Science of the Total Environment 752 (2021) 142264

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Comparation of the phytotoxicity between chemically and green

synthesized silver nanoparticles
Huiling Zhang a, Si Chen a, Xiaorong Jia a, Yuxiong Huang b,⁎, Rong Ji a, Lijuan Zhao a,⁎
a
State Key Laboratory of Pollution Control and Resource Reuse, School of Environment, Nanjing University, Nanjing 210023, China
b
Shenzhen Environmental Science and New Energy Technology Engineering Laboratory, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua Shenzhen International Graduate School, Tsinghua
University, Shenzhen 518055, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Green synthesized Ag NPs exhibit long- The green synthesized Ag NPs promote plant photosynthesis and exhibit at-
term antibacterial properties.
• Green synthesized Ag NPs can promote
tenuated toxicity to cucumber plants.
plant photosynthesis.
• Green synthesized Ag NPs exhibit atten-
uated toxicity to cucumber plants.
• Chemically synthesized Ag NPs induced
observed toxicity to cucumber plants.

a r t i c l e i n f o a b s t r a c t

Article history: Green synthesis of silver nanoparticles (Ag NPs) by using plants extracts has provided an eco-friendly alternation
Received 20 August 2020 for industry and agriculture application. Here, we prepared Ag NPs by using the cucumber leaves and rice husk
Received in revised form 4 September 2020 extracts, and further assessed the antimicrobial activity and phytotoxicity of green synthesized Ag NPs (g-Ag
Accepted 5 September 2020
NPs) comparing with chemically synthesized Ag NPs (chem-Ag NPs). The chem-Ag NPs had strong antibacterial
Available online 08 September 2020
activity on the growth of Escherichia coli, while g-Ag NPs by rice husks (gr-Ag NPs) exhibited long-term antibac-
Editor: Jay Gan terial effects. In terms of phytotoxicity, the chem-Ag NPs induced over-generation of ROS and activated plant an-
tioxidant defense systems, thus resulting in the upregulation of MDA and Zn contents and downregulation of
Keywords: antioxidant capacity, carotenoid, globulin and Mo contents. However, g-Ag NPs significantly promoted cucumber
Ag NPs photosynthesis by increasing chlorophyll contents. Besides, the green synthesized Ag NPs by cucumber extracts
Green synthesis (gc-Ag NPs) increased protein contents and gr-Ag NPs stimulated the upregulation of Mn and the downregula-
Cucumber tion of Al, which were all positive effects. Overall, compared with chem-Ag NPs, g-Ag NPs exhibited long-tern an-
Antimicrobial timicrobial properties and attenuated toxicity to plants, which could be used as potential nanopesticide or
Attenuated toxicity
nanoscale growth regulator in agriculture.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
⁎ Corresponding authors.
E-mail addresses: huang_yuxiong@sz.tsinghua.edu.cn (Y. Huang), ljzhao@nju.edu.cn The rapid development of nanotechnology provides new insights
(L. Zhao). into agricultural products revolution (Servin and White, 2016). Silver

