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org/est Article

Soil-Weathered CuO Nanoparticles Compromise Foliar Health and

Pigment Production in Spinach (Spinacia oleracea)
Swati Rawat, Keni Cota-Ruiz, Haijie Dou, Venkata L. R. Pullagurala, Nubia Zuverza-Mena,
Jason C. White, Genhua Niu, Nilesh Sharma, Jose A. Hernandez-Viezcas, Jose R. Peralta-Videa,
and Jorge L. Gardea-Torresdey*
Cite This: https://dx.doi.org/10.1021/acs.est.0c06548 Read Online

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Metrics & More Article Recommendations sı Supporting Information

ABSTRACT: In this study, spinach plants exposed to fresh/unweathered

(UW) or weathered (W) copper compounds in soil were analyzed for
Downloaded via BUTLER UNIV on May 15, 2021 at 13:07:36 (UTC).

growth and nutritional composition. Plants were exposed for 45 days to

freshly prepared or soil-aged (35 days) nanoparticulate CuO (nCuO),
bulk-scale CuO (bCuO), or CuSO4 at 0 (control), 400, 400, and 40 mg/kg
of soil, respectively. Foliar health, gas exchange, pigment content
(chlorophyll and carotenoid), catalase and ascorbate peroxidase enzymes,
gene expression, and Cu bioaccumulation were evaluated along with SEM
imagery for select samples. Foliar biomass was higher in UW control (84%)
and in UW ionic treatment (87%), compared to the corresponding W
treatments (p ≤ 0.1). Root catalase activity was increased by 110% in UW
bCuO treatment as compared to the W counterpart; the value for the W
ionic treatment was increased by 2167% compared to the UW counterpart
(p ≤ 0.05). At 20 days post-transplantation, W nCuO-exposed plants had
∼56% lower carotenoid content compared to both W control and the UW counterpart (p ≤ 0.05). The findings indicate that over
the full life cycle of spinach plant the weathering process significantly deteriorates leaf pigment production under CuO exposure in
particular and foliar health in general.
KEYWORDS: weathering/aging, nano and bulk copper oxide, bioaccumulation, physiology, foliar health, gene expression

■ INTRODUCTION
Healthy soils are not only critical to agriculture but also form
species in terms of physiology, biochemistry, essential nutrient
acquisition and distribution, and overall genetic response;
an important environmental sink for a range of contaminants. importantly, these effects may not be well-represented by
Copper-based engineered nanoparticles (ENPs) may enter soil toxicity studies focused on freshly applied ENPs.
through intentional use (agricultural amendments) as well as Few recent plant studies demonstrate the effect of aging of
through incidental exposure (biosolids application).1,2 As an ENPs on soil-grown crop plants. Servin et al.15 evaluated the
essential part of terrestrial ecosystems, plants are at significant effects of unweathered versus weathered CuO compounds on
risk of exposure to these materials.3,4 The potential for adverse lettuce (Lactuca sativa), including potential trophic transfer
effects depends on various factors such as the plant species, from the plants to insect and reptile species. The authors
time and duration of exposure, concentration of the ENPs, reported that weathering resulted in a 214% higher root Cu
composition of the soil matrix, and presence of co- concentration, whereas both ionic and bulk CuO treatments
contaminants, among others.5 The literature contains a rapidly had opposite trends with aging time. Micro (μ)-XRF analysis
growing number of studies investigating plant and ENP showed that the CuO in aged nanoscale treatments had been
interactions.6−11 One such condition or parameter that transformed to reduced Cu−S and oxide complexes in the root
remains largely unexplored is the impact of weathering/aging tissue, whereas that in the bulk-treated samples remained
of the ENP in soil on their particle activity.12 ENPs will
undergo various surface chemistry transformations in soil,
Special Issue: Environmental Implications of Nano-
including ionization, oxidation, sulfidation, aggregation, and
fertilizers
ROS formation, as well as interactions with organic matter and
other chemical species.13,14,11 Similarly, biologically derived Received: September 28, 2020
coatings or coronas may form and subsequently be altered over Revised: January 27, 2021
time through a range of dynamic processes. These changes Accepted: January 28, 2021
hold the potential for unique altered effects on exposed plant

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A Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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largely as CuO. Another study by Dimkpa et al.16 analyzed the PM temperature was 26.75 ± 0.11 °C. Additionally, the
effects of unaged versus aged ZnO NPs and ionic Zn on wheat average daylight was 13.74 ± 0.36 mol m−2 day−1. Gas
plant in terms of grain productivity and metal/nutrient exchange measurements were conducted, and enzyme and
accumulation. The freshly added compounds tended to have pigment extracts were prepared at regular growth intervals of
greater bioaccumulation in the shoots and the grain as the plants as described below.
compared to the aged particles. The treatment concentration of 400 mg/kg was selected
The influence of weathered or transformed ENPs on plants because it is at the borderline between safe and toxic for the
is poorly understood. This study adds to the existing body of plants.2 About 400−500 mg/kg has been used as maximum
literature on food safety and/or beneficial or detrimental concentration in previous studies as well.18,19 An approximate
effects from nanoparticulate CuO use in agriculture. These 10% particle dissolution was assumed in the CuO treatments;
findings can also inform efforts to assess the ecological and hence, the ionic Cu was applied at 40 mg/kg.15
human health risk associated with the use of these materials Gas Exchange Measurements. Gas exchange data were
and contribute to generating in silico predictive tools. collected at various intervals during plant growth (10, 20, and

