Uptake Of Copper And Cerium By Alfalfa, Lettuce And

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Uptake Of Copper And Cerium By Alfalfa, LettuceAnd Cucumber Exposed To nCeO2 And nCuOThrough The Foliage Or The Roots: Impacts OnFood Quality, Physiological And AgronomicalParametersJie HongUniversity of Texas at El Paso, [email protected]

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UPTAKE OF COPPER AND CERIUM BY ALFALFA, LETTUCE AND

CUCUMBER EXPOSED TO nCeO2 AND nCuO THROUGH THE FOLIAGE OR

THE ROOTS: IMPACTS ON FOOD QUALITY, PHYSIOLOGICAL AND

AGRONOMICAL PARAMETERS

JIE HONG

Department of Environmental Science and Engineering

APPROVED:

Jorge L. Gardea-Torresdey, Ph.D., Chair

Jose R. Peralta-Videa , Ph.D.

Jianying Zhang, Ph.D.

John Walton, PhD. PE.

Bess Sirmon-Taylor, Ph.D.

Interim Dean of the Graduate School

Copyright ©

by

Jie Hong

2014

Dedication

To my parents, for their support, encouragement, endless and great love they gave to me.

To my wife, for her joining my life and making it colorful.

UPTAKE OF COPPER AND CERIUM BY ALFALFA, LETTUCE AND

CUCUMBER EXPOSED TO nCeO2 AND nCuO THROUGH THE FOLIAGE OR

THE ROOTS: IMPACTS ON FOOD QUALITY, PHYSIOLOGICAL AND

AGRONOMICAL PARAMETERS

by

JIE HONG, MS

DISSERTATION

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Department of Environmental Science and Engineering

THE UNIVERSITY OF TEXAS AT EL PASO

August 2014

v

Acknowledgements

I would like to express the deepest appreciation to my supervisor, Dr. Jorge L. Gardea-

Torresdey. You are the most intelligent, productive and creative supervisor I have known and

any graduate student would ever want. I feel extremely lucky and proud that I can work in your

excellent research group. Your patience, guidance, and support helped me learned critical

thinking, scientific writing, and analytical techniques that are useful in my academic and

professional career. Without your help, I could not have accomplished what I have today. I am

truly thankful for the privilege of being mentored by you.

I would also like to thank Dr. Jose R. Peralta-Videa, for his valuable advices and

guidance on my research and papers. I appreciate the time we shared discussing my research

project and your strong participation and brilliant suggestions for the design and setting of my

experiments. I will never forget the patience and effort you put on revising and improving my

papers. Without your help, it is impossible to publish my papers. Thank you for taking the time

to teach me how to write and pay attention to details. You are not only a mentor but also a kind

grandfather to me; you taught me how to look at life in a positive way and face up life challenges

bravely. I will remember forever what I learned from you.

I would like to thank my committee members, Professor John Walton and Professor

Jianying Zhang, for their advice and guidance on my research, their precious times to read my

dissertation, and provide their critical and valuable comments. Thank you for all your support.

I would like to express my gratitude to Professor Genhua Niu and Dr. Youping Sun at

Texas A&M University for their assistance and encouragement that has enabled me to make

progress on the cucumber research project. Thank you for allowing me to use the facility,

providing experimental materials, and taking care of my plants.

vi

I also would like to thank the Environmental Science and Engineering Program, The

Graduate School, and Department of Chemistry for the financial support. Thank you for the

financial assistance that made my life in UTEP much easier.

I acknowledge the help, support, and friendship of my group members, especially Dr.

Lijuan Zhao and Cyren Rico. The friendship will last forever.

I also thank the financial support from UCCEIN, NSF, USEPA.

I greatly appreciate my parents, Jianping Hong and Yinghe Xie, for giving me life and

raising me up to reach this point of my life. Thank you for your deep love and encouragement,

which helped me overcome the loneliness and struggles of living in a foreign country. I love you

forever.

Finally, I give my deepest gratitude to my lovely wife, Lina Wang. Thank you for your

help and suggestion in my research, encouragement and support in my life, and your deep love.

You make my life wonderful and much more colorful. Getting married with you is the best

decision I have ever made. Thanks for making my life complete. I love you forever.

vii

Abstract

Nanotechnology is increasingly attracting attention not only for its variety of applications

in modern life, but for the potential negative effects that nanomaterials (NMs) can cause in the

environment and human health. Studies have shown varied effects of engineered nanoparticles

(ENPs) on plants; however, most of these studies focused on the interaction of NPs with plants at

root level. The increasing production and use of NPs have also increased the atmospheric

amounts of NPs, which could be taken up by plants through their leaves. Cucumbers (Cucumis

sativus L.) are broad leaf plants commonly grown both commercially and in home vegetable

gardens that can be easily impacted by atmospheric NPs. However, there is limited information

about the potential effects of these atmospheric NPs on cucumber. This research was aimed to

determine (I) the possible uptake and translocation of cerium (Ce) by cucumber plants exposed

to nCeO2 (cerium dioxide nanoparticles, nanoceria) through the foliage, (II) the impacts of the

NPs on physiological parameters of the plants and the effects on the nutritional value and quality

of the fruits, and (III) the effects of seven copper compounds/nanoparticles applied to the growth

medium of lettuce (Lactuca sativa) and alfalfa (Medicago sativa). For aim I, 15 day-old

hydroponically grown cucumber plants were exposed to nCeO2, either as powder at 0.98 and

2.94 g/m3 or suspensions at 20, 40, 80, 160, 320 mg/l. Ce uptake was analyzed by using

inductively coupled plasma-optical emission spectroscopy (ICP-OES) and transmission electron

microscope (TEM). The activity of three stress enzymes was measured by UV/Vis. Ce was

detected in all cucumber tissues and TEM images showed the presence of Ce in roots. Results

suggested nCeO2 penetrated plants through leaves and moved to other plant parts. The

biochemical assays showed nCeO2 also modified stress enzyme activities. For aim II, 15 day-

old soil grown cucumber plants were foliar treated, separately, with 50, 100, 200 mg/L of

viii

nCeO2, nCuO and the respective bulk material suspensions. Photosynthetic rate (Pn), stomatal

conductance (gs), and transpiration (E) of cucumber leaves were measured with a portable gas

exchange system. Nutritional elements and Ce/Cu uptake were determined by ICP-OES. Quality

of cucumber fruits was evaluated after harvest. Results showed that cucumber absorbed Ce and

Cu through foliar applied nCeO2 and nCuO and translocate them to new leaves and fruits.

Photosynthetic and transpiration rates were only affected in new leaves. None of the treatment

significantly affected cucumber, yield, length, and diameter of fruits. However, both nCeO2 and

nCuO significantly reduced the firmness of the fruit. Mineral element determination in fruit

showed that Zn decreased by 25% with 200 mg/L of both nCeO2 and bulk CeO2 and in fruit Mo

decreased by 51% and 44% with both nCuO and bulk CuO at 200 mg/L, respectively. For the

aim III, 15 day-old hydroponically grown lettuce and alfalfa were exposed to 0, 5, 10, and 20

mg/L nCu, bulk Cu, nCuO, bulk CuO, Cu(OH)2 (CuPRO 2005, Kocide 3000), and CuCl2. The

concentration of Cu, macro and microelements in plants were measured by using ICP-OES. The

size of the plants and the activity of catalase and ascorbate peroxidase were also determined.

Results showed that all Cu NPs/compounds reduced the root length by 49% in both plant species.

Under all treatments, Cu, P, and S were increased (>100%, >50%, and >20%, respectively) in

alfalfa shoots; while P and Fe were decreased (>50% and >50%, respectively) in lettuce shoot. In

addition, catalase activity was reduced in alfalfa (root and shoot) and ascorbate peroxidase

activity was increased in roots of both plant species. Our findings show that increasing

concentration of atmospheric nCeO2 can affect the nutritional value of crop plants with unknown

consequences for the food chain. In addition Cu NPs/compounds could impact the growth of

plants and altered the quality of crops as well. These results will help to understand the eco-

toxicity of NPs in food crops.

ix

Table of Contents

Acknowledgements ....................................................................................................................v

Abstract ................................................................................................................................... vii

Table of Contents ..................................................................................................................... ix

List of Tables ........................................................................................................................... xi

List of Figures ......................................................................................................................... xii

Chapter 1: Introduction ..............................................................................................................1

Chapter 2: Evidence of Translocation and Physiological Impacts of Foliar Applied CeO2

Nanoparticles on Cucumber (Cucumis sativus) Plants .....................................................6

2.1 Introduction ..............................................................................................................7

2.2 Experimental section ................................................................................................9

2.2.1 Seed germination and plant growth .............................................................9

2.2.2 CeO2 nanoparticles.......................................................................................9

2.2.3 Powder treatments ......................................................................................10

2.2.4 Suspension treatments ................................................................................10

2.2.5 Sample preparation for ICP and TEM analyses .........................................11

2.2.6 Enzyme assays .......................................................................................................12

2.2.7 Statistics Analysis .........................................................................................13

2.3 Results and Discussion ..........................................................................................14

2.3.1 Uptake and accumulation of Ce from powder treatments ............................14

2.3.2 Effect of blowing time ..................................................................................16

2.3.3 Uptake of Ce by cucumber tissues from nCeO2 suspension .........................17

2.3.4 TEM studies of cucumber tissues from plants treated with CeO2 powder ...18

2.3.5 Enzyme activity essays .................................................................................24

2.4 Conclusion .............................................................................................................25

Chapter 3: Foliar Applied CeO2 and CuO Nanoparticles/Bulk Materials Alter Firmness and

Nutrient Content in Cucumber (Cucumis sativus) ..........................................................27

3.1 INTRODUCTION ....................................................................................................28

3.2 MATERIALS AND METHODS ...........................................................................30

3.2.1 Characterization of NPs. ............................................................................30

x

3.2.2 Foliar application of NPs and bulk materials .............................................30

3.2.3 Gas Exchange.............................................................................................31

3.2.4 Growth Data and fruit quality ....................................................................31

3.2.5 Sample preparation for ICP-OES ...............................................................31

3.2.6 Statistics Analysis ......................................................................................32

3.3 Results and Discussion .............................................................................................32

3.3.1 Ce and Cu accumulation in cucumber plant tissues and fruits .....................32

3.3.2 Effects of NPs photosynthesis rate, transpiration rate, and stomata

conductance...................................................................................................34

3.3.3 Effects of NPs on cucumber growth, yield and fruit quality .....................36

3.3.4 Effects of aerial application of nCeO2 on Mineral content in cucumber ...37

3.4 Conclusion .............................................................................................................42

Chapter 4: Toxicity Effects of Seven Cu Nanoparticles/Compounds to Lettuce (Lactuca

sativa) and Alfalfa (Medicago sativa) ............................................................................43

4.1 INTRODUCTION .................................................................................................44

4.2 MATERIALS AND METHODS ...........................................................................45

4.2.1 Preparation of suspensions/solutions .........................................................45

4.2.2 Seed germination and plant growth ...........................................................46

4.2.3 Sample preparation for ICP-OES ...............................................................47

4.2.4 Enzyme assays ...........................................................................................47

4.2.5 Statistics Analysis ......................................................................................48

4.3 RESULTS AND DISCUSSION ............................................................................48

4.3.1 Effects of Cu NPs/compounds on root /shoot elongation ..........................48

4.3.2 Uptake of Cu by alfalfa and lettuce ...........................................................49

4.3.3 Effects of Cu NPs/compounds on nutrient elements uptake ......................51

4.3.4 Enzyme activities .......................................................................................55

4.4 conclusion ............................................................................................................57

Chapter 5: Conclusion..............................................................................................................59

References ................................................................................................................................61

Appendix ..................................................................................................................................79

Vita… .......................................................................................................................................81

xi

List of Tables

Table 2.1: Cerium concentration in cucumber leaves treated with nCeO2 at 2.94 g/m3 and

washed three times either with DI water, CaCl2, or HNO3. The plants were hydroponically grown

for 15 days in Hoagland nutrient solution before treatment. Data are means ± SE (n=4). ........... 15

Table 3.1. Photosynthetic parameters of cucumber seedling leaves. Fifteen days old plants were

sprayed with nCeO2 and bulk CeO2 suspensions and data was collected 15 days after treatment

application. Data are average of four replicates ± standard error. Values in a column followed by

different letters have significant difference (p<0.05). .................................................................. 35


sprayed with CuO NPs/bulk materials suspensions and data was collected 15 days after treatment

application. Data are average of four replicates ± standard error. Values in a column followed by

different letters have significant difference (p<0.05). .................................................................. 35

Table 3.3. Yield and characteristics of cucumber fruit harvested from plants foliarly treated with

nCeO2, nCuO, bulk CeO2 and bulk CuO suspensions. .................................................................. 37

Table 3.4. Mineral concentration in seedling leaves (mg/kg) from plants foliarly treated with


Table 3.5. Mineral concentration of cucumber fruit (mg/kg) from plants foliarly treated with


xii

List of Figures

Figure 2.1: Upper row: Hydroponic setup for cucumber plants treated with nCeO2. Lower row:

Nanoparticle application as powder (left) and suspension (right). The plants were cultivated for

15 days before NP treatment and harvested 15 days after treatment. Each treatment had four

replicates. ...................................................................................................................................... 15

Figure 2.2: A. Cerium concentrations in cucumber leaves after 12, 36, and 72 h of exposure to

0.98 g nCeO2/m3 and 2.94 g nCeO2/m

3. The samples were washed with 10 mM CaCl2. Data are

averages of four replicates ± SE. Different letters stand for statistical differences at p≤0.05. B.

Cerium concentration in roots, stems, and leaves of cucumber plants exposed to 2.94 nCeO2/m3

for 15 and 45 minutes of fan blowing in a closed environment. Values are means SE (n = 4).

Capital letters represent statistical differences within each blowing time period. ........................ 16

Figure 2.3: Cerium concentration in A. leaves, B. roots and stems, and C. flowers of cucumber

plants treated with nCeO2 suspensions. Values are means SE (n = 4). * Stands statistical

differences at p ≤0.05 ................................................................................................................. 20

Figure 2.4: A. TEM image of surface of cucumber leaf treated with 0.98 mg nCeO2/m3. B. EDS

of the spot highlight in A. C. TEM image of cucumber root treated with 0.98 mg nCeO2 /m3. D.