https://doi.org/10.1016/j.scitotenv.2020.142264
0048-9697/© 2020 Elsevier B.V. All rights reserved.
2 H. Zhang et al. / Science of the Total Environment 752 (2021) 142264
nanoparticles (Ag NPs) are potential ideal pesticides due to its wide
spectrum antimicrobial capacity. However, chemically synthesized Ag
NPs (chem-Ag NPs) are of great cost as well as posing a risk to the eco-
system and public health (Parandhaman et al., 2019). Therefore, green
synthesized Ag NPs (g-Ag NPs) has drawn more and more attention
due to its environmental-friendly nature (Singh et al., 2018). There
were quite a few studies reporting the biologically synthesis methods
of Ag NPs by using bacteria, fungi, actinomycetes, yeasts, viruses, and
plants (Hebbalalu et al., 2013; Singh et al., 2018; Tarannum et al.,
2019). Among them, plant-mediated g-Ag NPs were extensively inves-
tigated (Akhtar et al., 2013; Saratale et al., 2018) due to the various spe-
cies of plants which containing large quantities of functional
metabolites (e.g. reducing and capping agents) as the reactants for the
green synthesis.
Plant-mediated g-Ag NPs are of promising application prospects due
to its antioxidant, anticancer and antimicrobial capacity (Roy et al.,
2019). Plant-mediated g-Ag NPs were reported to have reducing
power and ROS scavenging ability (Alavi et al., 2019; Hajebi et al.,
2019; Hamelian et al., 2018; Samari et al., 2018) which can be used as
antioxidants. In addition, plant-mediated g-Ag NPs can inhibit the pro-
liferation of cancer cells, such as human cervical cancer (Sarkar and
Kotteeswaran, 2018), lung cancer (line A549) (Cyril et al., 2019),
human cervix carcinoma (hela NCBI-C115) (Hamelian et al., 2018),
breast cancer carcinogenesis (Hajebi et al., 2019), human gastric cancer
(Mortazavi-Derazkola et al., 2019) and human colon cancer cell (Samari
et al., 2018; Verma et al., 2017). More importantly, plant-mediated g-Ag
NPs have shown great inhibitory effects to microbial growth, including
bacteria (Pseudomonas nitroreducens (Ulaeto et al., 2019), Escherichia
coli (Ebrahimzadeh et al., 2019; Huang et al., 2011; Rao and Paria,
2015; Singh et al., 2015; Su et al., 2019), Staphylococcus aureus
(Asghar et al., 2018; Ebrahimzadeh et al., 2019; Huang et al., 2011;
Singh et al., 2015; Su et al., 2019), Enterococcus (Ebrahimzadeh et al.,
2019), Acinetobacter baumannii (Ebrahimzadeh et al., 2019), Proteus
mirabilis (Ebrahimzadeh et al., 2019), Pseudomonas (Ebrahimzadeh
et al., 2019), Klebsiella (Ebrahimzadeh et al., 2019), Bacillus cereus
(Siddiqui et al., 2017), Bacillus subtilis (Kathiravan, 2018) and Pseudo-
monas aeruginosa (Francis et al., 2017)) and fungus (Aspergillus unguis
(Ulaeto et al., 2019), Aspergillus flavus (Asghar et al., 2018),
A. parasiticus (Asghar et al., 2018), Aspergillus japonicas (Su et al.,
2019), Aspergillus nidulans and Penicillium chrysogenum (Francis et al.,
2017)). Given the strong inhibition of pathogens, plant-mediated g-Ag
NPs are ideal candidates for nanopesticides in agriculture.
A critical step to ensure the safe and sustainable use of Ag NPs is to
conduct the environmental implication assessment. Previous studies
have revealed that exposed to chemically synthesized NPs would in-
duce significantly negative effects on plants. Ag NPs exposure resulted
in growth (Lee et al., 2012; Rui et al., 2017) and reproductive (Geisler-
Lee et al., 2014) toxicity on plants due to the overproduction of ROS trig-
gered by Ag NPs (Zhang et al., 2018) or Ag+ released from Ag NPs (Yan
and Chen, 2019). The exposure to Ag NPs would alter the crop yield and
quality of plant on the whole growth period, which may be a potential
threat to food security (Yang et al., 2018). On the other hand, green syn-
thesized NPs have shown some positive effects rather than toxic effects
on plants. Acharya et al. (2019) found that green synthesized NPs en-
hanced seedling growth, yield, and quality of onion. It was suggested
that green synthesized NPs were more biocompatible and
environmental-friendly to plants compared to chemically synthesized
NPs, which might be due to the capping of amino acid, sugar, organic
acids or fatty acids onto the surface of NPs (Marslin et al., 2018). How-
ever, few studies paid attention to the phytotoxicity of green
synthesized NPs.
In this study, the antibacterial activity and phytotoxicity of green and
chemically synthesized Ag NPs were investigated by using Escherichia
coli (E. coli) and cucumber plant (Cucumis sativus) for model bacterium
and plant, respectively. The results will not only bridge the knowledge
gap of the response of plant and bacteria to green and chemically
synthesized Ag NPs but also provide valuable information about the
environmental implication assessment of green synthesized nanoparti-
cles, which will be conducive to the sustainable use of nanotechnology
and nanomaterials.
2. Experimental section
2.1. Silver nanoparticles synthesis and characterization
Chemically synthesized silver nanoparticles (20 nm, ≥99%) without
any coatings were purchased from Pantian nano Material Co., Ltd.
(Shanghai, China). The gc-Ag NPs (Zhang et al., 2019) with a size
range from 5 to 25 nm and gr-Ag NPs (Song et al., 2019) with a size
range from 20 to 40 nm was green synthesized by the extracts of cu-
cumber leaves and rice husk in our previous studies. The SEM (S-
4800, Hitachi, Japan) or TEM (JEM-200CX, JEOL, Japan) images were
shown in Fig. S1. As was shown in Table 1, the hydrodynamic diameter
of 50 mg/L chem-Ag NPs, gc-Ag NPs and gr-Ag NPs was 149.3 ±
39.3 nm, 75.7 ± 2.8 nm and 66.9 ± 0.9 nm, respectively. The zeta poten-
tial of 50 mg/L chem-Ag NPs, gc-Ag NPs and gr-Ag NPs was −30.8 ±
0.6 mV, −15.5 ± 0.4 mV and −17.7 ± 0.3 mV, respectively. The hydro-
dynamic diameter and zeta potential were measured through dynamic
light scattering (Zetasizer Nano ZS, Malvern).
2.2. Antimicrobial activity determination
2.2.1. Bacterial culture
E. coli was chosen as model bacterium to investigate the antimicro-
bial capacity of chemically and green synthesized Ag NPs. Bacterial sus-
pension of E. coli (106 CFU/mL) stored at −80 °C was deforested at 4 °C.
The suspension was then inoculated into LB culture medium and cul-
tured at 37 °C for 18 h. After three-passage cultivation, the bacterial sus-
pension was used for further experiments.
2.2.2. Growth curve determination
100 mg Ag/L chem-Ag NPs, gc-Ag NPs and gr-Ag NPs stock solution
were prepared with DI water. For growth curve determination, 100 μL
E. coli suspension was added in 96 well plate and then mixed with
100 μL Ag NPs suspensions (100 mg/L) to achieve a final concentration
of 50 mg/L Ag NPs, which was consistent with the concentration used
for phytotoxicity test. E. coli suspensions mixed with LB culture medium
were used as control. 100 μL LB culture medium without E. coli was
mixed with 100 μL Ag NPs suspension as blank. The plate was placed
at 37 °C for bacterial culture. The optical density at 600 nm was mea-
sured at set intervals by microplate reader (Synergy H4 Hybrid Reader,
Biotek, America).
2.2.3. Inhibition rate calculation
The optical density at 24 h was used for inhibition rate calculation.
The calculation was performed based on following equation:


Inhibition rate ð%Þ ¼ 1−ODAg =ODCK  100% ð1Þ
Table 1
Hydrodynamic diameter and zeta potential of all NPs (at pH = 6.5 ± 0.2).
Hydrodynamic diameter Zeta potential (mV)
(nm)
chem-Ag NPs 149.3 ± 39.3 −30.8 ± 0.6
gc-Ag NPs 75.7 ± 2.8 −15.5 ± 0.4
gr-Ag NPs 66.9 ± 0.9 −17.7 ± 0.3
 H. Zhang et al. / Science of the Total Environment 752 (2021) 142264
2.3. Phytotoxicity test
2.3.1. Plant cultivation
Cucumber (Cucumis sativus) seeds (Zhongnong No.28 F1), pur-
chased from Hezhiyuan Seed Corporation (Shandong, China), were
sown within 1 cm deep in potting soil (Miracle-Gro, Beijing) with a nu-
trient composition of 0.68% N, 0.27% P2O5, and 0.36% K2O. After 7 days'
germination at 25 °C, one cucumber was kept in one planter. Under
the condition of 28 °C/20 °C at day/night and 18 h/6 h light/dark cycle,
the seedlings were grown in the greenhouse for 3 weeks. The relative
humidity and illumination in the greenhouse were 60% and
180 μmol·m−2·s−1, respectively.
2.3.2. Exposure assay
The foliar exposure was started when the cucumber was 3-weeks
old. 50 mg Ag/L silver nanoparticles stock solutions were ultra-
sonicated in an ice-bath at 50 KHz (KH-100DB, Hechuang Ultrasonic,
China) for 30 min to achieve a well dispersed suspension. Four treat-
ments were established: control, chem-Ag NPs, gc-Ag NPs and gr-Ag
NPs. Each treatment had 4 replicates. The foliar application was made
2 times per day for a 7-day exposure period (from week 3 to week 4
of plant growth) using a hand-held spray bottle, with total volume of
100 mL per plant during 7 days applied, yielding approximate total de-
livered masses of 5 mg Ag per plant. The cucumber plant was harvested
after 4-week growth.
2.3.3. Biomass and photosynthetic pigments determination
At harvest (4-week old plant), cucumber leaves were rinsed with tap
water for 5 min and then washed by DI water for thrice. After wiped up
by Kim wipes, the leaves were weighed up and then grinded to powder
under liquid nitrogen protection for subsequent determination. The
stems were collected and weighed. Roots were separated from soil inte-
grally and washed by tap water and DI water. After dried in the air, roots
were weighed. For photosynthetic pigments determination, 0.1 g fresh
leaves were extracted by 5 mL 80% acetone and ethanol (v:v = 1:1).
After centrifuged at 4000 rpm for 10 min, the absorbance of chlorophyll
a, chlorophyll b and carotenoid in the supernatant were measured by
microplate reader at 663 nm, 645 nm and 470 nm.
2.3.4. Total phenolic content
Total phenolic content was determined based on the method of Sin-
gleton and Rossi (Singleton and Rossi, 1965). 50 μL extracts of fresh
leaves was mixed with 450 μL DI water, then added 250 mL Folin phenol
reagent and 1.25 mL 20 g/L Na2CO3 solution. The absorbance of the su-
pernatant was measured by microplate reader at 750 nm. The total phe-
nolic content was expressed as mg Gallic acid/equivalent g of fresh
weight.
2.3.5. Total antioxidant capacity determination
The total antioxidant capacity of cucumber leaves was determined
based on Ferric Reducing Ability of Plasma (FRAP) method (Benzie
and Strain, 1996). 50 μL extracts of fresh leaves was mixed with
1.5 mL FRAP solution (300 mM acetate buffer (pH 3.6), 10 mM
tripyridyl-striazine (TPTZ) and 20 mM FeCl3 mixed in 10:1:1 ratio).
After reaction at 37 °C for 10 min, the absorbance of the supernatant
was measured by microplate reader at 593 nm. The total phenolic con-
tent was expressed as μmol Fe2+/equivalent g of fresh weight.
2.3.6. Lipid peroxidation assay
Lipid peroxidation was determined according to the protocol of
Jambunathan (2010). Lipid peroxidation was expressed as μmol MDA/
equivalent g of fresh weight. Briefly, 0.1 g of cucumber leaves were
mixed with 4 mL of 0.1% TCA, then centrifuged at 10,000 rpm for
15 min. 1 mL of the supernatant was mixed with 2 mL of 20% TCA and
2 mL of 0.5% TBA, then the mixture was heated in a water bath at
3
95 °C for 30 min. After cooling, measured the absorbance of the superna-
tant at 532 nm and 600 nm with microplate reader.
2.3.7. Sugar and starch determination
The total sugar and starch of cucumber leaves were extracted based
on Verma and Dubey's method (Verma and Dubey, 2001). Cucumber
leaves was dried at 70 °C for 24 h, 0.1 g tissues was extracted with
80% ethanol at 80 °C for 30 min thrice. After centrifuged at 11,000 rpm
for 20 min, the supernatants were used for total sugar and reducing
sugar determination. The total sugar contents was measured based on
the protocol of DuBois et al. (1956). 1 mL extracts was mixed with
1 mL 5% phenol and 5 mL H2SO4. After cooling, the absorbance of the su-