■ EXPERIMENTAL SECTION
Characterization of Copper-Based Chemicals. Nano-
40 days post-transplantation) with a CIRAS-3 portable
photosynthesis system (PP Systems, Amesbury, MA). The
method followed is described in a previous study from the
particulate copper oxide (nCuO) and bulk copper oxide same lab.19
(bCuO) were purchased from Sigma-Aldrich (St. Louis, MO). Biochemical Analysis. Catalase and ascorbate peroxidase
The characterization data is given on Table S1. Copper sulfate enzymes in fresh leaf and root tissues were determined by the
(CuSO4) was used for ionic treatment and was purchased from method described by Gallego et al.20 and Murgia et al.21 using
Spectrum (New Brunswick, NJ). All compounds were used PerkinElmer Lambda 14 UV/vis Spectrophotometer (single-
without further purification. beam mode, PerkinElmer, Uberlinger, Germany). Spinach leaf
Growth Matrix and Greenhouse Plant Cultivation. extracts were prepared in 80% acetone, and leaf pigments were
The plant exposure experiments were conducted at the Texas estimated as suggested by Lichtenthaler and Wellburn22
A&M AgriLife Research Centre greenhouse (El Paso, TX) measuring respective absorbances in a plate reader (SPEC-
under controlled environmental conditions. The spinach seeds, TRAmax190 by Molecular Devices (Sunnyvale, CA)).
Seaside F1 (Johnny Selected Seeds, Winslow, ME), were tested Harvest and Elemental Analysis. The spinach plants
for germination on Petri dishes, and viability was estimated at were grown for 45 days post-transplantion under the
67%. The seeds were planted in Metro-Mix 360 (SunGro treatments noted above. The total foliar area, total number
Hort., Bellevue, WA) and were transplanted as 5 week old of leaves, and total biomass were recorded for each plant. The
seedlings into pots with treated soil; there was two kg soil and total foliar area was measured with a LI-3100C area meter (LI-
two seedlings in each pot. The soil used was a mixture of COR Biosciences, Lincoln, NE). The plant samples were
natural soil and potting mix (2:1). Natural soil was collected at further processed for acid digestion and elemental analysis as
a cotton-growing field in Socorro, TX (latitude: N described in a previous study by the group.19 They were
31°41′30.705′′ and longitude: W 106°17′13.936″, elevation: digested in a digiPrep hot block (SCP Science, Quebec,
1115 m above sea level) from a 0−30 cm soil depth and was Canada) using a mixture of plasma pure HNO3 and H2O2
characterized as a silty loam (65% silt, 20% sand, and 15% (1:4) using the standard method EPA 3050 and analyzed for
clay).17,18 It was fortified with SunGro Professional Mix elemental content by inductively coupled plasma−optical
(SunGro Hort., Bellevue, WA) for improved aeration. The mix emission spectroscopy.18,23,24
contains sphagnum peat moss, horticultural perlite, horticul- Standard reference material 1570a from the National
tural vermiculite, wetting agents, and starter nutrients. The Institute of Standards and Technology (NIST) was used to
mixture of soil and potting mix was characterized for its evaluate accuracy of the digestion protocol for the plant
physicochemical properties as given in Table S2. The samples; a recovery of 108.04 ± 1.67% was achieved.
treatments used were 0 (control), 400, 400, or 40 mg/kg of Additionally, aqua regia was used for soil sample digestion
nanoparticle CuO (nCuO), bulk CuO (bCuO), or CuSO4, and NIST standard reference material 2709a, San Jaoquin soil,
respectively. Pots were prepared with the soil media by was used for QA/QC; a recovery of 88.4 ± 1.85% was attained.
manually homogenizing it with measured amounts of the Cu RNA Extraction and qPCR Assays. RNA was extracted
compounds in a DI water suspension. Pots identical to the using the PureLink Plant RNA Reagent (Invitrogen) protocol
ones that underwent weathering were prepared as fresh/ for ≤0.1 g samples. The quantity and the quality were
unweathered treatments at the end of the 5 week period. Three evaluated using a Thermo Scientific NanoDrop spectropho-
replicate pots of each of treatment were established for the 0 or tometer. The cDNA was obtained with the use of the High-
5 week weathering period in the green house; during Capacity cDNA Reverse Transcription Kit with RNase
weathering, the soil was maintained at field capacity moisture. Inhibitor (ThermoFisher Scientific). The relative transcript
After the weathering period, spinach seedlings were trans- quantification for Cu superoxide dismutase (CuSOD)
planted to the replicate pots. The plants were regularly watered (GenBank accession no. D10244.1, forward primer 5′-
with 100 mL DI water/kg of soil media and occasionally TGACCCAAGAAGATGATGGT-3′ and reverse primer 5′-
treated uniformly with Avid 0.15 (Active ingredient, abamectin TGTAGACATGCACCCATTTG-3′) and Fe superoxide
2% v/v) for possible pests and insects. They were given the dismutase (FeSOD) (GenBank accession no.
designated amount of water every alternate day for the first half XM_021997993.1, forward primer 5′-ATTGGGGAGTGCA-
of the growth cycle, and everyday for the latter half. The ACATAGA-3′ and reverse primer 5′-ATCATTGAATTC-
average relative humidity at the green house during the 45 day AGGCAAGG-3′) was performed on a StepOnePlus Real-
growth period was 23.83 ± 0.18%. The average AM Time PCR using Actin as a reference gene (GenBank accession
temperature was recorded as 23.17 ± 0.12 °C and the average no. JN987183.1, forward primer 5′-TTTCCCTATAT-
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GCCAGTGGT-3′ and reverse primer 5′-TCTGCTGAAGTG-

