Supplementary Material
Silver sulfide nanoparticles (Ag2S-NPs) are taken up by plants and are
phytotoxic
Peng Wang, Neal W. Menzies, Enzo Lombi, Ryo Sekine, F. Pax C. Blamey, Maria C. HernandezSoriano, Miaomiao Cheng, Peter Kappen, Willie J.G.M. Peijnenburg, Caixian Tang, & Peter M.
Kopittke
Correspondence: Dr. Peng Wang, The University of Queensland, School of Agriculture and Food
Sciences, St Lucia, QLD 4072, Australia. Phone: +61 7 3365 4816, Fax: +61 7 3365 1177, Email:
[email protected]
Page S1
Synthesis of Ag2S-NPs
The Ag2S-NPs were synthesized by reaction of elemental sulfur with silver nitrate (AgNO3) in the
presence of polyvinylpyrrolidone (PVP) as a stabilizer (molar ratio Ag: S ~ 1: 2.6). Briefly, 500 mg PVP
(mean molecular mass = 40,000 Da) was dissolved in 500 mL of 1 mM AgNO3 in a 1 L Erlenmeyer
flask covered with aluminum foil on a hotplate with magnetic stirrer. Once dissolved, the solution was
heated to 60 °C and 42.0 mg of elemental sulfur dissolved in 50 mL of warm ethanol (60 °C) was added
at a rate of 2-3 drops per second and stirred continuously for 5 h. The solution was then heated to 80 °C
for 15 min to ensure complete evaporation of ethanol. Finally, the solution was cooled to room
temperature, and the Ag2S-NPs separated by centrifugation at 3400 g for 1.5 h. A washing procedure
was carried out with deionized water and the Ag2S-NPs were suspended in deionized water at a total Ag
concentration of 1118 mg Ag L-1.
Page S2
Table S1. Average particle size (nm), measured using scanning electron microscopy (SEM) and dynamic
light scattering (DLS), surface zeta potential (mV), and the ratio of Ag to S of Ag-NPs and Ag2S-NPs
dispersed in deionized water.
SEM (nm)
DLS d90 (nm)
Zeta potential (mV)
Ag-NPs
17.3 ± 3.2
45.3 ± 2.5
-7.54 ± 2.36
Ag2S-NPs
85.1 ± 12.2
110 ± 6.8
-35.7 ± 4.8
Page S3
Ag:S ratio
1.87:1
Table S2. Estimated effective concentrations of Ag reducing root growth by 50 % over 24 h expressed
as the measured total concentration (EC50 [Ag], µM) or the dissolved concentration (EC50 (Ag), µM).
The EC50 values were determined using a logistic function. Ag2S-NPs did not decrease root growth
precluding the calculation of EC50 values.
Cowpea (µM)
Wheat (µM)
EC50 [Ag]
EC50 (Ag)
EC50 [Ag]
EC50 (Ag)
AgNO3
0.112 a
(0.097-0.128)
0.112 a
(0.097-0.128)
0.077 a
(0.054-0.100)
0.077 a
(0.054-0.100)
Ag-NPs
0.710 b
(0.609-0.828)
0.042 c
(0.033-0.053)
1.856 b
(0.788-2.925)
0.070 a
(0.058-0.082)
--
--
--
--
Ag2S-NPs
Data are presented as the mean and the values in brackets show the 95 % confidence intervals; when
followed by a different letter, the means differ significantly (P < 0.05, t-test).
Page S4
Table S3. Target transformation SPOIL values of selected reference spectra obtained by principle
component analysis (PCA). Reference spectra are classified as excellent (SPOIL < 1.5), good (1.5-3.0),
acceptable (3.0-4.5), poor (4.5-6.0), or unacceptable (> 6.0). The standard compounds with SPOIL
values < 3 were included in the subsequent linear combination fitting (LCF) analyses.
References
SPOIL VALUES
Ag-glutathione
1.622
Ag2S-NPs
1.731
Ag-NPs
2.380
Ag-histidine
2.550
Ag2O
2.754
Ag2CO3
3.067
Ag3PO4
4.632
Ag2SO4
4.778
Ag-citrate
5.222
Ag-phytate
5.485
Ag-polygalaturonate
5.839
Ag-cysteine
5.839
AgNO3
5.993
Page S5
Table S4. The percentage speciation of Ag in freeze-dried and hydrated (frozen) shoots of wheat
exposed to Ag-NPs for two weeks as calculated using linear combination fitting (LCF) of the K-edge
XANES spectra.