pernatant was measured by microplate reader at 485 nm. The reducing
sugar contents was measured by 3,5- dinitrosalicylic acid (DNS) re-
agents (Ma et al., 2018). 1 mL extracts were mixed with 2 mL DNS re-
agent and boiled for 5 min. After cooling, the absorbance of the
supernatant was measured by microplate reader at 540 nm. After
sugar extraction, residues were oven dried at 80 °C for 24 h and then
used for starch extraction. Briefly, 2 mL water was added and then
boiled in water bath for 15 min. After cooling, 2 mL 9.2 M HClO4 was
added and the tubes were shaken for 15 min. The supernatants were
collected after centrifuging at 5000 rpm for 15 min. The residues were
added with 2 mL 4.6 M HClO4 and shaken for 15 min. After centrifuging
at 5000 rpm at 15 min, the supernatants were used for starch determi-
nation based on the protocol by DuBois et al. (1956). The contents of
sugar and starch were expressed as mg glucose/g dry weight.
2.3.8. Protein fractionation and determination
The protein fractionation of cucumber leaves were performed based
on the method of Chen and Bushuk (1970). 50 mg cucumber leaves
were extracted with water, 0.5 M NaCl and 70% ethanol subsequently.
After centrifugation at 10,000 rpm for 10 min, the supernatants were
collected separately and represent the fraction of albumin, globulin
and prolamin, respectively. The protein determination was performed
based on the method of Bradford (Bradford, 1976). 0.1 mL extracts
were mixed with 5 mL Coomassie Brilliant Blue reagent. The absorbance
of the supernatant was determined with microplate reader at 595 nm.
The protein contents were expressed as mg BSA/g dry weight.
2.3.9. Determination of macro and micro element in cucumber tissues
After weighed up, cucumber tissues were oven-dried at 70 °C for
72 h. Then tissues were smashed into powder and digested with 8 mL
H2O2 and 2 mL HNO3 using a microwave oven system (Milestone
Ethos Up, Italy) at 160 °C for 40 min. Macro- and micro elements were
determined by inductively coupled plasma mass spectrometry (ICP-
MS) (NexION-300, PerkinElmer, USA) and inductively coupled plasma
optical emission spectrometer (ICP-OES) (Optima 8300, Perkin Elmer,
USA). GBW07602 were used as standard reference with a recovery
varies from 90 to 103%.
2.4. Statistical analysis
One-way ANOVA (Tukey's test) and independent two sample t-test
were employed to conduct statistical analysis using SPSS 18.0. Signifi-
cance of data between treatments was evaluated at p ≤ 0.05. All data
were presented as the mean ± standard deviation of four replicates.
3. Results and discussion
3.1. Antimicrobial activity of chemically and green synthesized Ag NPs
The antimicrobial activity of chem-Ag and two different g-Ag NPs
were evaluated by the growth inhibition of E. coli. As indicated in
Fig. 1A, the growth curve of E. coli shown clear difference between nor-
mal culture condition and upon exposure to 50 mg/L either chemically
or green synthesized Ag NPs. It was evident that the growth of E. coli
4 H. Zhang et al. / Science of the Total Environment 752 (2021) 142264
Fig. 1. (A) Growth curve of E. coli under normal culture and with the exposure to 50 mg/L chem-Ag NPs, gc-Ag NPs and gr-Ag NPs culture; and (B) growth inhibited rate with the exposure
to 50 mg/L chem-Ag NPs, gc-Ag NPs and gr-Ag NPs in the culture for 24 h' cultivation. The data were expressed by mean ± S.D. of six replicates, and analyzed using t-test. ** represents the
significance is below 0.01.
was significantly inhibited by the exposure to all three Ag NPs. Com-
pared with g-Ag NPs, chem-Ag NPs showed most adverse effects on bac-
terial growth. As shown in Fig. 1A, when exposed to chem-Ag NPs, the
biomass of E. coli kept unchanged in the first 5-h culture. However,
when exposed to g- Ag NPs, E. coli showed significant biomass increase
after 3-h to 4-h culture under gc-Ag NPs and gr-Ag NPs stress, respec-
tively. The results demonstrated that chem-Ag NPs have stronger anti-
bacterial activity than g-Ag NPs at the very early stage of E. coli
growth, although the Ag contents in three Ag NPs was the same. Inter-
estingly, g-Ag NPs showed more significant antibacterial capacity after
24-h culture, as indicated by the inhibition rate in Fig. 1B. The loss of vi-
ability calculated at 24 h was the strong evidence that gr-Ag NPs have
long-term antibacterial capacity compared with chem-Ag NPs
(p ≤ 0.01, t-test). The gr-Ag NPs resulted in 42.6 ± 1.3% loss of viability
while chem-Ag NPs only resulted in 32.5 ± 5.7% loss of viability. The
reason responsible for the interesting results may be attributed to the