GTGAAAGA-3′). The PCR reactions were done using the
SYBR Select Master Mix (ThermoFisher Scientific) at the
following conditions: 95 °C for 5 min, followed by 40 cycles at
95 °C 15 s, 55 °C 15 s, and 72 °C 35 s, including a final
melting curve program from 60 to 90 °C. Data were analyzed
following the 2−ΔCt method.25
Scanning Electron Microscopy (SEM) Analysis. Root
and leaf samples were freeze-dried by warming to room
temperature in a bell jar evacuated with a rotary pump to 2.6
Pascal. Dried samples (root and leaf) were mounted on stubs.
Samples were viewed uncoated in a JEOL (Tokyo) 5400 LV
SEM at 15 kV low-vacuum modes using a back-scattered
electron detector. Elemental analysis was carried out using an
attached KEVEX Sigma Energy Dispersive X-ray Spectrometer
(EDS).
Statistical Analysis. All the data collected were analyzed
using SPSS. The mean values from the controls and the
treatments were compared across treatments using one-way
ANOVA followed by Tukey−Kramer multiple comparison test
(SPSS 19.0 package, Chicago, IL). Results are presented as
mean ± standard error (SE) and were observed for statistically
significant differences at p ≤ 0.05 and p ≤ 0.1.

■ RESULTS AND DISCUSSION

Physiological Effects (Agronomy, Copper Uptake, Figure 1. Fresh foliar biomass (a) and foliar area (b) of spinach
and Translocation). The physiological effects of unweath- (Spinacia oleracea) plants grown in unweathered (UW) and
ered (UW) and weathered (W) copper treatments on spinach weathered (W) control, nCuO, bCuO, and ionic Cu at 0, 400, 400,
plants were assessed after a 45 day exposure period. Figure 1a and 40 mg/kg treatments, respectively, in soil (natural soil: potting
depicts the fresh foliar biomass measurements. The UW mix; 2:1) for 45 days under greenhouse conditions. Data are averages
control had an average fresh mass of 56 g; this value was not of three replicates ± standard error (SE). The uppercase letters on the
columns signify statistically significant difference across the different
significantly different from that of the UW Cu treatments. The
Cu treatments in the UW or W categories individually. The lowercase
W control had an average fresh mass of 31g; this value was not letters on the columns signify statistically significant differences
statistically different than that of the W Cu treatments (p ≤ between the UW and W averages within each Cu treatment. These
0.1). However, the leaf fresh mass in the UW control and ionic differences are as per one-way ANOVA followed by Tukey−Kramer
treatments were significantly greater (by 84 and 87%, multiple comparison test (p ≤ 0.1).
respectively) than the corresponding W treatments (p ≤
0.1). This effect of aging was visible at 45 days of plant growth
for control (Figure S8) and for ionic Cu treatments (Figure bCuO on soil-grown zucchini squash (Cucurbita pepo) plants
S9). Similarly, Figure 1b represents the total foliar area in and saw no effect on plant growth. Weathering had an
spinach plants grown under the different treatments. The UW influence on the fresh biomass of the spinach leaves only for
control had an average 735 cm2 foliar area, which was not ionic treatment and controls in the current study. Dimkpa et
significantly different compared to the UW Cu treatments (p ≤ al.16 studied the effect of weathering of nZnO in soil on the
0.1). The W control gave an average 498 cm2 foliar area, which growth and nutrients of wheat plants. They saw no effects of
was again not significantly different compared to that in the W weathering on the dry biomass of the wheat plant. A general
Cu treatments (p ≤ 0.1). However, the foliar area in UW ionic lack of work on the effect of weathering on nanoparticle effects
treatment was higher than the corresponding W treatments by on exposed plants makes further discussion difficult.
77% (p ≤ 0.1). It is possible that the detrimental effect from No significant differences were found in the root Cu content
weathering is due to compaction and decreasing aeration of the of spinach plants grown under the Cu treatments and the
soil, although this does not explain the lack of a weathering controls including UW/W treatments (p ≤ 0.05) (Figure S1).
effect in the bulk and NP treatments. However, the amended Cu had a variable effect on the Cu
Trujillo-Reyes et al.26 cultured lettuce (Lactuca sativa) under content of spinach leaves across the treatments (Figure 2). A
hydroponic conditions in the presence of 10 and 20 mg L−1 noteworthy fact that surfaces in Figures S1 and S2 is that
nCu, as well as 10 mg L−1 CuSO4, for 15 days; the authors average Cu concentration in spinach roots is about 2- to 4-fold
reported that leaf biomass and water content were significantly higher than the average leaf Cu concentration. However,
lower in the Cu treatments than the control. Similarly, Nair et significant differences between treatments have been observed
al.27 cultivated mung bean (Vigna radiata L.) plants in nCuO in the leaf but not the root samples. This could be attributed to
amended hydroponic media for 21 days and reported reduced plant physiology and active translocation of added Cu to plant
shoot length and biomass at 200 and 500 mg L−1. The current shoot.29
study was conducted in a moderately high organic matter soil On Figure 2, the UW control leaves had a Cu content of
(8% OM, Table S2) that resulted in limited effects of Cu 10.6 mg/kg. For the 400 mg/kg nCuO UW treatment, the leaf
treatment at 400 mg kg−1 on plant biomass. Similarly, Tamez Cu concentration (39 mg/kg) was 270% higher than that of
et al.28 studied the influence of freshly applied nCuO and the corresponding control, 53% higher compared to that of the
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statistically significant (Figure 2). Reasoning out the