Treatment
Metallic Ag
Ag-glutathione
Ag-histidine
R-factor
Freeze-dried leaves
22 (3.9)
72 (3.2)
6 (2.3)
0.000129
Hydrated (frozen) leaves
55 (1.4)
46 (1.4)
0.000170
The values in brackets show the percentage variation in the calculated values. The goodness of fit is indicated by
the R-factor. R factor = ∑i(experimental – fit)2/∑i(experimental)2, where the sums are over the data points in the
fitted region.
Page S6
(A) (B)
Figure S1. Scanning electron micrograph (SEM) (A) and an energy dispersive X-ray spectroscopy (EDS)
spectrum (B) of the Ag2S-NP suspension.
Page S7
Normalised intensity
scan 1
scan 2
scan 3
scan 4
scan 5
scan 6
scan 7
25480 25500 25520 25540 25560 25580 25600 25620 25640
Energy (eV)
Figure S2. Normalized Ag K-edge XANES spectra for wheat roots exposed to 0.043 mg Ag L-1 as
AgNO3 for two weeks. The repeated rapid scans (conducted consecutively without moving the sample)
did not show changes in speciation induced by the X-ray beam. The total length of time required to
complete these seven rapid scans was approximately equal to the length required for a single scan of the
experimental samples.
Page S8
A
AgNO3
-1
Measured [Ag] (mg L )
1
0.1
0.01
0h
24h
0.001
0.001
0.01
0.1
1
-1
Nominal [Ag] (mg L )
B
Ag-NPs
-1
Measured [Ag] (mg L )
10
1
0.1
0.01
0.01
0.1
1
10
-1
Nominal [Ag] (mg L
C
Ag2S-NPs
-1
Measured [Ag] (mg L )
100
10
1
0.1
0.01
0.01
0.1
1
10
100
-1
Nominal [Ag] (mg L )
Figure S3. Relationships between nominal and measured total concentration of Ag at the start (0 h) and
end (24 h) of the experimental period in a short-term experiment for AgNO3 (A), Ag-NPs (B), and
Ag2S-NPs (C). The legend in (A) applies to all three panels. Data are presented as the mean ± standard
error.
Page S9
0h
24 h
-1
Measured dissolved Ag (mg L )
0.08
Y = 0.0016 + 0.0397 X
R2 = 0.933, P < 0.05
0.06
0.04
0.02
0.00
0.0
0.5
1.0
1.5
2.0
Measured total Ag (mg L-1)
Figure S4. Relationship between measured total Ag and dissolved Ag at the start (0 h) and end (24 h) of
the experimental period in a short-term experiment in which Ag-NPs were added. Total Ag is defined as
the combined concentration of dissolved Ag plus the solid-phase Ag-NPs in suspension.
Page S10
-1
Ag concentration (mg L )
0.12
0.10
0.08
0.06
0.04
PP (no plant)
Glass (no plant)
Glass (plants)
0.02
0.00
0
6
12
24
Time (h)
Figure S5. Measured concentrations of Ag at 0, 6, 12, and 24 h in polypropylene (PP) or glass beakers
with or without plants (i.e. seven cowpea seedlings). After 24 h, 54 % of 0.113 mg Ag L-1 as AgNO3 had
been lost from solution, with 46 % of the loss being due to plant roots and 8 % due to the containers.
The dotted line indicates the initial Ag concentration of 1.05 µM added as AgNO3.
Page S11
100
AgNO3 treatment
Ag-NPs treatment
Ag2S-NPs treatment
Ag loss (%)
80
60
40
20
0
0.001
0.01
0.1
1
10
100
-1
Intial Ag concentraion (mg L )
Figure S6. Relationship between the percentage Ag loss and the initial Ag in solution during the 24-h
toxicity assays.
Page S12
Figure S7. Light micrographs showing roots of cowpea and wheat grown for 3 d in nutrient solutions
containing no Ag (Control), and the discoloration and rupturing in solutions with nominal concentrations
of 0.043 mg Ag L-1 as AgNO3 or 0.6 mg Ag L-1 as Ag-NPs; discoloration was only slight and no
ruptures developed in roots at a nominal concentration of 6.0 mg Ag L-1 as Ag2S-NPs. The scale bar
applies to all micrographs.
Page S13
Ag-citrate
Ag-polygalaturonate
AgNO3
Ag-phytate
Normalized intensity
Ag-histidine
Ag-cysteine
Ag-glutathione
Ag2S-NPs
Ag foil
Ag-NPs
25480 25500 25520 25540 25560 25580 25600
Energy (eV)
Figu e S8. N o
a i ed A g K-edge X A N E S
co bi a io fi i g ( L C F ) a a
Page S14
e.
ec a of e Ag
a da d
a
e e u ed i
e i ea
Normalised intensity
25450
Ag-NPs
Ag foil
25500
25550
25600
25650
25700
Energy (eV)
Figure S9. Ag K-edge XANES spectra of the reference Ag-NPs and Ag foil. It was not feasible to
separate these two forms of Ag based upon these XANES spectra. Therefore, this component was
referred to as ‘metallic Ag’ (Table 2) where LCF analyses indicated Ag to be present in plant tissues as
Ag-NPs.