slow release of silver ion by gc-Ag NPs or the antibacterial metabolites
capping on Ag NPs, such as acetanilide, melatonin, acetylsalicylic acid,
scopoletin, and alizarin (Kuppusamy et al., 2016; Song et al., 2019).
Thus, although g-Ag NPs didn't exhibit as strong antibacterial activity
as chem-Ag NPs in the early stage of bacterial growth, the long-term an-
tibacterial effects could mitigate such deficiency, and could be applied
for long-term antibacterial purpose.
3.2. Phytotoxicity of chemically and green synthesized Ag NPs
In order to further explore the environmental impacts of chemically
and green synthesized silver nanoparticles, cucumber were used as
model plant to test phytotoxicity (Zhang et al., 2018). Overall, g-Ag
NPs didn't show toxicity to cucumber plants while chem-Ag NPs
showed significant adverse effects to cucumber plant, which is in con-
sistent with our previous studies (Zhang et al., 2018).
3.2.1. Biomass and photosynthetic pigments
The impact of Ag NPs on cucumber plant growth was investigated by
measuring the weight of biomass (leaves, stems and roots) and photo-
synthesis capacity of leaves. As showed in Fig. 2A, the fresh biomass of
cucumber tissues were not impacted by either chemically or green
synthesized Ag NPs exposure. However, the photosynthetic pigment
content (carotenoid) of cucumber leaves was significantly (p ≤ 0.01, t-
test) decreased by chem-Ag NPs (Fig. 2B). By contrast, g-Ag NPs expo-
sure significantly enhanced plant photosynthesis (Fig. 2B). As indicated
in Fig. 2B, the chlorophyll a content was significantly increased under
gr-Ag NPs exposure (p ≤ 0.05, t-test) while chlorophyll b contents
were significantly increased under both green synthesized Ag NPs
exposure (p ≤ 0.05, t-test). The plant-derived functional groups on the
surface of g-Ag NPs may play an important role in stimulating the up-
regulation of chlorophyll contents, Lucini et al. (2015) also found that
plant-derived biostimulant exhibited positive effects on plant PSII pho-
tochemical efficiency (Fv/Fm). Further study is still needed to explore
the underlying mechanism.
3.2.2. Lipid peroxidation and antioxidant defense systems activation
Malondialdehyde (MDA) is the end products of cell membrane lipid
peroxidation, which could be an indicator of the severity of oxidative
stress (Gaweł et al., 2004). As showed in Fig. 3A, MDA contents in-
creased approximately 3.7 folds upon exposure to chem-Ag NPs com-
pared to control, which indicate severe oxidative stress induced by
chem-Ag NPs. However, upon the exposure to g-Ag NPs, MDA contents
were unchanged, indicating no oxidative stress occurring. In addition,
total phenolics contents and total antioxidant capacity of cucumber
leaves, which are important components of plant antioxidant systems,
decreased significantly (p ≤ 0.05, t-test) upon exposure to chem-Ag
NPs (Fig. 3B and C). Again, these results further confirmed that
chem-Ag NPs induced oxidative stress and disturbed antioxidant de-
fense system in cucumber plants. However, the phenolic contents and
antioxidant capacity of cucumber leaves were unchanged upon expo-
sure to g-Ag NPs compared to control, indicating no oxidative stress oc-
curred. Taken together, chem-Ag NPs induced severe oxidative stress in
cucumber plants while g-Ag NPs showed no oxidative damage. These
results are quite interesting given that the original diameters of chem-
Ag NPs and g-Ag NPs were comparable. We speculate that the functional
groups at the surface of g-Ag NPs may contribute to the different toxic-
ity. In our previous study (Zhang et al., 2019), we found that metabolites
in plant extracts, e.g., reducing sugar, phenolics, organic acids, etc.
played important role in reducing the Ag ions to Ag NPs. In addition, a
number of metabolites act as coating agent during the formation of Ag
NPs. These biological molecules make g-Ag NPs more biocompatible
and less toxic. Further experiments are still needed to elucidate the
mechanism of the lesser toxicity of g-Ag NPs.
3.2.3. Sugar and starch contents
Sugar and starch are important carbohydrates for plant metabolism
(Börnke and Sonnewald, 2011). As showed in Table 2, upon exposure to
chemically and green synthesized Ag NPs, the total sugar, reducing
sugar and non-reducing sugar contents didn't show any significant
change compared to control. Interestingly, compared with chem-Ag
NPs, gr-Ag NPs significantly increased total sugar contents (35.2%) and
non-reducing sugar contents (53.2%) (Table 2). The reason might be
that the increased photosynthesis resulted in more sugar production
 H. Zhang et al. / Science of the Total Environment 752 (2021) 142264 5