similarities and differences in the nano and bulk CuO
weathering and uptake tendencies, we take a mass balance
approach. Both nano and bulk have apparently aggregated in
the available ∼8% soil organic matter (OM) with a soil pH of
7.13,30 Of the 400 mg/kg CuO amended samples, most of the
Cu has stayed in the soil (Table S4) and in the root tissue
(Figure S1), with only 7.5−10% translocated to the shoot. Cu
from bCuO treatments (averaging UW and W treatments
together) had approximately 7.5% translocation to the shoot,
while that from nCuO had about 10% translocate to the shoot.
Figure 2. Copper bioaccumulation in spinach (Spinacia oleracea) We suspect that nCuO has undergone much more aggregation
leaves of plants grown in unweathered (UW) vs weathered (W) in rhizosphere compared to the bulk because of smaller particle
control, nCuO, bCuO, and ionic Cu at 0, 400, 400, and 40 mg/kg size (Table S1).13,30 Nevertheless, nCuO particles used have a
treatments, respectively, in soil (natural soil: potting mix; 2:1) for 45
days under greenhouse conditions. Data are averages of 3 replicates ±
greater dissolution tendency in the soil solution versus the
SE. The uppercase letters on the columns signify statistically bCuO, as shown in a previous study from the group.18,19
significant difference across the different Cu treatments in the UW About 40% of Cu from ionic treatments has translocated to the
or W categories individually. The lowercase letters on the columns shoot because of the ease of dissolution of the ionic form.
signify statistically significant differences between the UW and W Therein, UW ionic Cu is uptaken more than the W
averages within each Cu treatment. These differences are as per one- counterpart because of temporal aggregation effects. Cu
way ANOVA followed by Tukey−Kramer multiple comparison test (p concentrations in the UW and W CuO soil samples (Table
≤ 0.05). S4) reflects a number higher than 400 mg/kg, with additional
Cu probably from the background and/or because of different
UW bCuO treatment (25.6 mg/kg), and 108% higher than biogeochemical processes and interactions in the soil
that of the UW ionic Cu treatment (19 mg/kg) (p ≤ 0.05). biota.31−33
Similarly, for the 400 mg/kg bCuO (UW) treatment, leaf Cu Figure S10 shows scanning electron microscopy (SEM)
concentration was higher by 141% compared to that of the images of the leaf samples from unweathered and weathered
corresponding control and 35% higher than that of the control and nCuO-treated spinach plants. These images are a
corresponding ionic treatment (p ≤ 0.05). For the W qualitative analysis of the incidence of Cu in spinach leaves.
treatments, the control leaves had a Cu concentration of 9.7 Figure S10a,b reflects the Cu uptake from natural soil, and
mg/kg. With the 400 mg/kg nCuO (W) treatment, the shoot Figure S10c,d reflects the Cu translocation from Cu spiked soil
Cu concentration (36 mg/kg) was significantly higher than to the leaf. Hence, a significant difference in the Cu count. In
that of the corresponding control by 275% and higher than addition, it is observable that the leaf samples from UW
that of the corresponding ionic W treatment (12.5 mg/kg) by treatments reflect higher Cu incidence than those from the W
188% (p ≤ 0.05). Similarly, the 400 mg/kg bCuO W treatment counterparts in terms of the Cu count. This agrees with the
had a shoot Cu concentration (32 mg/kg) that was quantitative analysis shown in Figure 2.
significantly higher than that of the corresponding control by Elemental concentrations in root and shoot plant tissues
232% and that of the corresponding ionic treatment by 155% have been represented in Tables S6 and S7, respectively. No
(p ≤ 0.05). There was no significant difference between the significant effects of the Cu treatments and/or weathering were
leaf Cu of the W nano and bulk CuO treatments. As shown in observed on the macro- and microelements in the spinach
Figures S1 and S2, weathering seems to have had little plant tissue, except for statistically significantly different Mn
influence on the plant tissue Cu concentration across the concentration in spinach leaf between 400 mg/kg W nCuO
different treatments. Additionally, Servin et al.15 reported and 400 mg/kg UW bCuO treatments (p ≤ 0.05).
findings contrary to ours with lettuce (Lactuca sativa) plants Gas Exchange. Various gas exchange parameters were
grown under similar treatments; weathering significantly analyzed at three different time points (10, 20, and 40 days)
increased root Cu content for nCuO treatment (2-fold) but during the exposure period, including stomatal conductance,
decreased it significantly for bCuO treatment (4-fold) photosynthesis, transpiration rate, water use efficiency, vapor
compared to that of the unweathered counterpart. However, pressure deficit, and intercellular CO2 concentration (Ci).
no significant differences were seen in plant shoot Cu content. These parameters are a quantification of carbon dioxide and
This difference could be attributed to the differences in soil moisture fluxes on the leaf surface under ideal conditions.34 Of
type, plant species, duration of weathering (35 vs 70 days), and all the gas exchange parameters estimated, only Ci had
growth conditions. Copper bioaccumulation in spinach plants significant differences in Cu treatments (Figure S3); however,
(barring the effect of weathering) in the current study aligns no effect of weathering was observed.
with that of Zuverza-Mena et al.,23 who studied Cu uptake into Biochemical Effects on Enzymes and Pigments. The
cilantro (Coriandrum sativum). There, the authors observed biochemical effects of exposure were quantified by measuring
that the Cu concentration in cilantro leaves was more than 2- root enzyme activity at plant harvest, as well as leaf enzyme
fold higher in the 80 mg/kg nCuO treatments compared to activity and pigment content (carotenoids and chlorophyll) at
that of the control plants. Similar statistically significant various time points over the growth period.
gradation was not seen in the root Cu content in cilantro plant. Enzymes. As shown in Figure 3, there was no significant
Weathering reduced the leaf Cu content in spinach plants effect of the UW Cu treatments, compared to control, on root
for nCuO and ionic Cu treatments but increased it in bCuO catalase activity in spinach plants measured at 45 days of plant
treatment compared to that in the unweathered counterparts. growth (cellular photosynthetic saturation). Under the W
Although these differences are noteworthy, they are not category, the nCuO treatment showed no effect on root
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(though not statistically significant) (Figure 2). Hence, it