EDS of the spot 1 highlight in C. .................................................................................................. 21

Figure 2.5: A. TEM image of control cucumber leaf sample showing a parenchymal cell (C-

chloroplast; V-vacuole); B. TEM image of cucumber leaf sample showing a parenchymal cell

(C-chloroplast; M- mitochondria; V-vacuole) treated with 320 ppm nCeO2 suspension; C. TEM

image of control cucumber root sample (I-interior of cell; B-between cells); D. TEM image of

cucumber root sample treated with 320 ppm nCeO2 suspension (I-interior of cell; B-between

cells); E. Elemental analysis of dark precipitation found in the interior of cell shown in D. ....... 23

Figure 2.6: Antioxidant enzyme activities in cucumber plants foliarly treated with CeO2 NP

powder at 0.98 and 2.94 g m-3 or 40, 80, 160, and 320 mg L-1

of nCeO2 suspensions. A, B, and

C correspond to APX, CAT, and DHAR activities, respectively, in plants treated with powder. D,

E, and F correspond to APX, CAT, and DHAR activities, respectively, in plants treated with

nCeO2 suspensions. Values are means± SE (n = 4). Means with the same letter are not

significantly different (Tukey test, p≤0.05). ............................................................................... 26

Figure 3.1. Ce (A) and Cu (B) concentration in seedling leaves, seedling roots, mature roots and

fruits. Data are average of four replicates± standard error. Data are averages of four replicates ±

SE. Different letters stand for statistical differences at p< 0.05. .................................................. 33

Figure 4.1: Root elongation of lettuce (A) and alfalfa (B) plants exposed for 15 days in

hydroponics to 0, 5, 10, 20 mg/L of Cu NPs/compounds. Plants were cultivated for 10 days in

Hoagland nutrient solution before exposure to Cu treatments. Data are averages of four replicates

± SE. * stands for statistic differences at p ≤ 0.05. ................................................................... 49

xiii

Figure 4.2: Copper concentration in lettuce root (A), lettuce shoot (B), alfalfa root (C), and

alfalfa shoot (D) exposed for 15 days in hydroponics to 0, 5, 10, 20 mg/L of Cu NPs/compounds.

Data are averages of four replicates ± SE. Different letters stand for statistical differences at p ≤0.05................................................................................................................................................ 51

Figure 4.3: Phosphorus concentration in lettuce root (A), lettuce shoot (B), alfalfa root (C), and

alfalfa shoot (D) exposed for 15 days in hydroponics to 0, 5, 10, 20 mg/L of Cu NPs/compounds.

Data are averages of four replicates ± SE. Different letters stand for statistical differences at p≤

0.05................................................................................................................................................ 52

Figure 4.4: Sulfur concentration in lettuce root (A), alfalfa root (B), and alfalfa shoot (C)

exposed for 15 days in hydroponics to 5, 10, 20 mg/L of Cu NPs/compounds. Data are averages

of four replicates ± SE. Different letters stand for statistical differences at p ≤ 0.05. .............. 54

Figure 4.5: Iron concentration in lettuce root (A), lettuce shoot (B), alfalfa root (C), and alfalfa

shoot (D) exposed for 15 days in hydroponics with 0, 5, 10, 20 mg/L of Cu NPs/compounds.

Data are averages of four replicates ± SE. Different letters stand for statistical differences at p ≤

0.05................................................................................................................................................ 55

Figure 4.6: Antioxidant enzyme activity in alfalfa and lettuce treated for 15 days in hydroponics

with 0, 5, 10, 20 mg/L Cu of NPs/compounds. CAT activity in alfalfa shoot (A) and alfalfa root

(B); APX activity in lettuce root (C) and alfalfa root (D). Values are averages of four replicates ±

SE. CAT activity in lettuce tissues and APX activity in shoots of both plant species were not

affected. ......................................................................................................................................... 57

Figure S1. Shoot elongation of 10 day-old lettuce treated for 15 days in hydroponics with 0, 5,

10, 20 mg/L Cu NPs/compounds. Data are averages of four replicates ± SE. Different letters

stand for statistic differences at p ≤ 0.05................................................................................... 79

1

Chapter 1: Introduction

According to the American Society for Testing and Materials (ASTM), nanotechnology

is “A term referring to a wide range of technologies that measure, manipulate, or incorporate

materials and/or features with at least one dimension between approximately 1 and 100

nanometers (nm).”1 Although in its infancy, nanotechnology is increasingly attracting attention

not only for its variety of applications in modern life, but also for the potential negative effects

that engineered nanomaterials (ENMs) can cause to the environment and to human beings.

The building blocks of nanotechnology are nanoparticles (NPs), which are “ultrafine

particle with lengths in two or three dimensions greater than 0.001 micrometer (1 nanometer)

and smaller than about 0.1 micrometer (100 nanometers) and which may or may not exhibit a

size-related intensive property.”1 The sources of NPs are numerous. Depending on the origin,

NPs are classified in three types: natural, incidental and engineered NPs.2 Natural NPs have

existed from the beginning of the earth and still occur in the environment (volcanic dust, lunar

dust, and mineral composites, among others). Incidental NPs, also defined as waste or

anthropogenic particles, are the result of manmade industrial processes (diesel exhaust, coal

combustion, welding fumes, etc.). Engineered NPs are made for special purposes and used in

various applications.

The small size and large surface area make NMs different from bulk materials in physical

strength, chemical reactivity, electrical conductivity, magnetism, and optical effects.3-4

These

properties enable the use of NMs in electronic engineering, energy, material applications,

pharmaceutics, cosmetics, textiles, food industry, and agriculture.5-7

Although nanotechnology has a lot of benefits, it could represent potential negative

impacts to ecosystems and environment that cannot be ignored.8 Current studies have shown that

2

some engineered NPs have negative effects on plant growth. For example, single walled carbon

nanotubes (SWCNTs) significantly affected root elongation of tomato (Lycopersicon

esculentum), cabbage (Brassica oleracea), carrot (Daucus carota) and lettuce (Lactuca sativa).9

ZnO NPs (nZnO) showed toxicity to Arabidopsis, Prosopis, and Glycine max.10-15

TiO2 NPs

(nTiO2) inhibited leaf growth and transpiration of maize seedlings due to the reduction of

hydraulic conductivities,16

and Ag NPs (nAg) disrupted the cell division process in Allium

cepa.17

Rico et al.15

analyzed the most recent literature related to the interaction of NPs with

edible plants and their possible implications in the food chain. These authors highlighted positive

and negative impacts and the potential risk of the bioaccumulation of NPs in the next plant

generation. In a more recent study, Zhao et al. 18

have shown that maize plants grown in sandy

loam soil treated with nZnO at 200 mg/kg can concentrate about 200 mg Zn/kg dry shoot

biomass. This could indicate that an unintentional or deliberate release of nZnO in the ecosystem

could put an excess of Zn in the food chain.

In most of the reported studies, researchers treated plants through adding NPs in the soil

or hydroponic solution. They focused on the root uptake of the NPs, not on the foliar uptake.

Although the mechanism of the foliar uptake is still unclear, and the literature on the topic is

limited, the foliar uptake of NPs and pollutants cannot be ignored. Nowadays, due to the widely

use of NPs in many fields, there are more NPs emitted into the atmosphere. They can be

transported over long distances in the troposphere19-20

and affect the biosphere due to specific

surface properties.21-22

There is high possibility that plants uptake atmospheric NPs through their

leaves.

Previous studies showed that plants have the ability to absorb nutrients through the

leaves,23

and NPs may follow the same pathway. The lipophilic and the hydrophilic pathways are

3

the two routes for crossing the cuticle, a film that covers and protects the epidermis of leaves.24-25

The lipophilic pathway is based on the diffusion of non-electrolytes in cutin and waxes, and the

hydrophilic pathway is penetration of plant cuticles via aqueous pores.24

Once the nutrients or

NPs have crossed the cuticle, they may remain in the apoplast or be transported inside cells.26

Beside these two ways of penetration, there is also a solid-state pathway. NPs may go into plant

leaves through stomata due to their large size exclusion limit of 10 nm-1 μm and high transport

velocity;27

of course, the penetration is strongly dependent on environmental conditions, plant

species, physiological status, and speciation of the element.28

There are some reports about the uptake of NPs through leaves. Corredor et al. treated

pumpkin plants with carbon coated magnetic nanoparticles through injection and spray.29

They

found that the NPs were capable of penetrating living plant tissues migrating to different regions

of the plant, although movements over short distances seemed to be favored.29

Eichert et al.

found that water-suspended hydrophilic polymeric NPs of 43 nm diameter penetrated the leaves

of Vicia faba (L.). However, particles of larger diameters (e.g., 1.1 μm) could not enter leaves.

Also, when the leaves were rewetted repeatedly, about 60% of the previously penetrated stomata

were penetrated again.27

Uzu et al.21

studied the foliar lead uptake by lettuce exposed to

atmosphere near industrial plants. After 43 days of exposure, they found 335 ± 50 mg Pb kg-1

(dry weight) in thoroughly washed leaves. Environmental scanning electron microscopy coupled

with energy dispersive X-ray microanalysis showed that smaller particles (a few micrometers in

diameter) were also present in the leaves, often located beneath the leaf surface, and sub-

micrometric particles were observed inside stomata openings. No translocation of CeO2 NPs

(nCeO2) in maize plants has been reported by Birbaum et al.30

They treated the plants with

nCeO2 either as aerosol or as suspension. The results showed that 50 μg of Ce/g of leaves was

4

either adsorbed or incorporated into maize leaves independently of closed or opened stomata, but

there was no detectable translocation of nCeO2 into newly grown leaves.

Very few studies have been focused on the effects of NPs in nutritional value and the

quality of crops. All of the reported studies correspond to exposure of NPs through plant roots.31-

36 While some studies report benefits, others report decrements. Kole et al.

32 reported that

fullerol greatly increased bitter melon (Momordica charantia) water content, fruit yield and

improved fruit quality by increasing the phytomedicine content. Zhao et al.34

found nCeO2 and

nZnO can be accumulated in cucumber fruit and decrease the yield, and nCeO2 have shown to

reduce leaf count and yield of soybean.37

To the best of the author’s knowledge, there are no

reports on the effects of foliar applied NPs on nutrition value and fruit quality of cucumber.

Cucumber has been chosen in this study because it is a worldwide consumed edible plant,

and the consumption is increasing annually. Cucumber is a low calorie vegetable, which contains

many nutrient elements, such as Ca, Fe, Mg, P, K, Na, Mn, S and Zn.38

In addition, cucumber

has large leaf area and big stoma size,39

which could allow the foliar deposition of NPs.

Besides CeO2, other engineered NPs are also widely used. For example, copper (nCu)

and copper oxide (nCuO) NPs are used in ceramics, films, polymers, inks, coatings, gas sensors,

heat transfer nano-fluids, catalysts and semiconductors.40-43

Some reports have shown the effects

of these two NPs to terrestrial plants.10, 44-53

However, very few studies focused on lettuce

(Lactuca sativa) and alfalfa (Medicago sativa),54

which are two important vegetable for humans

and cattle. The objectives of this research were: 1) to determine the possibility of Ce absorption

by cucumber plants from foliar application of nCeO2, 2) to determine Ce storage in plant tissues

and its translocation to newly formed structures, including fruits, 3) to study the toxicity effects

caused by foliar application of NPs, 4) to evaluate the effects of foliar application of nCeO2 on

5

the quality and nutritional value of cucumbers, and 5) to determine the effects of seven Cu

compounds/NPs on lettuce and alfalfa plants. The chemical elements’ determination in plant

tissues was performed by using inductively coupled plasma optical emission spectrometer (ICP-

OES), and the enzyme activities were measured by UV-vis spectroscopy. In addition,

transmission electron microscopy (STEM) and energy diffraction system (EDS) was used to find

out the localization/accumulation of Ce inside plant tissues. A portable gas exchange system was

used to collect the physiological data such as photosynthetic rate (Pn), stomatal conductance

(Gs), and transpiration (E).

6

Chapter 2: Evidence of Translocation and Physiological Impacts of Foliar

Applied CeO2 Nanoparticles on Cucumber (Cucumis sativus) Plants

Abstract

Currently, most of the nanotoxicity studies in plants involve exposure to nanoparticles

(NPs) through the roots. However, plants interact with atmospheric NPs through the leaves and

our knowledge on their response to this contact is limited. In this study, hydroponically grown

cucumber (Cucumis sativus) plants were aerially treated either with nano ceria oxide powder

(nCeO2) at 0.98 and 2.94 g/m3 or suspensions at 20, 40, 80, 160, 320 mg/l. Fifteen days after

treatment, plants were analyzed for Ce uptake by using ICP-OES and TEM. In addition, the

activity of three stress enzymes was measured. The ICP-OES results showed Ce in all tissues of

the nCeO2 treated plants, suggesting uptake through the leaves and translocation to the other

plant parts. The TEM results showed the presence of Ce in roots, which corroborates the ICP-

OES results. The biochemical assays showed that catalase activity increased in roots and

ascorbate peroxidase activity decreased in leaves. Our findings show that atmospheric NPs can

be taken up and distributed within plant tissues, which could represent a threat for environmental

and human health.

7

2.1 INTRODUCTION

Concerns about the release of excess NPs into the environment, with negative effects in

ecosystem components, including plants are increasing.1 Previous reports aimed to determine the

impacts of NPs in plants have shown varied results. While some studies report benefits, others

report decrements. For instance, carbon nanotubes (CNT) have been found to increase root and

shoot lengths in Chickpea (Cicer arietinum),2 but they reduce root elongation in tomato

(Lycopersicon esculentum), cabbage (Brassica oleracea), carrot (Daucus carota), and lettuce

(Lactuca sativa).3 Metal-based NPs have also shown different results. For example, reports

indicate that nZnO enhance root length in Chick pea (C. arietinum),4

but reduce root elongation

in some desert plants.5 TiO2 NPs decrease biomass in wheat

6 and inhibit maize leaf growth;

7

however, they increase root length in cucumber.8 Other negative effects of metal-based NPs

include disturbance on cell division in Allium cepa by nAg,9 genotoxic effects of nZnO and

nCeO2 on soybean,10

excess Zn accumulation in maize exposed to nZnO,11

and reduction in

squash (Cucurbita pepo) transpiration by nAg and nCu.12

In all above mentioned reports, plants were exposed to the NPs through the roots.

However, natural NPs from volcanic or eolic sources are in the troposphere and interact with

plants through the leaves.13

NPs have also been used as foliar antimicrobial products for

agricultural purposes.14

However, very few reports have mentioned the antimicrobial properties

of CeO2.15-16

Very likely, a portion of engineered NPs (ENPs) released to the environment will be

wind dispersed, reaching the leaves of plants.17-18

Although there is no exact data about the levels

of nCeO2 in the air, their release to the atmosphere from car exhaust is increasing. Johnson and

Park19

estimated that in the United Kingdom, there are 15.6~114.9 kg/year of nCeO2 released to

the environment through vehicles emissions. Thus, plants could be exposed to unusual high

concentrations of nCeO2.

8

Reports indicate that particles and chemical elements enter the leaves through the

cuticle20-23

or stomata.24-26

Other studies have shown that plants can also take up NPs through

leaves. For instance, Uzu et al.27

found sub-micrometric lead particles inside stomata openings of

lettuce leaves grown close to an industrial plant. In Vicia faba (L.), water-suspended hydrophilic

polymeric NPs were found to penetrate leaves.24

Carbon coated magnetic NPs, sprayed or

injected into pumpkin (Cucurbita pepo) leaves were taken up and translocated to different

regions of the plants.28

Recently, Larue et al.29

found that Lactuca sativa can take up nAg

through stomata and, after the penetration, some of these NPs can be transformed to ionic Ag

within the leaf. However, Birbaum et al.30

reported uptake but no translocation of Ce in maize

(Zea mays) plants treated with nCeO2, either as powder or in suspension.