Fig. 2. The fresh weight of cucumber leaves, stems and roots (A), and the photosynthetic pigments of cucumber leaves (B) for four treatments: control, chem-Ag NPs, gc-Ag NPs and gr-Ag
NPs. The data were expressed by mean ± S.D. of four replicates. The data are analyzed using one-way ANOVA, followed by Tukey-Honestly Significant Difference multiple comparisons
test. The letters represent statistical differences at p ≤ 0.05 compared with other groups.

Fig. 3. The MDA contents (A), total phenolics (B) and total antioxidant capacity (C) of cucumber leaves for four treatments: control, chem-Ag NPs, gc-Ag NPs and gr-Ag NPs. The data were
expressed by mean ± S.D. of four replicates. The data are analyzed using one-way ANOVA, followed by Tukey-Honestly Significant Difference multiple comparisons test. The letters
represent statistical differences at p ≤ 0.05 compared with other groups.

of cucumber leaves. Moreover, the starch contents of cucumber leaves
were unchanged upon foliar exposure to either green or chemically syn-
thesized Ag NPs compared with control. Putting the above results to-
gether, we found that the carbohydrate metabolism was not disturbed
by the either green or chemically synthesized Ag NPs. Furthermore,
compared with chem-Ag NPs, we observed gc-Ag NPs significantly de-
creased starch contents in cucumber leaves by 55.6%, which requires
more future investigation on the underlying mechanisms.
effects to plant protein metabolism. These results were in consistent
with our previous studies that revealed nitrogen metabolism perturba-
tion under Ag NPs stress, including the change of low molecular amino
acids like glutamine and asparagine as well as perturbation of nitrogen
related metabolic pathways like beta-alanine metabolism and arginine
and proline metabolism (Zhang et al., 2018). In summary, chem-Ag
NPs significantly inhibited protein metabolism of cucumber leaves
while g-Ag NPs didn't show any toxicity.