resulted in a significantly higher catalase activity in the root in
W ionic treatment, and the opposite results were seen in Figure
3. Similarly, Cu from W bCuO treatment was better
translocated to shoots than from its UW counterpart (though
not statistically significant) (Figure 2). The opposite effect was
observed in the bCuO catalase activity in the roots. A similar
observation can be made about the bCuO treatment,
compared with control under the W category. Catalase activity
in W bCuO treatment was similar to the observation made by
Ochao et al.35 in roots of pea plants tested after 45 days of
exposure to 50 and 100 mg/kg bCuO.
Additionally, nCuO treatments did not affect the root
Figure 3. Catalase activity in spinach (Spinacia oleracea) roots catalase activity. It is assumed that most of the nCuO
measured at 45 days post-transplantation. The plants were grown in aggregated with soil OM (∼8%) and/or was taken up and
uweathered (UW) vs weathered (W) control, nCuO, bCuO, and
ionic Cu treatments at 0, 400, 400, and 40 mg/kg, respectively, in soil
translocated to the aerial parts of the plant (Figure 2). Table
(natural soil: potting mix; 2:1) for 45 days under greenhouse S4 shows the Cu concentration in soil samples after the plants
conditions. Data are averages of 3 biological replicates and 3 were harvested. Cu in W nCuO treated soil samples was
subsequent technical replicates ± SE. The uppercase letters on the significantly higher by 39.69% as compared to that in soil
columns signify statistically significant difference across the different samples from W bCuO treatments (p ≤ 0.05). This
Cu treatments in the UW or W categories individually. The lowercase corroborates higher aggregation of nCuO compared to bulk
letters on the columns signify statistically significant differences and hence no effects on root enzyme activity were observed.
between the UW and W averages within each Cu treatment. These Ascorbate peroxidase (APOX) enzyme activity in the
differences are as per one-way ANOVA followed by Tukey−Kramer
multiple comparison test (p ≤ 0.05).
spinach roots was examined at the end of the growth cycle
and did not show any effect from weathering and/or the Cu
treatments (Figure S2). However, a gradual increase in the leaf
catalase compared to control. However, the root catalase APOX activity was evident over the growth cycle of the plant
activity for the W ionic treatment was increased by ∼151%, (Figure S6), although there were few significant differences as
compared to W control (p ≤ 0.05). Additionally, root catalase a function of Cu exposure or weathering. The APOX activity at
activity for the UW bCuO was 110% greater than the 10 days post-transplantation in the spinach leaf was found to
corresponding W counterpart. Conversely, activity was 216% be 219% higher in UW ionic treatment, compared to the W
greater for the W ionic treatment when compared to the UW ionic treatment (p ≤ 0.05). Of that, the UW ionic treatment
counterpart (p ≤ 0.05). The ionic treatment was apparently had significantly higher enzyme activity since it got little time
most potent in creating stress and toxicity in the spinach root in soil to aggregate, unlike the W one. Ionic Cu was apparently
under the W category. A comparison of the results for bCuO most actively absorbed by the plant since it was in ionic form
and ionic Cu treatments in Figures 2 and 3 showed that they and thus increased enzyme activity.36 The effect was
were relatable. Cu from W ionic Cu treatment was taken up prominent in the initial 10 days of exposure probably because
lesser in the spinach shoot as compared to its UW counterpart of minimal aggregation. Conversely, Cu ions from the CuO

Figure 4. Comparison of the leaf Chl a content at various time points during the growth cycle of spinach plants grown in unweathered (UW) vs
weathered (W) control, nCuO, bCuO, and ionic Cu treatments at 0, 400, 400, and 40 mg/kg, respectively, in soil (natural soil: potting mix; 2:1) for
45 days under greenhouse conditions. Data are averages of 3 biological replicates and 3 subsequent technical replicates ± SE. The uppercase letters
on the columns signify statistically significant difference across the different Cu treatments in the UW or W categories individually for an exposure
period. The lowercase letters on the columns signify statistically significant differences between the UW and W averages within each Cu treatment
for an exposure period, as per one-way ANOVA followed by Tukey−Kramer multiple comparison test (p ≤ 0.05).