Reactive oxygen species (ROS) produced in plants exposed to abiotic or biotic stressors

can damage cell structure.31

Antioxidant enzymes such as dehydroascorbate reductase (DHAR),

catalase (CAT), and ascorbate peroxidase (APX) protect plants from ROS damage. Previous

studies have shown that NPs applied to the root system can modulate the activity of stress

enzymes. Zhao et al.32

have shown that nCeO2 increased CAT and APX activity in maize plants.

In cilantro (Coriandrum sativum L.) plants, CAT and APX activity increased in plants treated

with nCeO2 at low concentration but decreased at high NP concentrations.33

DHAR activity in

rice was significantly higher in plants treated with high concentration of CeO2.34

To our

knowledge, very few studies have determined the physiological effects of foliar application of

ENPs.29

The aims of this study were to determine the Ce uptake and translocation in cucumber

plants exposed to nCeO2 n through the leaves and the effects of the nCeO2 on three antioxidant

enzymes. Hydroponically grown cucumber plants were foliarly treated with nCeO2 as powder or

9

aqueous suspension. The uptake was determined by inductively coupled plasma-optical emission

spectroscopy (ICP-OES) and the antioxidant enzyme activities were measured using UV-Vis

spectroscopy. The uptake and translocation of Ce were corroborated by TEM analysis. Results

from this research will contribute to understand the interaction of ENPs with terrestrial plants.

2.2 EXPERIMENTAL SECTION

2.2.1 Seed germination and plant growth

All materials used in the experiments, e.g. paper towels, tweezers and water, were

autoclaved to reduce contamination in the germinating seeds. Cucumber seeds were disinfected

using a 4% NaClO solution, stirred for 30 min, rinsed three times with distilled water, and

immersed in distilled water for 24 h. Then, seeds were placed on the edge of wet germination

paper towels, rolled, and added with 10 drops of antimycotic/antibiotic solution (Sigma 5955).

The rolls were put into Mason jars with distilled water in the bottom, set in the dark for four

days, and after that, exposed to light for one day. After the sixth day, the young plants were

transferred to the hydroponic jars (magenta boxes) containing 300 ml of a modified Hoagland’s

nutrient solution previously described in the literature.35

All the jars had plastic lids with six

small holes. One cucumber seedling was introduced in each hole and the lid covered with

aluminum foil to avoid contamination of the substrate with NPs (Figure 1). Aquarium pumps

were used to aerate the jars that were set in an Environmental Growth Chamber (TC2

Microcontroller, Chagrin falls, OH) with light intensity of 300 µmol m-2

s-1

, 25/20 ºC day/night

temperature, and 65% relative humidity.

2.2.2 CeO2 nanoparticles

The nCeO2 (Meliorum Technologies, NY) were obtained from the University of

California Center for Environmental Implications of Nanotechnology (UC CEIN). Previous

10

characterization at the UC CEIN showed that nCeO2 are rods with primary size of 8 ± 1 nm,

particle size of 231 16 nm in DI water, surface area of 93.8 m2g

-1 and 95.14% purity.

36

2.2.3 Powder treatments

After two weeks of growth, the young plants were transferred into three sealed chambers

of 0.45 m × 0.45 m × 0.74 m, similar to those described in a previous study.37

The plants were

treated with 0 (control), 0.98 g/m3 (low), and 2.94 g/m

3 (high) NP concentrations. The respective

amount of nCeO2 was put on a tray in front of a fan (120v, 0.25Amps, 60 Hz) into the chamber.

Two blowing times were used, 15 min and 45 min. Then, the cucumber leaf samples were

collected for analyses at 12, 24, 36, and 72 h. The Ce concentration in roots, stems and leaves of

the cucumber plants were measured by ICP-OES (Perkin-Elmer, Optima 4300 DV, Shelton, CT)

and samples were analyzed by transmission electron microscope (H-7650 Transmission electron

microscope (TEM) (Hitachi High-Technologies, Japan). The Ce content in the nutrient solution

was also measured to make sure there was no contamination. All the samples were collected 7

days after the treatment.

2.2.4 Suspension treatments

A second group of two week old cucumber plants was treated with CeO2 NP suspensions.

Enough nCeO2 were suspended in distilled water to obtain 0, 40, 80, 160 and 320 mg/l and

homogenized by sonication for 45 min (Crest Ultrasonics, Trenton, NJ). Then, the different CeO2

NP suspensions were sprayed on cucumber leaves with a hand-held sprayer bottle. A total

volume of 100 mL per treatment was split in three applications (every four h). Six, 12, and 18

days after treatment, samples of leaves, stems, and roots were collected and prepared for

analysis.

11

2.2.5 Sample preparation for ICP and TEM analyses

For the ICP-OES analyses, all the leaves in a treatment were severed by the half; a group

of halves was processed for ICP-OES analysis without washing and the other halves were

separated in three groups. A group of halves was firstly rinsed with DI water; then, immersed for

20 min in DI water, followed by another rinse with DI water. Another group was rinsed with DI

water, immersed for 20 min in 0.01 M CaCl2 and rinsed again with DI water. The third group of

halves was rinsed with DI water, immersed for 20 min in 0.01 M HNO3, followed by another

rinsing with DI water. Afterward, the samples were dried in an oven at 70 ºC for 72 h,

weighed, and microwave-assisted acid digested (CEM microwave accelerated reaction system,

MarsX, Mathews, NC) using a mixture of plasma pure HNO3 and H2O2 (1:4) following the U.S.

EPA method 3051. The digest were volume adjusted to 15 mL and analyzed for Ce concentration

using the ICP-OES. For quality control (QC) and quality assurance (QA), the NIST SRM 1547

(Gaithersburg, MD) and spiked samples with 5, 10, 20 mg/L of nCeO2 were processed and read

as samples. For TEM sample preparation, the plant samples were cut into small pieces around

1mm2 and transferred to fixation vials, which were 1/3 full with 3% glutaraldehyde in 0.12M

Millonig’s phosphate buffer (pH 7.4). The vials were agitated with a rotary agitator at room

temperature for 1 hour. The vials were rinsed three times (15 min each) with 1/2 full fresh cold

0.06M phosphate buffer. Then, 1% OsO4 in 0.12M phosphate buffer (enough to completely

cover the specimen) was injected into the vials, and these vials were put into an ice bucket (dark,

0-4°C). After four hours, the osmium was pipetted out and the vials were rinsed three times (15

min each) with cold 0.06M phosphate buffer followed by three rinses (10 min each) with

distilled water. A 0.5% aqueous uranyl acetate solution was added into the vials to cover the

samples. The labeled vials were wrapped with tin foil, and placed in the refrigerator for an hour.

Samples were rinsed three times with distilled water (5 min. each) and placed in the refrigerator

12

over the weekend. During the dehydration process, the samples were rinsed by 50%, 65%, 75%,

85%, 95%, 100% ethanol separately for 15min. Finally, the samples were rinsed with 100%

acetone twice (15min each). Slow plastic infiltration was done starting with a 7:3 mixture of

acetone and Poly/Bed 812 in a rotary agitator overnight. The 7:3 plastic mixture was replaced

with 5:5 acetone/Poly/Bed 812 mixtures for another night. This process was repeated again for

the last mixture (3:7). Poly/Bed 812 at 100% was added for 2 hours before the samples were

transferred into BEEM capsules, which were filled with 100% Poly/Bed 812. The BEEM

capsules were covered with lid, and placed in the embedding tray for at least 48 hour in a 60°C

oven. The embedded samples were prepared for thick (90nm) and thin sectioning (60nm) for

TEM analysis.

2.2.6 ENZYME ASSAYS

All the enzyme activities were calculated based on the changes of absorbance and Beer’s

Law. Total protein in the enzyme extracts was estimated based on the Bradford38 method and

the specific activity of enzymes was expressed as units per milligram protein. One unit of DHAR

was defined as 1 μmol of ascorbate formed per minute. One unit of CAT was defined as the

amount of enzyme required to degrade 1 μmol of H2O2 per minute. One unit of APX was defined

as the amount of enzyme that oxidises 1 μmol ascorbate per minute.

2.2.6.1 Determination of dehydroascorbate reductase activity

DHAR enzyme activity was determined as per Doulis et al.39

The assay mixture

contained 790 μl 100 mM phosphate buffer (pH 7.0), 100 μl 12.5 mM reduced glutathione

(GSH), and 10 μl extract solution. The reaction was initiated with the addition of freshly

prepared 100 μl 2.5 mM dehydroascorbate (DAsA) and the absorbance was recorded at 265 nm

13

using a Perkin Elmer Lambda 14 UV/VIS Spectrometer (single-beam mode, Perkin-Elmer,

Uberlinger, Germany).

2.2.6.2 Determination of catalase specific activity

The CAT enzyme activity was determined following the procedure previously described

by Gallego et al.40 Plants were homogenized in 0.1M KH2PO4 buffer at pH 7.0 and then

centrifuged at 4500 rpm during 5 min (Eppendorf AG bench centrifuge 5417 R, Hamburg,

Germany). The supernatant was placed in a quartz cuvette with 73 mM H2O2 in phosphate buffer

and the absorbance was recorded at 240 nm using a Perkin Elmer Lambda 14 UV/VIS

Spectrometer.

2.2.6.3 Determination of ascorbate peroxidase activity

The APX enzyme activity was measured according to Nakano and Asada.41 A ratio of

10% w/v of leaf sample was homogenized on phosphate buffer (0.100 mg of leaves mixed with

900 µL of 0.1 M KH2PO4 at pH 7.4). The mixture was centrifuged for 25 min at 14000 rpm on a

refrigerated centrifuge. The supernatant was transferred to micro tubes and assayed after

centrifugation. An aliquot of 886 µL of 0.1 M KH2PO4 buffer at pH 7.4, 10 µL of the 17 mM

H2O2, and 4 µL of 25 mM ascorbate were placed in a quartz cuvette. Afterward, 100 µL of the

sample were added in order to obtain a final volume of 1mL. The absorbance was recorded at

290 nm in a Perkin Elmer Lambda 14 UV/Vis Spectrometer.

2.2.7 Statistics Analysis

The reported data are means of four replicates ± standard error (SE). One-way ANOVA

was used to analyze the experiment variance (SPSS 19.0 package, Chicago, IL). Tukey Honestly

Significant Difference was used to determine statistical differences between treatment means.

14

2.3 RESULTS AND DISCUSSION

2.3.1 Uptake and accumulation of Ce from powder treatments

As it has been previously shown in the literature, NPs can be adsorbed in the leaf surface

without penetrating the leaf tissue.29

Thus, for better assessment of the NPs penetration into the

leaf tissue, the leaves were previously washed before preparation for Ce determination. Table 2.1

shows the efficiency of washing processes to remove nCeO2 from the leaf surface. As seen in

this table, CaCl2 removed more Ce from the leaf surface (81.3%) compared to HNO3 (76.5%) or

water (73.9%). The external surface of leaves of higher plants comprises a cuticular layer

covered by waxy deposits. The wax contains a wide range of organic compounds that can trap

the NPs.42

The surface wax of the leaves can protect against mechanical and pest damage,

ultraviolet irradiation, and air pollutants.43

In addition, the cucumber leaf is covered by many

glandular trichomes.44

These trichomes synthesize metabolites like terpenoids, phenylpropenes,

and fatty acids derivatives that can bind the NPs electrostatically.45

These NPs can be

internalized into the leaf tissues and translocated within the plant tissues.36

Figure 2.1 shows the experimental setting for the powder treatments and Figure 2.2

shows the Ce concentration in roots, stems, and leaves of cucumber plants. Figure 2.2 A shows

the concentration in leaves after washing with 10 mM CaCl2. As expected, the Ce concentration

in leaves treated with higher concentration of NPs (2.94 g/m3) were significantly higher than the

concentration found in leaves treated with 0.98 g/m3 (Figure 2.2 A). However, the uptake did not

increase with time. We hypothesize that the number of NPs on the surface saturated the routes of

entrance through the leaves avoiding penetration.

15

Table 2.1: Cerium concentration in cucumber leaves treated with nCeO2 at 2.94 g/m3 and

washed three times either with DI water, CaCl2, or HNO3. The plants were

hydroponically grown for 15 days in Hoagland nutrient solution before treatment.

Data are means ± SE (n=4).

Ce (mg kg-1

)

DI water 10 mM CaCl2 10 mM HNO3

Non-washed 745.5 ± 97.0 1364.8 ± 450.6 1244.6 ± 408.7

Washed 201.5 ± 56.1 259.1 ± 91.8 271.5 ± 67.9

Ce Removed 73.0% ± 7.5% 81.0% ± 1.0% 78.2% ± 6.4%

Figure 2.1: Upper row: Hydroponic setup for cucumber plants treated with nCeO2. Lower row:

Nanoparticle application as powder (left) and suspension (right). The plants were

cultivated for 15 days before NP treatment and harvested 15 days after treatment.

Each treatment had four replicates.

16

Figure 2.2: A. Cerium concentrations in cucumber leaves after 12, 36, and 72 h of exposure to

0.98 g nCeO2/m3 and 2.94 g nCeO2/m

3. The samples were washed with 10 mM

CaCl2. Data are averages of four replicates ± SE. Different letters stand for

statistical differences at p≤0.05. B. Cerium concentration in roots, stems, and leaves

of cucumber plants exposed to 2.94 nCeO2/m3 for 15 and 45 minutes of fan blowing

in a closed environment. Values are means SE (n = 4). Capital letters represent

statistical differences within each blowing time period.

2.3.2 Effect of blowing time

The effect of blowing time on foliar uptake of nCeO2 is shown in Figure 2.2 B. As seen

in this figure, the amount of Ce found in tissues of plants blown for 15 min was significantly

a

a a a

b

b b

b

0

100

200

300

400

500

600

700

12 24 36 72

μg

Ce

/g d

ry le

ave

s

Treatment time (h)

0.98 CeO2/m3

2.94 CeO2/m3 A

b

a

b

a

B

A

0

20

40

60

80

100

120

140

160

15 45

μg

Ce

/g d

ry p

lan

t ti

ssu

es

Blowing time (min)

Roots

Stems

Leaves

B

17

higher (p ≤ 0.05) compared to the amounts found in plants blown for 45 min. However, in both

blowing time periods, the Ce concentrations found in roots and stems were very similar, and

significantly lower compared to the Ce concentration found in leaves. It is possible that higher

blowing time removed the NPs from the surface of the leaves and the blowing velocity was not

enough to lift the particles deposited on the floor. After the exposure, no Ce content was detected

in the hydroponic solution. Thus, this suggets that the source for Ce in roots was due to

translocation of NPs from the leaves. According to Savvides et al.,46

stomata of cucumber have ~

21 µm length, ~13 µm width, pore length of ~12 µm, and pore aperture of ~1.23 µm. The size of

the nCeO2 powder used is 8 ± 1 nm,36

which is small enough for the NPs to enter into the

stomata chamber.