Page S15
Normalised intensity
(A)
Ag2S-NPs
Ag-GSH
25480
25500
25520
25540
25560
25580
25600
25620
25640
6
7
8
Energy (eV)
-2
k x(k) (A )
(B)
2
o
0
1
2
3
4
5
o -1
k (A )
2
Figure S10. Ag K-edge XANES spectra (A) and k -weighted EXAFS spectra (B) of reference Ag2S-NPs
and Ag-glutathione (Ag-GSH). The legend in (A) applies to (B) also. Page S16
Normalised intensity
A
Ag2S-NPs
Ag-glutathione
Wheat shoots in Ag2S-NP treatment
25500
25520
25540
25560
B
Normalised intensity
25580
25600
25620
25640
25660
Energy (eV)
(A)
Ag2S-NPs
Ag-glutathione
Cowpea stems in Ag2S-NP treatment
25500
25520
25540
25560
25580
25600
25620
25640
25660
Energy (eV)
Normalised intensity
C
Ag2S-NPs
Ag-glutathione
Cowpea cotyledon in Ag2S-NP treatment
25500
25520
25540
25560
25580
25600
25640
25660
Energy (eV)
Normalised intensity
D
25500
25620
Ag2S-NPs
Ag-glutathione
Cowpea leaves in Ag2S-NP treatment
25520
25540
25560
25580
25600
Energy (eV)
25620
25640
25660
Figure S11. Ag K-edge XANES spectra of reference Ag2S-NPs and Ag-glutathione plus those of the
wheat shoots (A), cowpea stems (B), cotyledons (C), and leaves (D) from plants exposed to 6.0 mg Ag
L-1 as in Ag2S-NPs for two weeks.
Page S17
Normalised intensity
(A)
Ag2S-NPs
Ag-GSH
Cowpea roots in AgNO3 treatment
Wheat roots in AgNO3 treatment
25480
25500
25520
25540
25560
25580
25600
25620
25640
6
7
8
Energy (eV)
-2
k x(k) (A )
(B)
2
o
0
1
2
3
4
5
o -1
k (A )
Figure S12. Ag K-edge XANES spectra (A) and k2-weighted EXAFS spectra (B) of reference Ag2S-NPs
and Ag-glutathione (Ag-GSH) plus those of the roots of wheat and cowpea plants exposed to AgNO3 for
two weeks. The legend in (A) applies to (B) also.
Page S18
Normalised intensity
Freeze dried shoots
Hydrated (frozen) shoots
25400
25500
25600
25700
25800
25900
Energy (eV)
Figure S13. Normalized Ag K-edge XANES spectra for leaves of wheat analysed after growth for two
weeks in nutrient solutions containing Ag-NPs. Samples were analysed after freeze-drying or after being
frozen in hydrated form. The results of LCF analyses are also presented in Table S3.
Page S19
A
D
0.05
0.05
AgNO3 treatment
AgNO3 treatment
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0.00
0.00
12
24
36
48
60
0
72
Time (h)
-1
B
0.6
Ag-NP treatment
0.5
0.4
0.3
0.2
0.1
0.0
0
12
24
36
48
60
72
84
Dissolved Ag concentraion (mg L
-1
Tota Ag concentration (mg L )
0
24
48
60
72
0.08
Ag-NP treatment
0.06
0.04
0.02
0.00
0
12
24
36
48
60
72
84
Time (h)
F
6
36
Time (h)
E
Time (h)
C
12
0.08
Ag2S-NP treatment
Ag2S-NP treatment
5
0.06
4
3
0.04
2
0.02
1
0
0.00
0
12
24
36
48
Time (h)
60
72
84
0
12
24
36
48
Time (h)
60
72
84
Figure S14. Measured total (A-C) and dissolved (D-F) Ag concentration over two weeks in nutrient
solutions containing Ag added at nominal concentrations of 0.043 mg Ag L-1 as AgNO3, 0.6 mg Ag L-1
as Ag-NPs, or 6.0 mg Ag L-1 as Ag2S-NPs. In this experiment, solutions were renewed every 3 d with
Page S20
the values plotted being the mean of the start and end of this period. No dissolved Ag was detected in
any of the Ag2S-NPs treatment solutions using ICP-MS with a detection limit of 0.001 mg Ag L-1.
Page S21