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treatments are likely to go into solution gradually over time.18 reported decreases in total chlorophyll in the leaves at 100,
Tamez et al.37 similarly saw increased APOX activity in sugar 200, and 500 mg L−1 treatments. Hong et al.44 treated soil-
cane leaves from a yearlong ionic Cu exposure at 20 and 60 grown cucumber (Cucumis sativus) plants with CuO NPs by
mg/kg in soil. foliar application. The authors observed a 30% decrease in
The leaf catalase activity data also examined over the photosynthesis and a 17% decrease in evapotranspiration at
different stages of plant growth did not show any effect of 200 mg L−1 nCuO treatment, suggesting compromised
weathering or the Cu treatments as shown in Figure S7. chlorophyll activity in the seedling leaves.
However, it exhibited a trend of gradually increasing enzyme For the W 20 day exposure period, Chl a under ionic
activity over time, although the changes were not of statistical treatment was 161% greater than the nCuO treatment. More
significance. importantly, Chl a from W nCuO treatment was lower than
Pigments. Leaf carotenoid content, as shown on Figure S4, that of the W control by 46%. The detrimental effect of
showed a clear effect of weathering in the 400 mg/kg nCuO nanosized Cu in the W treatments was thus significant at the
treatment at the 20 day exposure period, the mid of the plant 20 day exposure period. At the 30 day exposure period, the
life cycle. It was unaffected by weathering or by any of the effect of weathering on controls was reversed as compared to
treatments at the 10 day exposure period. Similarly, at the 20 the 10 day exposure period. Chl a under the W control was
and 30 day time points, there was no effect on carotenoid 91% greater (p ≤ 0.05) than that under the UW control. The
content in the UW treatment groups. However, at the end of probable rationale behind this observation is not clear.
the 20 day exposure period, the carotenoid content in the W Additionally, under W treatment at 30 days, Chl a in the
nCuO treatment was reduced by 56% compared with W nCuO treatment was 53% lower, and that in bCuO treatment
control (p ≤ 0.05). The value was also lower than the UW was 62% lower compared to that in the control (p ≤ 0.05). At
counterpart by 57% (p ≤ 0.05), clearly underlining the effect of the 30 day exposure point, both nano and bulk CuO
weathering at the middle of the plant life cycle. However, these significantly reduced the leaf chlorophyll and were toxic to
differences were no longer evident at the 30 day time period, the plant. This suggests a gradual release of Cu ions from the
perhaps suggesting that the effect was countered by the plant’s rhizosphere, likely negatively affecting plant photosynthetic
internal defense mechanisms.38 Nevertheless, at the 30 day electron transport.42 Post-weathering, the CuO could have
time point the carotenoid content in the W bCuO treatment either aggregated with the soil OM or been adsorbed on the
was reduced by 62.2% relative to the W controls. As noted root or other surfaces, becoming a somewhat stable source of
previously, nCuO dissolves more rapidly into solution as slow ion release over time.45,46,40 Additionally, both carotenoid
compared to bCuO, and this seems to correspond to the earlier content (Figure S4) and Chl a (Figure 4) showed a significant
response in the spinach leaf for the nCuO (20 days) and the reduction under W bCuO treatment compared to those of the
later response for the bCuO (30 days).18,19 More specifically, it W control at the end of the 30 day exposure interval. The
is possible that CuO releases ions more effectively into the observation of reduced overall chlorophyll content aligns with
rhizosphere solution (post-weathering) and becomes bioavail- Ouzounidou et al.,47 where chlorophyll reduction in spinach
able to the plant over the 20−30 days of its growth (Spinacia oleracea) leaves under 160 μM Cu exposure (ionic
cycle.39,31,40 However, the aforementioned works18,19 demon- source) was reported. Additionally, Da Costa and Sharma48
strated the Cu compounds dissolution characteristics in a soil observed significant decreases in the photosynthetic and
environment with 2% organic matter. Our current study was transpiration rates in rice (Oryza sativa) plants cultivated
dealing with 8% soil organic matter with a pH of 7; hence, the under nCuO hydroponic treatment at 1000 mg L−1.
aggregation and agglomeration characteristics of CuO likely Expression of CuSOD and FeSOD. The relative
have a stronger influence, especially for the weathered expression of CuSOD did not significantly differ in leaves
samples.41 Relating back to Figure 1, CuO treatments did from control the Spinacia oleracea plants grown in weathered
not show an effect of weathering on plant foliar health. We and unweathered soils. However, CuSOD transcripts from
hypothesize that the W CuO agglomeration characteristics leaves were significantly reduced in all treatments by ∼30-fold
were strong enough to influence microproperties like leaf against the controls, except in the plants that grew under
pigments (Figure 4) but not macro properties like foliar health. nCuO weathered conditions. At this latter condition, the
Likewise, UW and W nano and bulk CuO had no significant CuSOD mRNA was unaffected in comparison with controls
spikes in the root catalase activity (Figure 3) for similar (Figure 5). A similar pattern was observed when the FeSOD
reasons. levels were tested in leaves. The FeSOD mRNA levels showed
Cu as an essential micronutrient in plants with defined roles no statistical differences between controls and weathered
in photosynthetic electron transport, including both photo- nCuO. Meanwhile, the FeSOD expression was downregulated
systems I and II, as well as mitochondrial respiration.42 in the rest of the treatments by ∼20-fold (Figure 5).
However, excess Cu influences photosynthesis negatively by Additionally, no changes in CuSOD nor in FeSOD mRNA
inhibiting lipid and pigment biosynthesis.43 We note that Chl a levels were encountered in roots that were exposed to the same
was differently affected across the treatments with some clear treatments (data not shown).
effects of weathering of Cu compounds on soil-grown spinach In this study, Cu bioaccumulation in spinach was not
plants (Figure 4). At the 10 day interval, Chl a under the UW significantly different under bulk or nanoweathered treatments
control was 72% higher than that with the W control (p ≤ (Figure 2); however, both FeSOD and CuSOD transcript
0.05). An evident effect of weathering among the controls was levels were only down-regulated under bulk Cu treatment,
observed. Additionally, the UW control Chl a value was 43, 94, which suggests that possible elemental speciation may elicit a
and 129% higher than the UW nCuO, bCuO, and ionic Cu different transcriptional response. In line with this, a previous
treatments, respectively (p ≤ 0.05). Nair et al.27 cultured mung study in lettuce (Lactuca sativa) showed that plants exposed to
bean (Vigna radiata L.) seedlings in CuO NPs treated weathered CuO nanoparticles accumulated Cu (I) sulfur and
Murashige and Skoog medium for 21 days. The authors oxide compounds in tissues, whereas the tissues exposed to
F https://dx.doi.org/10.1021/acs.est.0c06548
Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology pubs.acs.org/est Article