Birbaum et al.30

reported that nCeO2 did not translocate in maize plants; however, Wang

et al.47

pointed out that watermelon plant can take up aerosol NPs with diameter lower than 100

nm through their leaves and transport them to the root system through the phloem. All these

results suggest that foliar uptake of NPs are affected by different factors, including size and type

of nanoparticles, environmental conditions (light, wind, and moisture), plant species, mode of

application, and others.47

2.3.3 Uptake of Ce by cucumber tissues from nCeO2 suspension

The concentration of Ce in leaves, roots, stems, and flowers of cucumber plants sprayed

with nCeO2 suspensions is shown in Figure 2.3. As seen in Figure 2.3A, Ce was not detected in

control leaves and the concentration with 40 mg L-1

(~700 µg/g) was significantly lower than the

other treatments. Cerium was also detected in stems and roots of NP treated plants (Figure 2.3B).

Results of this study have shown that nCeO2 may penetrate leaf surface when applied

either as powder or suspension. Corredor et al.28

proved that magnetic nanoparticles can get into

18

the cells of pumpkin plants through the stomata and the vascular system. It has also been

reported that water-suspended NPs can be taken up through stomata in Vicia faba (L.).24

Eichert

et al. observed that in leaves rewetted with DI water, about 60% of all stomata were penetrated

again by NPs.24

In the present experiment, the spray treatment was repeated three times in one

day, which means the leaves were dampened three times with NPs suspension. This increased the

chance of NPs to enter through the stomata. Even though the Ce concentration in leaves was

much higher in suspension treatments, the Ce concentration in the roots and stems were similar

as in plants treated with NP powder. This suggests that nCeO2 penetrate the leaf surface but their

translocation through the leaf tissue is limited.

Ce concentrations in stems and flowers increased as the external concentration of nCeO2

increased. Flower structures appeared several days after treatment application. This may indicate

an effective uptake and translocation of the nCeO2 n through the cucumber leaves. As shown in

Figure 2.3C, Ce in flowers increased as the concentration of NPs in treatments increased. The Ce

concentration at 80 mg/L (~6 µg/g) and above was statistically higher than at 40 mg/L. In this

study, we did not determine the speciation of Ce in cucumber tissues; however, previous studies

have shown that most of the Ce in tissues of plants treated with nCeO2 remains in the

nanoparticulate form.47-48

This suggests that atmospheric nCeO2 can be absorbed by plant leaves.

Further studies are needed in order to determine if these NPs will reach the fruit, which will be

significant for the risk assessment of air pollution with nCeO2.

2.3.4 TEM studies of cucumber tissues from plants treated with CeO2 powder

Figure 2.4 shows TEM micrographs and corresponding EDS spectra of cucumber tissues

from plants treated with 0.98 mg nCeO2/m3. The micrograph in Figure 2.4A shows a white spot

in the epidermal surface and the corresponding EDS spectrum shows the presence of Ce in the

19

spot (Figure 2.4B). In addition, Figure 2.4C shows the cross section of the cucumber root from a

nCeO2 treated plant. Two spots (1 and 2) were selected from one of the phloem areas and the

corresponding EDS spectrum shows the presence of Ce in phloem (Figure 2.4D). Cerium dioxide

NPs are very stable in a variety of environments.43-44

Previous studies have shown that a portion

of the Ce taken up by the roots of plants treated with nCeO2 is transported to the aboveground

tissues and remain as nCeO2 in vegetative and reproductive organs.48-49

The presence of nCeO2

in reproductive organs48

suggests phloem transportation. In our work, the TEM image (Figure

2.4C), suggest that small nCeO2 penetrated leaf surface through hydathodes (cuticle free

segments) and stomata,50

traverse cell walls of palisade parenchyma, reach the leaf phloem and

distribute within the plant body.

Figure 2.5A displays the electron micrograph of a control cucumber leaf sample showing

healthy chloroplasts filled with starch granules. While Figure 2.5B shows a structurally normal

parenchymal cell from a cucumber leaf sample treated with 320 ppm nCeO2 suspension.

Parenchyma cells are the only photosynthetic cells in leaves and stems. Their primary cell walls

are thin, which allow light, water, gases, and metabolites to pass through.

20

Figure 2.3: Cerium concentration in A. leaves, B. roots and stems, and C. flowers of cucumber

plants treated with nCeO2 suspensions. Values are means SE (n = 4). * Stands

statistical differences at p ≤0.05

21

Figure 2.4: A. TEM image of surface of cucumber leaf treated with 0.98 mg nCeO2/m3. B. EDS

of the spot highlight in A. C. TEM image of cucumber root treated with 0.98 mg

nCeO2 /m3. D. EDS of the spot 1 highlight in C.

In Figure 2.5B, the parenchymal cell shows an increase of vacuole space, which has been

shown to be an ultra-structural sign of metal toxicity among various algae, such as Laminaria

saccharina,51

Chlamydomonas reinhardtii,52

and Crypthecodinium cohnii.53

Meristematic cells

contain many small vacuoles that will form the large central vacuole when the cell is matured.54

Epimashko et al.55

found that Mesembryanthemum cystallinum L. can form two vacuoles in one

22

cell under salt stress. These two vacuoles can help the plant to separate the contrasting vacuolar

functions of salt storing and malate cycling. There is no information on physiological or ultra-

structural changes due to CeO2 NPs. Although the mechanism of two vacuoles co-existing in one

cell is still poorly understood, this abnormal phenomenon is hypothesized to be caused by

nCeO2, since this was not observed in the control. In Figure 2.5B, dark precipitates are present

inside the vacuole, near mitochondria and in the tonoplast (circles). Also, there is only one

chloroplast shown in the cell. It has been proven that the number of chloroplasts is reduced by

metal toxicity.56-57

Figure 2.5C shows control root cells, while Figure 2.5D shows cucumber root

sample treated with 320 ppm nCeO2 suspension. It clearly displays dark precipitates in both the

interior of root cells and between root cells. The elemental analysis spectrum identified the dark

precipitation as Ce. The dark precipitates were only observed in the treated plant tissues; thus

corroborating the uptake and translocation of the nCeO2 through the leaves.

23

Figure 2.5: A. TEM image of control cucumber leaf sample showing a parenchymal cell (C-

chloroplast; V-vacuole); B. TEM image of cucumber leaf sample showing a

parenchymal cell (C-chloroplast; M- mitochondria; V-vacuole) treated with 320

ppm nCeO2 suspension; C. TEM image of control cucumber root sample (I-interior

of cell; B-between cells); D. TEM image of cucumber root sample treated with 320

ppm nCeO2 suspension (I-interior of cell; B-between cells); E. Elemental analysis

of dark precipitation found in the interior of cell shown in D.

24

2.3.5 Enzyme activity essays

Figure 2.6 shows the activity of three antioxidant enzymes in cucumber leaves, stems,

and roots treated with either nCeO2 powder or suspension. As seen in Figure 2.6A, in powder

treatments the APX activity decreased in stems and increased in roots as the external nCeO2

increased. The activity of CAT significantly increased in leaves and roots at the higher NP

concentration, but no changes were observed in stems (Figure 2.6B). On the other hand, DHAR

did not show changes in activity at any concentration of the nCeO2 powder (Figure2.6C).

On the other hand, in plants treated with the CeO2 suspensions, the APX activity

decreased in leaves at all NP concentrations. However, in stems and roots, only the 40 mg/L

produced a significant increase in APX activity (Figure 2.6D). The activity of CAT increased in

leaves treated with 160 and 320 mg/L, it also increased in stems of plants treated with 80 and

160 mg/L, and in roots of plants treated with 40 to 160 mg/L (Figure 2.6E). In addition, the

activity of DHAR showed increase in leaves only in plants treated with 80 mg/L, but it increased

in stems and roots of plants treated with 80 to 320 mg/L (Figure 2.6F). The increase in DHAR in

roots and stems could lead to an increase in APX in these organs; however, the result was

contrary to the expected. Previous studies58-59

have shown that metals can interfere with APX

activity. As shown in Figures 2 and 3, the Ce uptake from suspensions was higher compared to

powder applications. Thus, this could explain the different response in enzyme activity in plants

treated with NP suspensions. Over all, CAT activity increased and APX activity decreased in the

treated samples compared to control.

The increase in CAT activity in all plants treated either with powder or suspension at low

concentrations suggests that the nCeO2 in contact with leaves can modify this enzyme activity in

cucumber plants. On the other hand, the decrease of CAT in stems and roots of plants treated

with 320 mg/L suggests that at this concentration nCeO2 caused toxicity. Morales et al.33

25

reported that in cilantro, 62.5 and 125 mg/kg of nCeO2 produced increase in CAT activity but at

250 and 500 mg/kg the NPs caused decreased in CAT activity. In addition, Zhao et al.32

found

that in 10 day-old maize plants, APX activity was higher at 400 mg CeO2 NPs/kg, but lower at

800 mg/kg. In the present study, the APX activity decreased in leaves of nCeO2 treated plants but

increased in stems and roots of plants treated with 40 mg/L. As previously reported, APX utilizes

ascorbic acid (AsA) as the electron donor to remove H2O2 in the ASA- glutathione (GSH)

cycle.60

In chloroplasts, APX isoenzymes prevent oxidative stress, maintaining the antioxidant

system.61-62

In Figure 5B, we observed the cell with only one chloroplast, and perhaps the

number of chloroplasts was reduced maybe because of nCeO2 toxicity. This could explain the

decrease of APX activity in leaves. As shown in Figure 2.6D, the activity of DHAR was higher

in stems and roots of plants treated with 80 to 320 mg/L of CeO2. In rice plants, DHAR activity

was also enhanced as the CeO2 NP concentration in the treatments increased.34

2.4 CONCLUSION

In summary, this study has shown that cucumber plants can take up Ce from nCeO2

applied to the leaves as powder or suspension. In addition, the TEM studies have shown that the

Ce taken up by the leaves is translocated to other plant tissues. The nCeO2treatments, even at

concentrations of 40 mg/L or 0.98 g/m3, can modify the enzyme activity in cucumber plants.

Although the speciation of Ce in tissues was not determined, previous studies have shown that

most of the Ce in tissue of plants treated with nCeO2 remains in nanoparticulate form. This

suggests that atmospheric nCeO2 could be stored in the fruit of cucumber plants. Further studies

are needed in order determine the risk of atmospheric nCeO2 to human and environmental health.

26

Figure 2.6: Antioxidant enzyme activities in cucumber plants foliarly treated with CeO2 NP

powder at 0.98 and 2.94 g m-3 or 40, 80, 160, and 320 mg L-1

of nCeO2

suspensions. A, B, and C correspond to APX, CAT, and DHAR activities,

respectively, in plants treated with powder. D, E, and F correspond to APX, CAT,

and DHAR activities, respectively, in plants treated with nCeO2 suspensions.

Values are means± SE (n = 4). Means with the same letter are not significantly

different (Tukey test, p≤0.05).

27

Chapter 3: Foliar Applied CeO2 and CuO Nanoparticles/Bulk Materials Alter

Firmness and Nutrient Content in Cucumber (Cucumis sativus)

Abstract

There is lack of information about the effects of foliar applied nanoparticles on fruit

quality. In this study, soil grown cucumber seedlings were foliarly treated with nCeO2, nCuO,

and corresponding bulk materials at 50, 100, and 200 mg/L. Net photosynthesis rate (Pn),

stomatal conductance (Gs), and transpiration rate (E), were measured 15 days after treatment

application and in mature plants. Nutrient elements and fruit parameters were also measured.

Results showed that at 200 mg/L, nCeO2 and nCuO treatments decreased Pn by 22% and 30.2%

and E by 11% and 17% in seedling leaves, respectively. nCuO at 200 mg/L and bulk CuO at 50

mg/L increased fruit fresh weight. However, nCeO2 at 50 mg/L, bulk CeO2 at 200 mg/L, and all

CuO treatments, except nCuO at 100 mg/L, significantly reduced fruit firmness. Compared to

control, fruit Zn decreased by 25% with 200 mg/L of both nCeO2 and bulk CeO2 and fruit Mo

decreased by 51% and 44% with both nCuO and bulk CuO at 200 mg/L, respectively. The data

suggest that nCeO2 and nCuO could reduce the quality and marketability of cucumber fruit. To

the author’s knowledge, this is the first report on the effect of nCeO2 and nCuO in the nutritional

quality of cucumber fruit.

28

3.1 INTRODUCTION

Cerium oxide (nCeO2) and copper oxide (nCuO) are widely used ENPs. nCeO2 are used

in UV coatings, fuel catalysis, polishing, and mechanical planarization. By 2012, the annual

global production of nCeO2 was estimated to be 1000 t/year.1 nCuO is used in gas sensors,

catalysis, lithium ion batteries, high-temperature superconductors, solar energy conversion, and

field emission guns.2 Such profuse use enables the release of nanoparticles (NPs) to the soil,

atmosphere and water. Additionally, they can end up on crop plants.3-4,6

Soil-root interface is the major pathway for plants to uptake nutrients and other soil

components. Reports indicate that certain nanomaterials can be taken up by roots and

translocated to the fruits causing different effects.5-10

For instance, fullerol [C60(OH)20] has been

found to increase the biomass and fruit yield of bitter melon (Momordica charantia) by 54% and

128% , respectively.9 Wang et al. reported that tomato (Solanum lycopersicum L.) plants exposed

to 1 and 10 mg/L nCeO2 produced less fruits with slight delay in maturing time, which were

relatively big and heavy compared to fruit from control and 0.1 mg/L nCeO2 treated plants.7 On

the other hand, nCeO2 have been reported to decrease Fe, S, prolamin, glutelin, lauric and valeric

acid, and starch in rice (Oryza sativa L.) grains.8 Zhao et al. reported nCeO2 did not affect

cucumber plant growth, gas exchange, and chlorophyll content, but impacted the nonreducing

sugar content, carbohydrate pattern, and phenolic content.5-6

nCeO2 have also been found to alter

the nutritional value of soybean.11

nCuO, repetitive, have also been found to reduce water

content, root length, and dry biomass of lettuce (Lactuca sativa) plants.12

In addition, nCuO have

been reported to reduce shoot and root growth of sand-grown wheat (Triticum aestivum) and

significantly increased Cu accumulation in plant tissues, which may cause potential harmful

health effects in the food chain.13-14

29

Previous studies have shown that plants can also take up NPs through the leaves. nAg

were found to penetrate lettuce leaves when applied at 100 µg nAg g-1

FW. TEM images showed

that carbon-coated iron NPs were able to penetrate pumpkin (Cucurbita pepo L.) leaves and

migrate to different plant tissues. Wang et al. found that TiO2 NPs, MgO NPs, ZnO, and Fe2O3

NPs with size less than 100 nm could enter watermelon (Citrullus lanatus) leaves through

stomata and translocate to root through stem. In addition, Eichert et al. also found that small size

(43 nm) polystyrene NPs could enter leaves of broad bean (Vicia faba L.), while larger particles

(1.1µm) could not pass through stomata. nCeO2 were also reported to penetrate cucumber

(Cucumis sativus L) leaves and to be transferred to flowers.15-20

However, in these studies,

authors only reported the uptake of NPs through leaves. To the author’s knowledge, fruit quality

and changes in physiological markers such as leaf net photosynthetic rate (Pn), stomatal

conductance (Gs), and transpiration rate (E) in plants foliarly treated with NPs have not been

reported yet.

