0.01
300
Proportions are expressed as mean percentage (%) calculated for these fits. Residual between fit and experimental data:
301
NSS NSS = Σ [k χexp − k χfit] / Σ[k χexp] x 100).
3
3
2
3
2
302
303
304
305
306
S18
307
3.4. Silver distribution
308
309
310
Figure S8: Elemental distribution in the final dried sludge thin section by µXRF. Silver in red, sulfur in green and silicon in
311
blue. Map b) detaill of the squared area in map a).
312
313
Figure S9: Elemental distribution in the non planted soil thin section by µXRF. Silver in red, sulfur in green and silicon in
314
blue. Squares in a) and c) maps indicate the areas were higher resolution maps b) and d) respectively were recorded.
315
E)Temperature map of Ag distribution collected by nanoXRF.
316
317
S19
318
319
Figure S10: Elemental distribution in the wheat planted soil thin section by µXRF. Silver in red, sulfur in green and silicon in
320
blue. c) higher resolution map of a specific area in map a), F1, F2 and F3 indicate the points where the fluorescence spectra
321
mark in d) were collected.
S20
322
323
Figure S11: STEM-EDX observations of a nanoparticle present in the sludge: a)DF4 image, b) Ag distribution, c) Sulfur
324
distribution, d) Iron distribution, e) EDX spectra.
325
326
327
S21
328
329
Figure S12: STEM-EDX observations of a nanoparticle present in the sludge: a)DF4 image, b) Ag distribution map, c) Sulfur
330
distribution map, d) Iron distribution map, e) EDX spectra.
331
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332
333
334
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338
339
340
341
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344
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Rodriguez-Carvajal, J. R., T; ́, FullProf, WinPLOTR, and accompanying programs. 2015.
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Levard, C.; Reinsch, B. C.; Michel, F. M.; Oumahi, C.; Lowry, G. V.; Brown, G. E., Sulfidation
Processes of PVP-Coated Silver Nanoparticles in Aqueous Solution: Impact on Dissolution Rate.
Environ. Sci. Technol. 2011, 45 (12), 5260-5266.
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Ravel, B.; Newville, M., ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption
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Ressler, T.; Wong, J.; Roos, J.; Smith, I. L., Quantitative speciation of Mn-bearing particulates
emitted from autos burning (methylcyclopentadienyl)manganese tricarbonyl-added gasolines using
XANES spectroscopy. Environ. Sci. Technol. 2000, 34 (6), 950-958.
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Kirpichtchikova, T. A.; Manceau, A.; Spadini, L.; Panfili, F.; Marcus, M. A.; Jacquet, T.,
Speciation and solubility of heavy metals in contaminated soil using X-ray microfluorescence, EXAFS
spectroscopy, chemical extraction, and thermodynamic modeling. Geochimica Et Cosmochimica Acta
2006, 70 (9), 2163-2190.
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6.
Manceau, A.; Lanson, B.; Schlegel, M. L.; Harge, J. C.; Musso, M.; Eybert-Berard, L.;
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soils by EXAFS spectroscopy. American Journal of Science 2000, 300 (4), 289-343.
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Kaegi, R.; Voegelin, A.; Sinnet, B.; Zuleeg, S.; Hagendorfer, H.; Burkhardt, M.; Siegrist, H.,
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