To summarize, data from this study demonstrates that

weathering of nanoscale and bulk CuO treatments yields
observable biochemical changes only in the aerial parts of root-
exposed spinach plants. In addition, weathering did not
influence Cu bioaccumulation in the plant tissue; the shoot
Cu concentrations were exclusively influenced by the Cu
treatment type. However, the effect of weathering was evident
in leaf health and pigment production. The levels of both
pigments suggest that W CuO treatments exerted greater
negative influence on plant foliar health at the middle and
toward the end of the plant life cycle. Nano and bulk CuO
aggregation tendencies in the high OM soil used, and the
variation in their dissolution and ion production tendencies,
likely had a bearing on their influence in leaf pigment but not
in the Cu concentration and foliar health. The transcriptomic
analysis showed that CuSOD and FeSOD expression were
subdued by all the (UW and W) Cu treatments except for W
nCuO.
Overall, a variable influence of weathered Cu compounds on
spinach plants grown in organic soil in a controlled greenhouse
environment was observed. Given the lack of overt toxicity as
measured by biomass and leaf area, additional study should
include more sensitive biochemical end points to better
Figure 5. Effect of the weathering and unweathering of Cu understand the more subtle but potentially significant impacts
compounds on the expression of SOD isoforms in Spinacia oleracea of the exposure. In addition, further detailed nutritional
leaves. Panels a and b show the relative expression of CuSOD and analysis could help elucidate the effects of weathered Cu
FeSOD genes, respectively. The transcripts levels were normalized to compounds on spinach plants. Variation in the soil type with
the actin expression levels. Bars represent the means + SE (n = 3).
The uppercase letters on the columns signify significant differences
variable organic matter content would further widen our
across the different Cu treatments in the UW or W categories understanding of nanosoil geochemical interactions under
individually. The lowercase letters on the columns signify statistically weathering conditions and the resultant effects on plants
differences between the UW and W averages within each Cu cultivated therein.

■
treatment. These differences are as per one-way ANOVA followed by
Tukey−Kramer multiple comparison test (p ≤ 0.05).
ASSOCIATED CONTENT
unweathered CuO stored Cu mainly as CuO.15 In the same *
sı Supporting Information

study, the authors also found that the expression of genes The Supporting Information is available free of charge at
involved in Cu uptake were differentially regulated depending https://pubs.acs.org/doi/10.1021/acs.est.0c06548.
on Cu treatment. Accordingly, we also observed that catalase
Soil and CuO compound characterization, ratio of
activity was reduced when the plants were treated with
chlorophyll a and chlorophyll b for all leaf samples,
weathered bCuO as compared to that in the controls (not
elemental concentration from all the UW and W Cu-
statistically significant), while no changes were observed for
treated soil samples before and after plant harvest,
weathered nCuO compared to that in the control (Figure 3).
elemental content from all the leaf and root samples,
Additionally, in this study we found that the weathered nCuO
spinach root Cu concentration, root APOX data, gas
caused a greater level of disturbance on leaf physiology. Thus,
exchange (intercellular CO2) leaf data, leaf carotenoid
it is possible that under nCuO weathered conditions, the
data, leaf Chl b content, leaf APOX data, leaf catalase
spinach maintains “stable” CuSOD and FeSOD levels in leaf
activity data, comparative images of W and UW control
tissue to combat possible ROS production.
and ionic-treated spinach plants at the greenhouse, SEM
It has been documented that the antioxidant responses of
and corresponding EDS graphs (UW and W control and
plants are complex since many genes, such as Cat, Apx, Prx,
400 mg kg−1 nCuO-treated root and leaf samples)
and Sod, among others, may be involved in the systemic
(PDF)
responses against exposure to stress.49,50 In this study, it was

■
found that SOD levels in leaves were downregulated in almost
all treatments; however, it should be taken into consideration AUTHOR INFORMATION
that the expression of other antioxidant genes may be
upregulated as a compensatory strategy. For instance, Gupta Corresponding Author
et al.51 reported that the expression of CuSOD was reduced Jorge L. Gardea-Torresdey − Environmental Science and
when plants were exposed to AgNPs, while the levels of Engineering Ph.D. Program, University of California Center
catalase and APOX were increased as compared to controls. for Environmental Implications of Nanotechnology (UC
Furthermore, the downregulation process may represent a CEIN), and Department of Chemistry and Biochemistry, The
strategy to save energy, which could then eventually be used in University of Texas at El Paso, El Paso, Texas 79968, United
processes such as protein/metabolite synthesis to combat ROS States; orcid.org/0000-0002-9467-0536; Phone: 915-
production. 747-5359; Email: jgardea@utep.edu; Fax: 915-747-5748
G https://dx.doi.org/10.1021/acs.est.0c06548
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Environmental Science & Technology pubs.acs.org/est Article