In this study, cucumber was selected not only because of its high nutrition value and

increasing consumption around the world,6 but also because in a previous study, it was found

that cucumber can take up and translocate Ce from leaves to flowers when exposed to nCeO2

through the leaves.20

However, no reports were found about nCuO. Thus, we apply nCeO2 and

nCuO to cucumber seedling leaves and allow the plants to growth to full maturity. At harvest,

fruit and other tissues were analyzed for nutrient, Ce, and Cu content by inductively coupled

plasma optical emission spectrometer (ICP-OES). Biochemical markers such as Pn, Gs, and E

were measured with a portable gas exchange system.

30

3.2 MATERIALS AND METHODS

3.2.1 Characterization of NPs.

The nCeO2 (Meliorum Technologies, NY) and nCuO (Sigma- Aldrich Chemical Co., St.

Louis, MO) were obtained from the University of California Center for Environmental

Implications of Nanotechnology (UC CEIN). Previous studies showed that nCeO2 are rods with

primary size of 8 ± 1 nm, particle size of 231 ± 16 nm in DI water, surface area of 93.8 m2g

-1 and

95.14% purity.22

nCuO were found to be irregularly shaped, 84.8 ± 2.7% pure with surface

area 29 m2 g

−1 and their size were less than 50 nm.

23-24 CeO2 (< 5 µm) and CuO bulk materials (<

5 µm) (Sigma- Aldrich Chemical Co., St. Louis, MO) were also used in this experiment to

compare with the NPs.

3.2.2 Foliar application of NPs and bulk materials

Cucumber plants were grown in 5.8 L Poly-Tainer containers filled with a substrate

previously described in the literature.5 Each container had two plants. When plants were two

weeks old they were sprayed either with nCeO2, nCuO, bulk CeO2 or bulk CuO suspended in

distilled deionized water at 0, 50, 100, and 200 mg/l and homogenized by sonication (Crest

Ultrasonics, Trenton, NJ) in ice-cooled water for 45 min. Suspensions were sprayed with a hand-

held sprayer bottle when the plants had two true leaves. Incipient leaves, soil, and neighbor

plants were protected with plastic sheet to prevent contamination and plant surface sticking of

NPs. A total volume of 250 mL per treatment was split in three applications (every four h). There

were four replicates for each treatment. Two weeks after treatment, one plant in each pot was

harvested to measure the Ce, Cu and mineral content in the tissues. The roots were labeled as

seedling roots. The rest of the plants were treated with 250 ml suspensions every three days for

12 days (four times). Plants were grown for 74 days until 80% of fruits obtained marketable size.

At harvest, the roots were labeled as mature roots.

31

3.2.3 Gas Exchange

In this study, leaf net photosynthetic rate (Pn), stomatal conductance (Gs), and

transpiration rate (E), were measured in seedling leaves and mature leaves. These data were

measured by using a portable gas exchange system (CIRAS-2; PP Systems, Amesbury, MA).

During the measurement, the fully expanded leaf was placed in the cuvette, where the

environment conditions were controlled at a leaf temperature of 25 °C, photosynthetic photon

flux (PPF) of 1000 μmol•m−2

s−1

, and CO2 concentration of 375 μmol•mol−1

. The data was

collected when the readings were stable. In order to eliminate the light and water effects, the

measurements were only taken between 10 am and 1pm, and the plants were well watered one

hour before the reading to eliminate the water stress effects.

3.2.4 Growth Data and fruit quality

At harvest, the firmness (kg/cm2), length (cm), diameter (cm) and fresh weight (g) of

cucumbers were measured. The firmness of the pulp was measured in terms of maximum

resistance by hand penetrometer. In addition, total yield (kg/plot), fruit number (fruit/plot) and

average fruit weight (g/fruit) were measured.

The dry weight (d wt) of stems, leaves, and fruits were determined after oven-drying at

65 °C for three days. Before drying, all the plant samples were cleaned with running tap water,

followed by rinsing (1 min, three times) with deionized water.

3.2.5 Sample preparation for ICP-OES

Nutrients, Cu, and Ce content in tissues, including fruits, were measured by ICP-OES

(Perkin- Elmer Optima 4300 DV). Dried plant and fruit samples were ground to pass a 40- mesh

screen (0.425 mm) with a stainless Wiley mill (Thomas Scientific, Swedesboro, NJ). Then, 0.2 g

32

powder samples were digested following the EPA method 3051 using microwave oven (CEM

Corp, Mathews, NC). A mixture of 30% H2O2 and plasma pure HNO3 (v/v: 4:1) was used in the

digestion process. The standard reference materials NIST 1547 was also digested and analyzed

as sample for quality control. The recoveries for all elements were between 90 and 99%.


The reported data are means of four replicates ± standard error (SE). One-way ANOVA

was used to analyze the experiment variance (SPSS 19.0 package, Chicago, IL). Tukey Honestly

Significant Difference was used to determine statistical differences between treatment means.


3.3.1 Ce and Cu accumulation in cucumber plant tissues and fruits

In a previous study we determined Ce in flowers of cucumber plants exposed through the

leaves to nCeO2 at a very early growth stage.20

In this study, we exposed the leaves of soil grown

cucumber seedlings to nCeO2 and nCuO and measured their effects on fruit production and

nutritional quality of fruit. Figure 3.1 showed Ce (A) and Cu (B) concentration in seedling leaves

and roots, mature roots, and fruit of cucumber plants. As shown in Figure 3.1, Ce concentration

in seedling leaves of nCeO2 and bulk CeO2 treatments was significantly higher, compared to

control. Since these leaves were formed after treatment application, it is concluded that this

difference comes from nCeO2/Ce ions translocated from treated leaves.

33

Figure 3.1. Ce (A) and Cu (B) concentration in seedling leaves, seedling roots, mature roots and

fruits. Data are average of four replicates± standard error. Data are averages of four

replicates ± SE. Different letters stand for statistical differences at p< 0.05.

a

ab

b

b

ab ab

b

a ab

ab b

a ab

ab

a a a b

a a a

0

2

4

6

8

10

12

Control 50 nCeO2 100 nCeO2 200 nCeO2 50 Bulk CeO2 100 BulkCeO2

200 BulkCeO2

Ce

co

nte

nt

in c

ucu

mb

er

tiss

ue

(m

g/kg

)

CeO2 treatment (mg/L)

Seedling Leaf Seedling Root Mature Root FruitA

a

b

ab

ab

ab ab ab

ab

a

ab

ab

b

ab ab b

a

ab ab ab

ab ab

0

2

4

6

8

10

12

14

Control 50 nCuO 100 nCuO 200 nCuO 50 Bulk CuO 100 Bulk CuO 200 Bulk CuO

Cu

co

nte

nt

in c

ucu

mb

er

tiss

ue

(m

g/kg

)

CuO treatment (mg/L)

Seedling Leaf Seedling Root Mature Root FruitB

34

This result corroborates our previous finding in 1-month-old cucumber plants.20

The highest Ce

concentration in the seedling roots and fruit occurred under 200 mg/L nCeO2 treatment, but no

effects were found in mature roots. Wang et al. also reported that watermelon plants can

transport MgO NPs from leaves to roots through phloem.16

Photosynthetic products such as

sucrose, proteins, and mineral ions are translocated to shoots and roots via phloem.21

NPs could

follow the same pathway to plant root and fruit. In addition, cucumber and tomato plants have

been reported to accumulate more Ce in the shoot and fruit when they were treated with nCeO2.5,

7 Wang et al. reported that nCuO in maize (Zea mays L.) could be translocated from roots to

shoots via xylem and translocated from shoots back to roots via phloem.22

This may explain why

Ce concentration in mature roots became lower compared with seedling roots. Unlike nCeO2

treatment, nCuO/bulk CuO only caused Cu increase in seedling leaves, but no obvious effects on

seedlings roots, mature roots, and fruit were observed. This could be explained by nutrient

mobility, which has been known for a long time: plant can “remobilize nutrients from leaves and

transport them in the phloem to other organs.”23

According to Bukovac and Wittwer, Cu moves

rapidly to other leaves when it is foliar applied.24

The rate of Cu movement is faster in Cu-

sufficient plants compared to Cu-deficient plants.23

3.3.2 Effects of NPs photosynthesis rate, transpiration rate, and stomata conductance

Photosynthetic parameters such as Pn, E and Gs are indicators of plant response to

changes in the environment. As shown in Table 3.1 and 3.2, at 200 mg/L, nCeO2 and nCuO

treatments decreased Pn by 22% and 30.2% and E by 11% and 17% in seedling leaves,

respectively. Gs was significantly higher in plants treated with 50 mg/L nCeO2 and 100 mg/L

CuO bulk treatment, compared to the other treatments. However, photosynthetic parameters were

35

not affected on mature leaves (data not shown). Also, there was no damage or sign of toxicity in

seedling leaves. Usually, low Pn values have been found in old trees and old leaves.25


sprayed with nCeO2 and bulk CeO2 suspensions and data was collected 15 days

after treatment application. Data are average of four replicates ± standard error.

Values in a column followed by different letters have significant difference

(p<0.05).

Treatment Pn (µmol·m-2

·s-1

) E (mmol·m-2

·s-1

) Gs (mol·m-2

·s-1

)

Control 17.3±2.6 d 4.8±0.4 b 360.0±125.0 a

50 mg/L nCeO2 16.0±1.9 cd 5.4±0.6 b 566.4±139.3 b

100 mg/L nCeO2 10.8±1. ab 3.5±0.8 ab 273.9±86.1 a

200 mg/L nCeO2 13.4±1.5 c 4.3±0.5 ab 339.4±96.8 a

50 mg/L CeO2 Bulk 13.0±1.9 bc 5.4±1.1 b 365.0±97.7 a

100 mg/L CeO2 Bulk 10.0±2.0 ab 3.7±1.1 a 270.1±122.8 a

200 mg/L CeO2 Bulk 8.8±2.1 a 3.3±0.9 a 226.4±59.1 a


sprayed with CuO NPs/bulk materials suspensions and data was collected 15 days

after treatment application. Data are average of four replicates ± standard error.

Values in a column followed by different letters have significant difference

(p<0.05).

Pn(µmol·m-2

·s-1

) E (mmol·m-2

·s-1

) Gs (mol·m-2

·s-1

)

Control 17.3±2.6 c 4.8±0.4 bc 360.0±125.0 a

50 mg/L nCuO 9.1±1.5 a 3.6±0.6 a 243.7±90.6 a

100 mg/L nCuO 11.8±1.6 ab 3.7±0.4 a 239.0±59.3 a

200 mg/L nCuO 10.7±2.3 a 4.0±0.9 ab 283.3±91.8 a

50 mg/L CuO Bulk 12.4±3.2 ab 3.6±1.1 a 204.4±95.6 a

100 mg/L CuO Bulk 15.3±2.4 bc 5.2±0.6 c 467.1±150.8 b

200 mg/L CuO Bulk 11.9±2.1 a 3.6±0.5 a 294.4±91.5 a

Heavy metals such as, Cu and Cd have been found to decrease Pn and Gs in plants.26

Bazzaz et al.27

reported the Pn and E decreased in corn and soybean treated with Pb. The authors

concluded that “transpiration exhibited similar trends to photosynthesis, especially in corn,

suggesting that an appreciable part of the inhibition of the two processes is related to increased

36

stomata resistances with increased Pb concentrations.” In this study, the possible reason for

stomata resistances is that NPs blocked the stomata of cucumber leaves.

Few studies have evaluated the effects of NPs on gas exchange in plants. Zhao et al.5

monitored Pn, Gs, and E of cucumber plants every 10 days after soil amendment with ZnO and

nCeO2. They found no significant effects on these parameters throughout the life cycle of the

plants. However, Alidoust and Isoda28

observed that foliar applied nFe2O3 significantly enhanced

photosynthetic parameters in soybean, compared with soil applied nFe2O3. Stomata opening

played a more important role in these enhancements rather than increased CO2 uptake at

chloroplast level. In the present study, the decrease in Pn and Gs could indicate that stomata are

a major pathway for NP entrance into cucumber leaves.

3.3.3 Effects of NPs on cucumber growth, yield and fruit quality

Table 3.3 shows some physical characteristics of cucumber fruits harvested from plants

grown in soil and aerially treated with nCeO2, nCuO, and bulk material suspensions. As seen in

this table, none of the treatments significantly affected the yield, length, and diameter of

cucumbers. However, 50 mg/L nCeO2, 200 mg/L bulk CeO2, and all CuO treatments, except

nCuO at 100 mg/L, significantly reduced the firmness of cucumbers. This suggests that, even

though Ce and Cu were at low concentration in fruits, the treatments could be affecting fruit

texture. Pulp texture is an important fruit characteristic,29

which can be affected by the type of

fruit, gibberellic acid and storage time, among others.30-32 The mechanism of NPs’ effect on fruit

firmness is still not clear and further studies need to be performed.

Cerium is a rare earth element that has positive effects on plant growth and yield

enhancement.33

Cerium has been used as fertilizer in China for a long time.34

It has been reported

37

that nCeO2 increased tomato size and weight when applied as low as 10 mg/L to soil.7 In the

present study, foliar application of nCeO2 had no effects on the number, size, and biomass of

fruits. Zhao et al.5 reported that at 800 mg/kg, nCeO2 decreased yield by 31.6%, compared with

control. Also, Priester et al.35

reported that nCeO2 applied to soil reduced soybean yield by 17-

23%. All these studies suggested that the effects of NPs on crop yield depend on plant species,

types of NP, and concentrations.

Table 3.3. Yield and characteristics of cucumber fruit harvested from plants foliarly treated with

nCeO2, nCuO, bulk CeO2 and bulk CuO suspensions.