3.2.4. Protein contents
Proteins are critical components of plant primary metabolism and of
vital importance in plant physiological process. Among them, albumin is
water soluble fraction, globulin is salt soluble fraction and prolamin is
ethanol soluble fraction (Horax et al., 2010). As showed in Fig. 4, com-
pared to control, chem-Ag NPs significantly decreased globulin contents
(p ≤ 0.01, t-test) while gc-Ag NPs significantly increased albumin con-
tents (p ≤ 0.05, t-test). Obviously, chem-Ag NPs exhibited adverse
3.2.5. Macro- and micro- elements of cucumber tissues
Macro- and micro-elements, acting as important components of en-
zymes (Hänsch and Mendel, 2009), are critical to plant physiology. As
showed in Tables 3, S1 and S2, Ag contents in cucumber leaves and
stems were significantly increased upon foliar exposure to either chem-
ically or green synthesized Ag NPs, except for roots. It suggested that Ag
NPs were taken up by leaves, but were not translocated from leaves and
stems to roots. Being exposed to either chemically or green synthesized

Table 2
Sugar and starch in cucumber leaves (mg glucose/g dry weight).

Total sugar Reducing sugar Non-reducing sugar Starch

Control 52.3 ± 7.6 ab 14.6 ± 1.9 a 38.4 ± 8.5 ab 74.5 ± 21.9 ab

chem-Ag NPs 44.7 ± 5.0 b 13.9 ± 0.4 a 30.8 ± 5.4 b 97.1 ± 24.2 a
gc-Ag NPs 48.6 ± 8.2 ab 14.5 ± 1.7 a 34.1 ± 8.0 ab 43.2 ± 9.4 b
gr-Ag NPs 60.3 ± 5.4 a 13.1 ± 0.7 a 47.2 ± 5.9 a 69.4 ± 12.5 ab

Note: Different letters (a, b, ab) stand for statistical differences at p ≤ 0.05 (ANOVA).
6 H. Zhang et al. / Science of the Total Environment 752 (2021) 142264

significantly upon exposure to gr-Ag NPs (Table 3). This is reasonable

since Mn is critical to chlorophyll synthesis. The elevation of Mn
contents might result in the chlorophyll increasing, which was corrobo-
rated by the observed increase of chlorophyll upon exposure to rice-
husk-Ag NPs. We also observed the reduction of Al contents in cucum-
ber stems when exposed to rice-husk Ag NPs (Table S1), which might
be beneficial to plant growth.
Overall, chem-Ag NPs induced oxidative stress that resulted in the
up-regulation of Zn and down-regulation of Mo. The gc-Ag NPs might
induce minimal toxic effects that resulted in Mo down-regulation and
Al up-regulation. Although gr-Ag NPs also resulted in Mo reduction,
given the upregulation of Mn in leaves and downregulation of Al in
stems, gr-Ag NPs exhibited positive effects to cucumber plants.

4. Conclusion

Fig. 4. The protein contents of cucumber leaves for four treatments: control, chem-Ag NPs,
gc-Ag NPs and gr-Ag NPs. The data were expressed by mean ± S.D. of four replicates. The
data are analyzed using one-way ANOVA, followed by Tukey-Honestly Significant
Difference multiple comparisons test. The letters represent statistical differences at
p ≤ 0.05 compared with other groups.
Ag NPs didn't change the contents of macro-elements of cucumber
leaves, stems and roots, such as K, Ca, P, Mg, Na and Fe, indicating no ev-
ident damage to plant inorganic nutrient absorption. However, Mo con-
tents were significantly decreased upon exposure to both chemically
and green synthesized Ag NPs. Mo deficiency might result in significant
disturbance in plant physiology since Mo is an essential component of
xanthine oxidase/dehydrogenase, aldehyde oxidase and sulfite oxidase
(Bray, 1975; Coughlan, 1980). Besides, Mo is believed to have antioxi-
dant capacity due to the transformation of Mo (V) to Mo (VI). The re-
duction of Mo contents under chem-Ag NPs and green synthesized Ag
NPs exposure indicate that both Ag NPs may induce physiological toxic-
ity to cucumber leaves. However, given that g-Ag NPs did not induce the
reduction of MDA contents and total antioxidant capacity. It was specu-
lated that g-Ag NPs induced very minimal oxidative stress onto cucum-
ber plants compared to chem-Ag NPs.
Upon exposure to chem-Ag NPs, Zn contents were significantly in-
creased by 39.9% (Table 3). Zn is a key component of the Cu-Zn SOD.
Given that chem-Ag NPs induced over-generation of ROS, SOD might
be stimulated to scavenge those radicals. Zn contents increased as a re-
sult of plant antioxidant systems activation. Besides, when exposed to
gc-Ag NPs, Al content increased significantly (Table 3), indicating po-
tential toxic effects to cucumber leaves. While Mn contents increased
Plant enabled g-Ag NPs can be ideal substitutes for chem-Ag NPs due
to its' eco-friendly properties. Therefore, environmental impacts must
be evaluated to ensure the sustainable use in agriculture. In this study,
the antimicrobial activity and phytotoxicity of chem-Ag NPs and plant
(cucumber leaves and rice husk extracts) mediated Ag NPs were
assessed. We found that chem-Ag NPs had strong antibacterial activity
while gr-Ag NPs exhibited long-term antibacterial effects. In addition,
chem-Ag NPs showed toxic effects on cucumber plants by inducing
over-generation of ROS, thus resulting in the upregulation of MDA con-
tents and Zn, downregulation of antioxidant capacity, carotenoid, glob-
ulin and Mo contents. However, g-Ag NPs significantly promoted
cucumber photosynthesis. Besides, gc-Ag NPs increased protein con-
tents and gr-Ag NPs stimulated the upregulation of Mn and downregu-
lation of Al, which were all positive effects. Nevertheless, the
downregulation of Mo upon exposure to green synthesized Ag NPs
and the upregulation of Al in gc-Ag NPs treatment indicated minimal
toxicity to cucumber plants. Overall, compared with chem-Ag NPs, g-
Ag NPs showed limited toxic effects to cucumber plants. Given the bio-
compatibility and well-dispersion of g-Ag NPs due to the functional
groups, we believed that g-Ag NPs with high efficiency and low toxicity
could be used as nanopesticides in agriculture.
CRediT authorship contribution statement
Huiling Zhang: Conceptualization, Methodology, Formal
analysis, Investigation, Data curation, Writing - original draft, Visual-
ization. Si Chen: Methodology, Validation, Formal analysis,
Visualization. Xiaorong Jia: Methodology, Validation, Formal analy-
sis, Visualization. Yuxiong Huang: Conceptualization, Writing -