Authors (2) Reddy Pullagurala, V. L.; Adisa, I. O.; Rawat, S.; Kim, B.; Barrios,
Swati Rawat − Environmental Science and Engineering Ph.D. A. C.; Medina-Velo, I. A.; Hernandez-Viezcas, J. A.; Peralta-Videa, J.
Program and University of California Center for R.; Gardea-Torresdey, J. L. Finding the conditions for the beneficial
Environmental Implications of Nanotechnology (UC CEIN), use of ZnO nanoparticles towards plants-A review. Environ. Pollut.
The University of Texas at El Paso, El Paso, Texas 79968, 2018, 241, 1175−1181.
(3) Reddy, P. V. L.; Hernandez-Viezcas, J.; Peralta-Videa, J.; Gardea-
United States Torresdey, J. Lessons learned: are engineered nanomaterials toxic to
Keni Cota-Ruiz − Department of Chemistry and Biochemistry, terrestrial plants? Sci. Total Environ. 2016, 568, 470−479.
The University of Texas at El Paso, El Paso, Texas 79968, (4) Ochoa, L.; Zuverza-Mena, N.; Medina-Velo, I. A.; Flores-
United States; orcid.org/0000-0003-4025-8383 Margez, J. P.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Copper
Haijie Dou − Texas A&M AgriLife Research and Extension oxide nanoparticles and bulk copper oxide, combined with indole-3-
Centre at Dallas, Dallas TX-75252, United States acetic acid, alter aluminum, boron, and iron in Pisum sativum seeds.
Venkata L. R. Pullagurala − Environmental Science and Sci. Total Environ. 2018, 634, 1238−1245.
Engineering Ph.D. Program and University of California (5) Cota-Ruiz, K.; Delgado-Rios, M.; Martínez-Martínez, A.; Núñez-
Center for Environmental Implications of Nanotechnology Gastelum, J. A.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Current
(UC CEIN), The University of Texas at El Paso, El Paso, findings on terrestrial plants-engineered nanomaterial interactions: are
Texas 79968, United States plants capable of phytoremediating nanomaterials from soil? Current
Opinion in Environmental Science & Health 2018, 6, 9−16.
Nubia Zuverza-Mena − Connecticut Agricultural Experiment (6) Schwab, F.; Zhai, G.; Kern, M.; Turner, A.; Schnoor, J. L.;
Station, New Haven, Connecticut 06511, United States; Wiesner, M. R. Barriers, pathways and processes for uptake,
orcid.org/0000-0003-2721-7691 translocation and accumulation of nanomaterials in plants-Critical
Jason C. White − Connecticut Agricultural Experiment review. Nanotoxicology 2016, 10, 257−278.
Station, New Haven, Connecticut 06511, United States; (7) Zuverza-Mena, N.; Martínez-Fernández, D.; Du, W.; Hernandez-
orcid.org/0000-0001-5001-8143 Viezcas, J. A.; Bonilla-Bird, N.; López-Moreno, M. L.; Komárek, M.;
Genhua Niu − Texas A&M AgriLife Research and Extension Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Exposure of engineered
Centre at Dallas, Dallas TX-75252, United States nanomaterials to plants: Insights into the physiological and
Nilesh Sharma − Department of Biology, Western Kentucky biochemical responses-A review. Plant Physiol. Biochem. 2017, 110,
University, Bowling Green, Kentucky 42101, United States 236−264.
Jose A. Hernandez-Viezcas − University of California Center (8) Du, W.; Tan, W.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L.; Ji,
R.; Yin, Y.; Guo, H. Interaction of metal oxide nanoparticles with
for Environmental Implications of Nanotechnology (UC
higher terrestrial plants: physiological and biochemical aspects. Plant
CEIN) and Department of Chemistry and Biochemistry, The Physiol. Biochem. 2017, 110, 210−225.
University of Texas at El Paso, El Paso, Texas 79968, United (9) Pullagurala, V. L. R.; Rawat, S.; Adisa, I. O.; Hernandez-Viezcas,
States J. A.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Plant uptake and
Jose R. Peralta-Videa − Environmental Science and translocation of contaminants of emerging concern in soil. Sci. Total
Engineering Ph.D. Program, University of California Center Environ. 2018, 636, 1585−1596.
for Environmental Implications of Nanotechnology (UC (10) Rajput, V.; Minkina, T.; Suskova, S.; Mandzhieva, S.;
CEIN), and Department of Chemistry and Biochemistry, The Tsitsuashvili, V.; Chapligin, V.; Fedorenko, A. Effects of copper
University of Texas at El Paso, El Paso, Texas 79968, United nanoparticles (CuO NPs) on crop plants: a mini review.
States; orcid.org/0000-0002-7424-8637 BioNanoScience 2018, 8, 36−42.
(11) Lv, J.; Christie, P.; Zhang, S. Uptake, translocation, and
Complete contact information is available at: transformation of metal-based nanoparticles in plants: recent advances
https://pubs.acs.org/10.1021/acs.est.0c06548 and methodological challenges. Environ. Sci.: Nano 2019, 6, 41−59.
(12) Servin, A. D.; White, J. C. Nanotechnology in agriculture: next
Notes steps for understanding engineered nanoparticle exposure and risk.
The authors declare no competing financial interest. NanoImpact 2016, 1, 9−12.

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(13) Rawat, S.; Pullagurala, V. L. R.; Adisa, I. O.; Wang, Y.; Peralta-
ACKNOWLEDGMENTS Videa, J. R.; Gardea-Torresdey, J. L. Factors affecting fate and
transport of engineered nanomaterials in terrestrial environments.
The authors acknowledge the USDA grant 2016-67021-24985 Current Opinion in Environmental Science & Health 2018, 6, 47−53.
and the NSF Grant CHE-0840525. Partial funding was also (14) Dimkpa, C. O. Soil properties influence the response of
provided by the NSF ERC on Nanotechnology-Enabled Water terrestrial plants to metallic nanoparticles exposure. Current Opinion in
Treatment (ERC-1449500). This work was also supported by Environmental Science & Health 2018, 6, 1−8.
the National Science Foundation and the Environmental (15) Servin, A. D.; Pagano, L.; Castillo-Michel, H.; De la Torre-
Protection Agency under Cooperative Agreement Number Roche, R.; Hawthorne, J.; Hernandez-Viezcas, J. A.; Loredo-Portales,
DBI-1266377. This work has not been subjected to EPA R.; Majumdar, S.; Gardea-Torresday, J.; Dhankher, O. P.; et al.
review, and no official endorsement should be inferred. J.L.G.- Weathering in soil increases nanoparticle CuO bioaccumulation
T. acknowledges the Dudley family for the Endowed Research within a terrestrial food chain. Nanotoxicology 2017, 11, 98−111.
(16) Dimkpa, C. O.; Singh, U.; Bindraban, P. S.; Elmer, W. H.;
Professorship. Also, J.L.G.-T. acknowledges the LERR and
Gardea-Torresdey, J. L.; White, J. C. Exposure to weathered and fresh
STARs Retention Award (2018) of the University of Texas nanoparticle and ionic Zn in soil promotes grain yield and modulates
System.

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nutrient acquisition in wheat (Triticum aestivum L.). J. Agric. Food
Chem. 2018, 66, 9645−9656.
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