Fruit number Fresh weight (g) Dry weight (g) Length (cm) Diameter (cm) Firmness

(kg/cm2)

control 10 735.7±132.5 ab A 17.3±3.0 20.7±1.3 52.4±3.2 9.1±0.9 b D

50 mg/L nCeO2 10 565.3±81.4 a 15.2±2.2 20.3±1.0 52.6±5.3 7.5±0.5 a

100 mg/L nCeO2 9 634.3±233.1 a 22.9±3.2 21.1±1.7 51.7±2.9 9.0±0.7 b

200 mg/L nCeO2 10 840.3±103.4 a 23.3±1.2 22.0±0.8 54.9±2.5 8.8± 0.9 b

50 mg/L Bulk CeO2 13 982.3±101.0 a 25.6±1.2 18.9±0.7 51.0±3.3 8.8±1.1 b

100 mg/L Bulk CeO2 8 922.3±19.9 a 31.3±2.6 21.1±2.1 53.5±5.4 7.9±0.8 ab

200 mg/L Bulk CeO2 10 932.3±388.0 a 30.0±9.4 19.8±1.5 50.0±4.5 7.5±0.6 a

50 mg/L nCuO 8 620.3±131.6 A 22.6±1.2 19.5±1.2 51.5±4.2 7.6±1.0 BC

100 mg/L nCuO 10 640.3±88.4 A 19.7±1.6 21.1±1.4 54.0±5.0 8.3±1.2 CD

200 mg/L nCuO 13 950.0±140.3 BC 15.6±1.2 21.0±1.2 53.1±2.9 7.8±0.4 BC

50 mg/L Bulk CuO 13 1076.3±108.7 C 33.3±5.7 21.2±1.0 52.4±2.0 7.2±0.3 ABC

100 mg/L Bulk CuO 14 627.7±63.58 A 19.8±1.9 21.1±0.5 54.3±2.5 6.2±0.8 A

200 mg/L Bulk CuO 12 539.7±68.7 A 23.0±7.0 22.1±0.7 54.2±2.3 6.8±0.4 ABC

Data are average of four replicates ± standard error. Values in a column followed by different

letters have significant difference (p < 0.05). Lower case letters stand for significant differences

between control and CeO2 treatments, and upper case letters stand for significant differences

between control CuO treatments.

3.3.4 Effects of aerial application of nCeO2 on Mineral content in cucumber

Crop plants are major sources of essential minerals for human beings. Uptake of these

minerals by plants depends on soil condition (including composition, cation exchange capacity,

38

and pH), microorganisms, and metal mobilization, among others.36

The seedling stage is very

important for plant growth; the NP treatment at seedling stage may change mineral contents in

mature plants and finally affect plant health, yield, and fruit quality. As shown in Table 3.4, at

200 mg/L, nCeO2, bulk CeO2, and all CuO treatments significantly increased P concentration but

reduced Ca, Fe, Mn, and B concentration in seedling leaves. In addition, 200 mg/L nCeO2 and all

CuO treatments decreased Fe and Mn concentration in seedling roots (data not shown).

According to Table 3.4, there was no difference between NPs and their respective bulk

counterpart in elements’ accumulation, except in P accumulation. At 100 and 200 mg/L, bulk

CuO significantly decreased P concentration in seedling leaves, compared to 100 and 200 mg/L

nCuO treatments. Since there was no difference in P concentration between nCuO and bulk

treatment in seedling roots, we hypothesize that CuO bulk only affected the P transfer inside the

cucumber plant but did not impact P uptake. Jeschke et al.37

reported that in P-sufficient plants,

most of the P is absorbed by the roots and transported though the xylem to the younger leaves

and in P-deficient plants P will be recycled back to the roots through phloem. Considering the

particle size, CuO bulk materials may cause some blockage in these pathways, reducing P in

seedling leaves. On the other hand, mineral elements in cucumber fruit (Table 5) were not

affected by treatments, except Zn under CeO2 treatment and Mo under CuO treatment. Zinc

concentration decreased by about 25% at 200 mg/L of both nCeO2 and bulk CeO2, compared to

control. Mo decreased by 51%, 40%, and 44% under 200 mg/L nCuO, 100 mg/L, and 200 mg/L

bulk CuO treatments, respectively, compared to control. Mo uptake by plants could be affected

by soil type and texture, plant species, and organic matter in the soil.38

Under most condition, Mo

uptake by plants increases with an increase in soil pH. Also, SO42-

could reduce uptake of Mo,

since Mo uptake may be affected by one of the sulfate transporter.39-40

In our study, S and Mo

39

uptake was not affected in seedling leaves, seedling roots, and mature roots. We cannot conclude

that NPs affect the uptake of Mo, but they may affect the transfer/accumulation of Mo in fruit. In

soybean, root applied nCeO2 also decreased Mo concentration in stems and roots, but increased

Mo in nodules by134% to164% compared to control.11

Also, Zhao et al. reported that nCeO2

reduced Mo by 40-53% in cucumber fruit.6 However, it seems that the level of decrease in Mo

accumulation should not cause health issues. According to USDA Beltsville Human Nutrition

Research Center: “Mo deficiency and toxicity are rare, possibly because of the body’s ability to

adapt to a wide range of molybdenum intake levels.” 41

The mechanism as to how nCuO affects

Mo uptake still needs more study.

According to the Food and Nutrition Board of the Institute of Medicine at the National

Academies, the recommended dietary allowance of Zn is 8 and 11 mg for female and males

adults, respectively.42

Considering small difference on Zn concentration in fruit between controls

and treated with nCeO2 or bulk CeO2, a reduction of Zn would not significantly decrease

cucumber nutrition value and cause health concern. Although reduction of Zn and Mo in

cucumber fruit would not affect human health, NP treatments may affect other nutrition

components, such as carbohydrates, starches, sugars, protein, and fibers. These may impact

cucumber flavor and nutrition value. More studies are needed in order to fully comprehend the

effects of NPs on the quality and nutritional value of cucumbers and the potential impact on

human health.

40

Table 3.4. Mineral concentration in seedling leaves (mg/kg) from plants foliarly treated with nCeO2, nCuO, bulk CeO2 and bulk CuO

suspensions.

P Ca Mg Fe Mn B

control 4379.8±83.6 a A 24830.2±823.6 b B 4339.2±61.8 a B 87.5±5.7 b B 29.3±2.3 b B 22.2±1.4 b B

50 mg/L nCeO2 6342.0±285.7 b 13237.1±873.1 a 3777.6±119.4 ab 70.5±2.1 ab 24.3±1.7 ab 15.8±1.5 a

100 mg/L nCeO2 5709.6±303.9 ab 12009.2±1098.8 a 3599.2±133.3 a 55.2±1.2 a 18.4±1.2 a 13.0±1.0 a

200 mg/L nCeO2 6075.0±376.8 b 9488.3±287.0 a 3519.9±122.6 a 73.5±4.4 ab 19.4±0.7 a 12.1±0.5 a

50 mg/L CeO2 Bulk 5575.8±169.9 ab 12962.1±1163.8 a 3766.9±123.1 ab 63.8±2.0 a 22.8±1.0 ab 14.5±1.5 a

100 mg/L CeO2 Bulk 5714.6±540.4 ab 12274.4±166.0 a 3643.7±297.3 a 67.5±8.6 ab 21.2±3.6 ab 14.8±2.2 a

200 mg/L CeO2 Bulk 6337.7±420.9 b 11082.0±859.6 a 3472.5±165.4 a 57.6±5.4 a 19.9±1.4 a 13.7±1.7 a

50 mg/L nCuO 6154.7±286.3 C 13499.3±634.9 A 3772.2±161.4 A 62.4±4.7 A 23.1±1.4 AB 13.8±13. A

100 mg/L nCuO 5883.9±407.7 C 13171.9±521.0 A 3595.7±25.6 A 57.3±5.7 A 21.8±1.2 A 12.8±0.8 A

200 mg/L nCuO 6103.0±128.5 C 13402.9±1624.2 A 3806.2±152.5 A 64.1±5.1 A 22.2±0.5 A 12.5±1.5 A

50 mg/L CuO Bulk 5922.6±83.9 C 13768.4±1293.8 A 3862.0±166.2 AB 59.4±5.0 A 23.9±1.8 A B 12.2±1.3 A

100 mg/L CuO Bulk 5489.7±130.4 BC 12713.1±812.1 A 3653.1±177.0 A 62.8±7.2 A 21.5±2.2 A 9.5±1.1 A

200 mg/L CuO Bulk 4794.2±149.0 AB 11388.2±603.7 A 3301.1±48.5 A 48.9±2.0 A 18.6±0.5 A 10.4±0.7 A

Data are average of four replicates ± standard error. Values in a column followed by different letters have significant difference

(p<0.05). Lower case letters means the significant difference between control and CeO2 treatment, and upper case letters for CuO

treatment.

41

Table 3.5. Mineral concentration of cucumber fruit (mg/kg) from plants foliarly treated with nCeO2, nCuO, bulk CeO2 and bulk CuO

suspensions.

Treatment P Ca S Mg Mo Fe Mn Zn B

control 7737.7±238.9 3983.4±220.9 2879.1±70.6 2983.7±125.7 2.7±0.2 41.5±2.2 17.6±0.8 35.8±0.7 b 5.9±0.4

50 mg/L nCeO2 7499.0±122.5 4378.5±173.3 2999.6±64.1 3030.4±24.8 2.3±0.1 43.4±2.7 15.9±0.2 35.2±1.0 b 4.2±0.3

100 mg/L nCeO2 6947.5±301.7 5117.6±75.5 2926.3±86.0 2995.5±75.9 2.20±0.1 40.3±1.4 18.1±0.6 34.1±1.3 ab 3.2±0.2

200 mg/L nCeO2 6265.7±39.5 4343.7±110.5 2559.4±52.2 2615.4±27.5 1.8±0.1 54.9±6.6 14.8±0.5 26.1±0.6 a 2.3±0.1

50 mg/L CeO2 Bulk 7175.3±225.9 4969.2±194.2 2901.1±92.3 2930.6±70.9 2.6±0.2 37.6±1.7 17.0±0.5 33.6±1.3 ab 5.6±1.2

100 mg/L CeO2 Bulk 6498.0±163.5 4438.5±144.7 2708.3±78.3 2816.2±65.1 2.3±0.2 33.3±1.3 15.2±0.7 29.8±0.6 ab 2.4±0.6

200 mg/L CeO2 Bulk 6472.2±220.8 4362.5±104.3 2494.5±85.7 2791.2±59.7 1.6±0.1 30.3±0.7 14.0±0.3 26.4±0.5 a 3.3±0.6

Control 7737.7±238.9 3983.4±220.9 2879.1±70.6 2983.7±125.7 2.7±0.2 b 41.7±2.2 17.5±0.8 35.8±0.7 5.9±0.4

50 mg/L nCuO 5967.6±301.4 3983.5±146.2 2536.7±137.7 2669.6±94.3 1.8±0.1 ab 29.0±1.3 13.8±0.4 26.9±1.6 2.5±0.0

100 mg/L nCuO 6481.6±210.1 4311.7±96.5 2731.7±94.4 2874.7±96.7 1.9±0.1 ab 35.6±1.2 15.2±0.8 29.7±1.2 2.5±0.1

200 mg/L nCuO 6091.8±164.5 4308.7±143.8 2525.9±24.5 2681.8±35.5 1.3±0.1 a 39.6±1.4 17.2±0.5 28.6±0.9 3.3±0.5

50 mg/L CuO Bulk 6568.9±184.7 4559.3±155.4 2664.1±83.5 2886.8±78.2 1.73±0.1 ab 39.7±1.8 16.1±0.6 30.2±0.8 2.3±0.2

100 mg/L CuO Bulk 6023.8±349.1 3830.4±138.3 2567.5±104.2 2581.0±95.6 1.6±0.1 a 57.4±10.8 14.3±0.6 30.9±2.2 2.9±0.2

200 mg/L CuO Bulk 6296.0±482.8 3887.5±293.8 2472.6±158.0 2694.9±171.2 1.5±0.2 a 32.8±2.3 14.9±1.4 28.7±1.9 3.8±0.5

Data are average of four replicates± standard error. Values in a column followed by different letters have significant difference

(p<0.05).

42

3.4 CONCLUSION

In summary, foliar applied nCeO2 and nCuO significantly increased Ce concentration in

seedling leaves and fruit, and Cu concentration in seedling leaves. There was no significant

effect on Cu accumulation in fruit under nCuO treatment. In this study, photosynthetic

parameters in mature leaves, cucumber growth, fruit size, and yield were not affected by

treatments, except fruit firmness, which may reduce quality and marketability of cucumbers.

Although mineral (Ca, Mg, Fe, Mn and B) concentrations in seedling leaves significantly

reduced under 200 mg/L of both nCeO2 and nCuO treatment, there was no significant effect on

fruit elements’ accumulation. However, the results from two NPs are not enough to state that

atmospheric NPs have no effects on crop plant; since NPs have distinct properties and plant

species have different degree of susceptibility to external stimuli.

43

Chapter 4: Toxicity Effects of Seven Cu Nanoparticles/Compounds to Lettuce

(Lactuca sativa) and Alfalfa (Medicago sativa)

Abstract

The increase in production and use of nanoparticles (NPs) has generated concerns about

their impacts in organisms, especially plants. In this study, nCu, Cu Bulk, nCuO, CuO bulk,

Cu(OH)2 (CuPRO 2005, CuPRO 3000), and CuCl2 were exposed for 15 days to 10 day-old

hydroponically grown lettuce (Lactuca sativa) and alfalfa (Medicago sativa). Each compound

was applied at 0, 5, 10, and 20 mg/L. At harvest, we measured the size of the plants and

determined the concentration of Cu, macro and microelements by using ICP-OES. Catalase and

ascorbate peroxidase activity was also determined. Results showed that all Cu NPs/compounds

reduced the root length by 49% in both plant species. All Cu NPs/compounds increased Cu, P,

and S (>100%, >50%, and >20%, respectively) in alfalfa shoots and decreased P and Fe in

lettuce shoot (>50% and >50%, respectively, except Fe with CuCl2). Biochemical assays showed

reduced catalase activity in alfalfa (root and shoot) and increased ascorbate peroxidase activity in

roots of both plant species. Results suggest that Cu NPs/compounds not only reduced the size of

the plants but altered the ionome in both plant species. This could have unknown impacts in the

food chain and human health.