Table 3
Macro- and micro-elements in cucumber leaves (mg/kg dry weight).

K Ca P Mg

Control 55,713 ± 23,773 a 23,252 ± 3736 a 10,315 ± 1341 ab 8156 ± 1188 a

chem-Ag NPs 51,921 ± 8222 a 25,773 ± 2684 a 11,796 ± 172 a 11,463 ± 1287 a
gc-Ag NPs 52,519 ± 9788 a 20,982 ± 2553 a 9577 ± 1479 ab 10,418 ± 2421 a
gr-Ag NPs 52,485 ± 11,971 a 20,876 ± 4019 a 8846 ± 1334 b 9688 ± 1124 a

Na Fe Al Mn

Control 1570 ± 619 a 196.3 ± 15.0 a 209.0 ± 10.0 b 86.2 ± 13.2 b

chem-Ag NPs 2160 ± 460 a 225.6 ± 22.6 a 258.7 ± 31.4 ab 115.4 ± 17.5 b
gc-Ag NPs 1893 ± 304 a 242.7 ± 25.8 a 323.1 ± 75.1 a 100.2 ± 9.3 b
gr-Ag NPs 2044 ± 492 a 180.7 ± 42.9 a 192.6 ± 28.9 b 238.4 ± 52.0 a

Zn Ag Cu Mo

Control 93.5 ± 27.2 b 2.4 ± 1.8 b 6.12 ± 0.91 a 0.34 ± 0.09 a

chem-Ag NPs 130.8 ± 19.8 a 307.9 ± 86.3 a 6.59 ± 0.34 a 0.18 ± 0.04 b
gc-Ag NPs 107.4 ± 7.7 ab 402.2 ± 203.2 a 6.26 ± 0.85 a 0.17 ± 0.01 b
gr-Ag NPs 112.7 ± 9.6 ab 318.8 ± 170.5 a 5.60 ± 1.85 a 0.12 ± 0.01 b

Note: Different letters (a, b, ab) stand for statistical differences at p ≤ 0.05 (ANOVA).
 H. Zhang et al. / Science of the Total Environment 752 (2021) 142264
review & editing, Resources, Project administration, Funding acquisi-
tion. Rong Ji: Resources, Supervision, Project administration,
Funding acquisition. Lijuan Zhao: Conceptualization, Writing - re-
view & editing, Resources, Supervision, Project administration,
Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was funded by the National Natural Science Foundation of
China under 21876081 and 21906081; and Natural Science Foundation
of Guangdong, China under 2019A1515011692 and 2019QN01L797.
Any opinions, finding, and conclusions or recommendations expressed
in this material are those of authors and do not necessarily reflect the
views of National Science Foundation of China.
Appendix A. Supplementary data
Macro- and micro-elements in cucumber stems (Table S1) and roots
(Table S2); TEM image of chem-Ag NPs (Fig. S1A) and gc-Ag NPs
(Fig. S1B), SEM image of gr-Ag NPs (Fig. S1C) are provided in the
Supporting information. Supplementary data to this article can be
found online at https://doi.org/10.1016/j.scitotenv.2020.142264.
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