Key words: Nanoparticles, copper, alfalfa, lettuce, nutrient uptake

44

4.1 INTRODUCTION

Nanoparticles (NPs) are materials with at least two dimensions between 1 and 100 nm. 1

The small size and large surface area provide NPs different physical strength, chemical

reactivity, electrical conductivity, magnetism, and optical effects, compared to bulk materials. 2-3

These special properties allow NPs utilization in many fields.4-6

It is reported that 1,317 products

containing NPs were on the market in 2010,7 and it is predicted that manufactured nanomaterials

(NMs) will increase from 2,300 to 100,000 tons yearly from 2010 to 2020.8 The increase in NP

utilization has raised concerns about their release in to the environment and possible impacts on

living organisms.9-10

Copper (Cu) and copper oxide (CuO) NPs are widely used in ceramics, films, polymers,

inks, coatings, gas sensors, heat transfer nano-fluids, catalysts and semiconductors.11-14

Previous

reports have shown the effects of these two NPs to terrestrial plants.15-22

Zshah and Belozerova15

found that the shoot/root ratio in lettuce plants treated for 15 days with 0.013% (w/w) nCu in soil

was 2.7, while in control plants the ratio was 1.4. Another report indicates that, compared to

control, 1000 mg nCu/L reduced by 77% the root length and by 90% the biomass of zucchini

(Cucurbita pepo) grown in hydroponic.16

Similarly, Musante and White17

observed that nCu not

only affected root length and biomass of zucchini, but also reduced transpiration volume from 59

ml (control) to 29 and 23 ml in plants treated with 100 and 500 mg nCu/L, respectively. Lee et

al. 18

reported that the seedlings’ length of Mung bean (Phaseolus radiatus) and wheat (Triticum

aestivum L.) were reduced by 60 % and 75%, respectively, when exposed to 1000 mg nCu/L.

They also observed less reduction in shoot growth compared to root, which could be associated

with low translocation of NPs from roots to shoots.19

It has also been reported that nCuO did not

affect seed germination but significantly reduced root length, surface area, and biomass

accumulation in maize (Zea mays L.).19

Other reports indicate that nCuO do not affected

45

zucchini seed germination (16) but reduce root length in wheat (T. aestivum )20

and chlorophyll

content and root length in duckweed (Landoltia punctata).21

In addition, nCuO have shown to

damage DNA in radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual

ryegrass (Lolium rigidum).22

The activity of antioxidant enzymes such as catalase (CAT) and ascorbate peroxidase

(APX) can be affected in Cu exposed plants. These enzymes protect plants from reactive oxygen

species (ROS) damage, which are overproduced under abiotic or biotic stresses.23

Hou et al. 24

reported that Cu2+

, up to 10 mg/L, increased CAT activity in duckweed in a concentration-

dependent manner; however, a reduction in CAT activity was observed when Cu2+

was higher

than 10 mg/L. A similar trend was observed in APX activity on duckweed treated with copper

sulfate.25

nCuO were also found to increase CAT activity in cucumber (Cucumis sativus) and

wheat.26-27

Beside the effects of nCuO on seedlings growth and CAT and APX activity, no reports

were found about the effects of Cu NPs/compounds on nutrient uptake by crop plants. In this

study, the effects of seven Cu NPs/compounds were evaluated in lettuce (Lactuca sativa) and

alfalfa (Medicago sativa). Root and shoot elongation, macro/micro nutrients uptake, and enzyme

activities were determined in hydroponically grown plants by using different spectroscopic and

biochemical techniques.

4.2 MATERIALS AND METHODS

4.2.1 Preparation of suspensions/solutions

All Cu NPs/compounds were obtained from the University of California Center for

Environmental Implications of Nanotechnology (UC CEIN). The particle size of nCu, nCuO, Cu

bulk and CuO bulk were 40 nm, <50 nm, <60 µm, and < 5 µm, respectively. Cu NPs/compounds

46

suspensions/solutions were prepared at 0, 5, 10, 20 mg L−1

in modified Hoagland’s nutrient

solution and homogenized by sonication in a water bath (Crest Ultrasonics, Trenton, NJ) at 25 °C

for 30 min. There were four replicates per treatment.

4.2.2 Seed germination and plant growth

Seeds of alfalfa (Medicago sativa L.) Mesa variety and black seeded Simpson lettuce

(Lactuca sativa L.) were stirred in 4% NaClO solution for 30 min and rinsed with distilled (DI)

water three times and kept in DI water for 24 h. Subsequently, seeds were rolled in autoclaved

wet germination paper towels. Ten drops of antimycotic/antibiotic solution (Sigma 5955) were

added to the seeds before the paper was rolled. The rolls were put into Mason jars containing

approximately 10 mL of DI, incubated in the dark for four days and exposed to light for one day.

After that, the young plants were transferred into magenta boxes containing 300 ml of modified

Hoagland’s nutrient solution.28

All the boxes and lids were covered with aluminum foil to

prevent algae growth. Aquarium pumps were used to aerate boxes, which were put in an

Environmental Growth Chamber (TC2 Microcontroller, Chagrin falls, OH) with light intensity of

300 µmol m-2

s-1

, 25/20 ºC day/night temperature, and 65% relative humidity.

After 10 days of growth in the nutrient solution, seedlings were transferred to magenta

boxes containing the Cu NPs/compounds suspended in nutrient solution and were cultivated for

15 days. Subsequently, plants were removed from the growth medium, washed with tap water

and rinsed with DI water. The length of the primary root and shoot for each seedling was

determined and the samples were saved for further analyses. There were 20 young plants per

replicate.

47

4.2.3 Sample preparation for ICP-OES

At the end of exposure time, plants were removed from the growth medium, washed with

tap water and rinsed with DI water, as previously described in the literature.19,20, 22

Subsequently,

the samples were put in paper envelops, oven dried at 70 ℃ for 72 h, weighed, and digested with

plasma pure HNO3 (65%) and H2O2 (30%) (1:4). Standard Reference Material NIST 1547

(Gaithersburg, MD) and samples spiked with 5, 10, 20 mg/L of nCu were processed and read as

samples for quality control (QC) and quality assurance (QA). Digestion was performed in a

CEM microwave accelerated reaction system (MarsX, Mathews, NC). The digests were adjusted

to 15 mL and analyzed for Cu, micro and macroelement concentrations using inductively

coupled plasma-optical emission spectroscopy (ICP-OES, PerkinElmer Optima 4300 DV,

Shelton, CT)

4.2.4 Enzyme assays

4.2.4.1 Determination of catalase specific activity

The CAT enzyme activity was measured according to Gallego et al.29

Plant samples were

grinded in 0.1M KH2PO4 buffer (pH 7.0) and centrifuged at 4500 rpm for 5 min (Eppendorf AG

bench centrifuge 5417 R, Hamburg, Germany). The supernatant was separated, added with 73

mM H2O2, and read at 240 nm through UV/Vis (Perkin Elmer Lambda 14 Spectrometer).

4.2.4.2 Determination of ascorbate peroxidase activity

The APX enzyme activity was determined following the method of Nakano and Asada.30

Tissue samples were homogenized in phosphate buffer (pH 7.4) with a ratio of 10% w/v. The

homogenate was centrifuged at 4 ºC for 25 min at 14000 rpm. Then, 100 µL of the supernatant

was added with 886 µL of KH2PO4 buffer (0.1 M, pH 7.4), 4 µL of 25 mM ascorbate, and 10 µL

of 17 mM H2O2. Absorbance was recorded at 290 nm using UV/Vis Spectrometer.

48


The data (means from four replicate/treatments) was analyzed using One-way ANOVA

(SPSS 19.0 package, Chicago, IL). Statistical differences between treatments’ means were

determined by using the Tukey’s Honestly Significant Difference test.


4.3.1 Effects of Cu NPs/compounds on root /shoot elongation

The size of roots and shoots of alfalfa and lettuce plants treated for 15 days with Cu

NPs/compounds is shown in Figure 4.1. After 15 days of treatment, the roots of plants treated

with Cu NPs/compounds, showed brown color compared to controls. Also, leaves of control

plants were greener than Cu treated plants. As seen in Figure 4.1, at all concentrations, all Cu

NPs/compounds significantly reduced root length in both plant species. The shortest root in

lettuce (15.88 ± 2.4 cm) and alfalfa (16.2 ± 0.2 cm) occurred in plants treated with 20 mg/L

nCuO and nCu, respectively. The reduction of root length was 49.5% ± 7.8% in lettuce and

47.6%±1% in alfalfa. Similar results have been reported for Mung bean, zucchini, and wheat

(16-18); however, in these studies, plants were exposed to 1000 mg/L,16

100-500 mg/L17

and 200

mg/L,18

respectively.

Lee et al.18

reported that nCu at 200 mg/L affected the roots of wheat, while only the 800

mg/L treatment reduced shoot length. Another report indicates that nCuO at 10 mg/L reduced

root and shoot length in radish seedlings by 46% and 4%, respectively.22

However, at 1000 mg

nCuO/L, the root and shoot length of radish decreased by 97% and 79%, respectively. In our

study, none of the treatments significantly reduced alfalfa shoot elongation; however, Cu bulk,

KC 2005, KC 3000, and CuCl2 at 20 mg/L significantly reduced lettuce shoot length (Appendix

Figure S1). This suggests a species specific response that could be associated to the Cu form.

49

Figure 4.1: Root elongation of lettuce (A) and alfalfa (B) plants exposed for 15 days in

hydroponics to 0, 5, 10, 20 mg/L of Cu NPs/compounds. Plants were cultivated for

10 days in Hoagland nutrient solution before exposure to Cu treatments. Data are

averages of four replicates ± SE. * stands for statistic differences at p ≤ 0.05.

4.3.2 Uptake of Cu by alfalfa and lettuce

Figure 4.2 shows Cu concentrations in roots and shoots of alfalfa and lettuce

hydroponically grown for 15 days with Cu NPs/compounds at 0, 5, 10, 20 mg/L. As shown in

this figure, Cu increased in roots as the treatment concentration in the medium increased. The

50

highest Cu concentration (20,000 mg/kg and 12,000 mg/kg for lettuce and alfalfa root,

respectively) occurred in plants treated with 20 mg/L CuCl2, which was very likely due to the

bioavailability of the Cu ions. The second highest Cu concentration for both plant species was in

nCu treated plants. Cu accumulation from nCuO at 10 and 20 mg/L was ~ 11000 mg/kg in the

lettuce root, while accumulation from Cu bulk was ~7500 mg/Kg. Wang et al.19

reported 1.8

times more Cu in maize roots treated with nCuO compared to bulk CuO.

The accumulation of Cu in alfalfa shoot followed a similar trend than in root (Figure

4.2D). Except for Kocide 2005 and bulk CuO, all compounds at all concentrations significant

increased Cu accumulation alfalfa shoots, compared to control. In the case of lettuce shoot, only

nCu at 10 and 20 mg/L significantly increased Cu accumulation, respect to control (Figure 4.2B).

More studies are needed in order to explain the higher Cu translocation from root to shoot in Cu

NP treated lettuce plants. Differences in Cu translocation from diverse Cu compounds have been

previously reported. The shoots of wheat exposed to nCuO accumulated more Cu (375 mg/kg)

than plants treated with CuO bulk (254 mg/kg).27

Perhaps the smaller size of NPs allowed higher

uptake and translocated within the plant. In our experiment, under all treatments, alfalfa

translocated about 3 to 5% Cu from root to shoot, while only 0.5 to 0.6% was translocated in

lettuce. This suggests the uptake and translocation of Cu is associated to the copper compound

and plant species.

51

Figure 4.2: Copper concentration in lettuce root (A), lettuce shoot (B), alfalfa root (C), and

alfalfa shoot (D) exposed for 15 days in hydroponics to 0, 5, 10, 20 mg/L of Cu

NPs/compounds. Data are averages of four replicates ± SE. Different letters stand

for statistical differences at p ≤0.05.

4.3.3 Effects of Cu NPs/compounds on nutrient elements uptake

Under the conditions of this research, Cu NPs/compound did not alter the uptake of Ca,

Mg, Mo, Mn, Zn, and Na in lettuce and alfalfa exposed for 15 days to the Cu treatments.

However, the uptake of P (Figure 4.3), S (Figure 4.4), and Fe (Figure 4.5) was significantly

affected.

4.3.3.1 Phosphorus uptake

At all concentrations, all Cu compounds reduced P uptake in roots of both plant species

(Figure 4.3A and 4.3C). Moreover, P was not detected in roots of both plant species treated with

52

at 10 and 20 mg/L. Similar results were reported by Ali et al. who found that P content decreased

in root of maize exposed for 15 days to Cu at 15.7, 78.5, and 157 μM (31). This could be due to

the formation of Cu phosphate, which cannot be taken up by plant roots32

or to damage on root

cell plasmalemma, resulting in loss of ions.31

Phosphorus was also reduced in shoots of Cu

treated lettuce plants (Figure 4.3B) but it increased in alfalfa shoot (Figure 4.3D). Phosphorus is

key element in antioxidant enzymes, which remove ROS excess induced by copper.33

In this

study, Cu concentration in alfalfa shoot was much higher than in lettuce shoot (Figure 4.2),

which suggests higher enzyme activity. This may explain why P concentration was higher in

alfalfa shoots.

Figure 4.3: Phosphorus concentration in lettuce root (A), lettuce shoot (B), alfalfa root (C), and

alfalfa shoot (D) exposed for 15 days in hydroponics to 0, 5, 10, 20 mg/L of Cu


for statistical differences at p≤ 0.05.

53

4.3.3.2 Sulfur uptake

As shown in Figure 3.4, all Cu treatments increased S concentration in lettuce root

(Figure 4.4A), alfalfa root (Figure 4.4B), and alfalfa shoot (Figure 4.4C), but did not affect S

accumulation in lettuce shoot (results not shown). There were no obvious differences in S

accumulation between NPs and bulk material treatments in lettuce root. However, highest S

accumulation was found in alfalfa root treated with the highest concentration of nCu, nCuO, and

CuCl2. In alfalfa shoot, Kocide 2005 and CuCl2 at 10 and 20 mg/L exhibited the highest S

accumulation, except for nCu at 10 mg/L. Exposure of plants to toxic metals may affect the

uptake and assimilation of S due to the formation of sulfur-rich phytochelatins and

metallothioneins.34-35

According to Shahbaz et al.36

plants increase S uptake to mitigate Cu

toxicity; but Cu could also induce over expression of sulfate transporters by affecting the signal

transduction pathway, resulting in higher S accumulation. More studies are needed in order to

clarify what mechanisms induced the increase of S accumulation in lettuce and alfalfa treated

with Cu NPs/compounds.

4.3.3.3 Iron uptake

As shown in Figure 4.5, except for CuCl2, all the Cu treatments significantly reduced Fe

concentration in shoots and roots of alfalfa and lettuce. Kocide 2005 and Kocide 3000 at 20

mg/L caused the lowest Fe accumulation in the shoot of lettuce (512 ±10 mg/kg) and alfalfa

(203 ±13 mg/kg), respectively. CuCl2 did not change Fe concentration in both plants, except at

20 mg/L in alfalfa shoot. Previous studies have shown that copper sulfate can cause Fe

deficiency in rice (Oryzasativa L.)37

and beans (Phaseolus vulgaris L.).38

Patsikka et al.38

also

reported that the Cu treatment reduced leaf chlorophyll concentration and the thylakoid

membrane network in beans. However, the adverse effects were reverted by adding excess Fe.

54

Ouzounidou et al. 39

reported that the increase in Fe concentration in spinach leaves reduced the

toxic effects of Cu on photosynthesis.

Figure 4.4: Sulfur concentration in lettuce root (A), alfalfa root (B), and alfalfa shoot (C)

exposed for 15 days in hydroponics to 5, 10, 20 mg/L of Cu NPs/compounds. Data

are averages of four replicates ± SE. Different letters stand for statistical differences

at p ≤ 0.05.

* a * * a a a

c c b

b b bc

c b

b b c

c

0

1000

2000

3000

Co

nce

ntr

atio

n (

mg/

kg

DW

)

0 5 mg/L

a * a * * * a ab ab

b b

b

bc

b b

c

0

500

1000

1500

2000

2500

Co

nce

ntr

atio

n (

mg/

kg D

W)

0 5 mg/L 10 mg/L 20 mg/L

a * a * a a a

b

b b a b

c

c c ab

c ab

b c b

c

0

1000

2000

3000

4000

5000

6000

7000

Cu NPS Cu Bulk CuONPs

CuOBulk

KC2005 KC3000 CuCl2

Co

nce

ntr

atio

n (

mg/

kg D

W)

Treatment

0 5 mg/L 10 mg/L 20 mg/L

55

In addition, Schmidt et al. stated that Cu can displace Fe from chelating molecules in the growth

medium, inhibiting the induction of Fe stress response.40

This suggests that Cu and Fe could

share the same uptake pathway.41

In recent study, the Cu form was not a key factor in the

reduction of Fe accumulation in the shoot of both plants.

Figure 4.5: Iron concentration in lettuce root (A), lettuce shoot (B), alfalfa root (C), and alfalfa

shoot (D) exposed for 15 days in hydroponics with 0, 5, 10, 20 mg/L of Cu


for statistical differences at p ≤ 0.05.

4.3.4 Enzyme activities

After entering the root cell, CuNPs/compounds and the released Cu ions may cause the

formation of reactive oxygen species (ROS), changing the activity of stress enzymes. Figure 3.6

shows the activity of CAT in alfalfa tissues (there was no effect on CAT in lettuce tissues) and

APX in lettuce and alfalfa root (there was no effect on shoot APX activity in both plant species).

As seen in this figure, all Cu treatments, at all concentrations, significantly reduced CAT activity

56

in alfalfa shoot, compared to control (Figure 4.6 A). The highest reduction was found with nCuO

at 10 mg/L. A similar trend was observed in alfalfa root where the most striking results were

produced by Kocide 2005 and bulk CuO at 5 mg/L (Figure 4.6B). At 10 mg/L bulk Cu, nCuO,

and Kocide 3000 did not reduce CAT activity in alfalfa root, compared to control; while at 20

mg/L, all the treatments, except nCuO, significantly reduced CAT activity, compared to control.

Kim et al.26

reported that nCuO at 500 and 1000 mg/L increase CAT activity in Cucumis sativus.

This suggests that nCuO could have low effects on CAT activity. A concentration-dependent

reduction on alfalfa root CAT activity was observed with CaCl2, which at 20 mg/L reduced CAT

activity by 87% (Figure 4.6B). Hou et al.24

reported that Cu2+

at 5 mg/L increased CAT in

duckweed, but at 10 mg/L inhibited CAT activity. This suggests the effects of Cu on CAT

activity are associated to the plant species and the form of Cu.

The APX activity in the roots of lettuce (Figure 4.6C) and alfalfa (Figure 4.6D) showed

contrasting results, compared to CAT activity in alfalfa root (Figure 4.6B). At all concentrations,

all Cu treatments significantly increased APX activity in lettuce root, compared to control,

except for bulk CuO (Figure 4.6C). In lettuce root (Figure 4.6C), APX activity showed a sharp

decrease with CuCl2 at 20 mg/L, but still the difference was significant (p ≤ 0.05), compared to

control. In alfalfa root (Figure 4.6D), APX activity significantly increased at all Cu treatments,

except with both Cu(OH)2. Notoriously, in alfalfa root, four compounds at 20 mg/L presented a

decrease in APX activity; however, the activity was still significantly higher, compared to

control. Similar results were reported in Brassica juncea (42) exposed to Cu; but in duckweed, it

was found that APX activity was inhibited by Cu at 0.25 µM.25

These results suggest that to

some extent, in lettuce and alfalfa roots, CAT and APX are down regulated and up regulated,

respectively, by the same external stimulus, in this case the Cu treatment. The data also suggests

57

that in roots, APX is more effective to remove ROS. Additionally, in both plants species, the

highest APX activity was recorded with nCu, which indicates this form of Cu is more toxic to

alfalfa and lettuce.

Figure 4.6: Antioxidant enzyme activity in alfalfa and lettuce treated for 15 days in hydroponics

with 0, 5, 10, 20 mg/L Cu of NPs/compounds. CAT activity in alfalfa shoot (A) and

alfalfa root (B); APX activity in lettuce root (C) and alfalfa root (D). Values are

averages of four replicates ± SE. CAT activity in lettuce tissues and APX activity in

shoots of both plant species were not affected.

4.4 CONCLUSION

In summary, this study has shown that all Cu treatments, even at low concentration (5

mg/L), reduce root length in hydroponically grown lettuce and alfalfa. Both plant species can

take up more Cu when they are treated with nCu than bulk materials, except CuCl2. Cu

NPs/compounds reduced P and Fe accumulation plant tissues, while S concentration increased in

alfalfa shoot and in the roots of both plant species. The activity of stress enzyme CAT was not

58

affected in lettuce and sharply reduced in alfalfa shoot. The APX activity in lettuce and alfalfa

roots increased at all Cu treatments, except for bulk Cu in lettuce and Cu(OH)2 in alfalfa root.

Results from APX activity in roots suggest higher toxicity from nCu.

59

Chapter 5: Conclusion

To the author’s knowledge, this is the first study evaluating the potential effects of foliar

applied NPs on fruit quality and nutrition value of cucumber. In part I, based on ICP-OES data

and TEM images, we proved that cucumber plants can take up Ce from nCeO2 applied to the

leaves as powder or suspension. In addition, these NPs also caused increased Ce concentration in

other plant tissues, such as stems and roots. The increase in CAT activity in all plants treated

either with powder or suspension at low concentrations (40 mg/L or 0.98 g/m3)

suggests that the

nCeO2 in contact with leaves alter ROS balance in cucumber plants. On the other hand, the

decrease on CAT activity in stems and roots of plants treated with 320 mg nCeO2/L suggests that

at this concentration nCeO2 caused toxicity. Although the speciation of Ce in tissues was not

determined, previous studies have shown that most of the Ce in tissue of plants treated with

nCeO2 remains in nanoparticulate form. This suggests that atmospheric nCeO2 could be stored in

the fruit of cucumber plants.

In part II, we found that foliar application of nCeO2 allowed Ce accumulation in old

cucumber leaves, new leaves, and fruit. However, nCuO treatment did not increase Cu in tissues.

Gas exchange in old leaves, plant growth, yield, and fruit quality were not affected by treatment,

except fruit firmness. Although minerals (Ca, Mg, Fe, Mn and B) concentration in new leaves

significantly reduced under the treatment, there was no effect on nutrient elements in fruit,

except Zn and Mo. Fruit Zn decreased by about 25% at 200 mg/L nCeO2 and bulk CeO2. Fruit

Mo decreased by 51% and 44% under 200 mg/L nCuO and bulk CuO treatments, respectively.

In part III, we found that all Cu treatments, even at low concentration (5 mg/L), reduce

root length in hydroponically grown lettuce and alfalfa. Both plant species can take up more Cu

when they are treated with nCu than bulk Cu, except CuCl2. Results showed that all Cu

60

NPs/compounds reduced the root length by 49% in both plant species. Under all treatments, Cu,

P, and S increased (>100%, >50%, and >20%, respectively) in alfalfa shoots; while P and Fe

decreased (>50% and >50%, respectively) in lettuce shoot. In addition, CAT activity was

reduced in alfalfa (root and shoot) and APX activity was increased in roots of both plant species.

Results from APX activity in roots suggest higher toxicity from nCu.

Results of this research have shown that foliar applied nCeO2 allowed Ce accumulation

in cucumber tissues, including fruit. In addition, these NPs have shown to affect fruit quality and

nutrient content with unknown impacts for human nutrition. Results also showed that nCuO

affect the growth of alfalfa and lettuce and altered the content of minerals in plant tissues. These

results expand our knowledge on the interaction of ENPs with crop plants.

Acknowledgments

This material is based upon work supported by the National Science Foundation and the

Environmental Protection Agency under Cooperative Agreement Number DBI-0830117. Any

opinions, findings, and conclusions or recommendations expressed in this material are those of

the author(s) and do not necessarily reflect the views of the National Science Foundation or the

Environmental Protection Agency. This work has not been subjected to EPA review and no

official endorsement should be inferred. The authors also acknowledge the USDA grant number

2011-38422-30835 and the NSF Grant # CHE-0840525. J. L. Gardea-Torresdey acknowledges

the Dudley family for the Endowed Research Professorship and the Academy of Applied

Science/US Army Research Office, Research and Engineering Apprenticeship program (REAP)

at UTEP, grant # W11NF-10-2-0076, sub-grant 13-7.

61

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Appendix

Figure S1. Shoot elongation of 10 day-old lettuce treated for 15 days in hydroponics with 0, 5,

10, 20 mg/L Cu NPs/compounds. Data are averages of four replicates ± SE.

Different letters stand for statistic differences at p ≤ 0.05.

b c b b

b

c

b

ab

b

ab a

ab a

a

a

a

0

2

4

6

8

10

12

Cu NPs Cu Bulk CuO NPs CuO Bulk KC2005 KC3000 CuCl2

Sho

ot

Len

gth

(cm

)

Treatments

control

5 mg/L

10 mg/L

20 mg/L

80

81

Vita

Jie Hong received his Bachelor of Engineering in Food Science and Technology in

Henan University of Technology in China, 2008. He earned his Master degree from Southern

Illinois University Edwardsville in Environmental Science in 2010. Then, he joined the doctoral

program in Environmental Science and Engineering at The University of Texas at El Paso under

the supervision of Dr. Jorge Gardea Torresdey.

During these 4 years, he published 8 peer-review journal papers (Environmental Science

& Technology, Journal of Hazardous Materials, Journal of Agricultural and Food Chemistry,

Journal of Nano Research, Analytica Chimica Acta,) and 2 book chapters (IWA Publish and

ACS Symposium Series) as first author and co-author. Also, he has two other manuscripts which

are almost ready to submit. In addition, he has presented his research at national conference

meetings including the American Chemical Society’s National Meeting, Society of

Environmental Toxicology and Chemistry North America Annual Meeting. Annual Conference

of Sustainable Nanotechnology Organization.

Based on his previous project, Mr. Jie Hong has been awarded the Honorable Mention of

the EPA P3 (People, Prosperity and the Planet) award, which is a Student Design Competition

for Sustainability held in 2012 at Washington, D.C by U.S. Environmental Protection Agency.

From Sep, 2012 to Dec, 2012, he was selected by Chemistry Department of UTEP as an

exchange student to do research in Nagaoka University of Technology in Japan. Also, he

received the scholarship from Japan Student Services Organization (JASSO) Student Exchange

Support Program of Japanese government.

While pursuing his PhD, Mr. Jie Hong worked as a research assistant and teaching

assistant in the chemistry department. Also, he was the vice president (2010-2011) and president

82

(2011-2012) of UTEP Chinese Students and Scholars Association (CSSA). Recently, he has been

offered a full time position of research associate in University of South Carolina.

Publications

1. Hong, J., Bañuelos, G.S., Fowler, T., Lin, Z-Q. 2011. Use of selenium-laden plant

materials from phytoremediation to produce selenium-biofortified edible mushrooms.

ICOBTE 2011, 11th International Conference on the Biogeochemistry of Trace Elements.

Conference Proceedings Parte I, Congress Center, Florence, Italy, July 3–7, pp. 349–350.

2. Peralta-Videa, J.R., Zhao, L., Lopez-Moreno, M.L., de la Rosa, G., Hong, J., Gardea-

Torresdey, J.L. 2011. Nanomaterials and the environment: A review for the biennium

2008-2010. Journal of Hazardous Materials 186. 1–15.

3. Zhao, L., Peralta-Videa, J.R., Hernandez-Viezcas, J.A., Hong, J., Gardea-Torresdey, J.L.

2012. Transport and retention behavior of ZnO nanoparticles in two natural soils: Effect

of surface coating and soil composition. Journal of Nano Research 17, 229-242.

4. Majumdar, S., Rico, C.M., Hong, J., Castillo-Michel, H., Peralta-Videa, J.R., Gardea-

Torresdey, J.L. 2012. Applications of Synchrotron µ-XRF to Study the Distribution of

Biologically Important Elements and Nanoparticles in Environmental and Biological

Samples. Analytica Chimica Acta 775, 1-16.This article made the cover page of this

issue.

5. Hong, J., Peralta-Videa, J.R., Gardea-Torresdey, J.L. 2012. Plant-based nanoparticle

manufacturing. In: Virkutyte, J., Thilo, H., Jegatheesan, V., Alvarez, P., Lens, P.

Nanotechnology for water wastewater treatment. IWA Publishing, Chapter 18, 409-435.

6. Hong, J., Peralta-Videa, J.R., Gardea-Torresdey, J.L. 2012. Nanomaterials in agricultural

production: benefits and possible threats. In: Green Nanotechnology and the Environment

Edited by Sharma V., and Sham A.N. ACS Symposium Series Vol. 1124, 73-90

(DOI:10.1021/bk-2013-1124).

7. Rico, C. M.; Hong, J.; Morales, M.I.; Zhao, L.; Barrios, A.C.; Zhang, J.; Peralta-Videa,

J.R.; Gardea-Torresdey, J.L. Effect of cerium oxide nanoparticles on rice: A study

involving the antioxidant defense system and in vivo fluorescence imaging. Environ. Sci.

Technol. 2013. 47, (11), 5635-5642.

8. Rico, C.; Morales, M.; McCreary, R.; Castillo-Michel, H.; Barrios, A.; Hong, J.; Tafoya,

A.; Wee-Yee, L.; Varela-Ramirez, A.; Peralta-Videa, J.; Gardea-Torresdey, J.L. Cerium

Oxide nanoparticles modify the antioxidative stress enzyme activities and macromolecule

composition in rice seedlings. Environ. Sci. Technol., 2013, 47 (24), 14110–14118.

9. Zhao, L.; Youping, S.; Hernandez-Viezcas, J.; Servin, A.; Hong, J.; Genhua, N.; Peralta-

Videa, J.R.; Duarte-Gardea, M.; Gardea-Torresdey, J.L. Influence of CeO2 and ZnO

Nanoparticles on Cucumber Physiological and Bioaccumulation of Ce and Zn; A life

Cycle Study. J. Agric. Food Chem., 2013, 61 (49), 11945–11951.

10. Rico, C.; Morales, M.; Barrios, A.; McCreary, R.; Hong, J.; Lee, W.; Nunez, J.; Peralta-

Videa, J.; Gardea-Torresdey, J. 2013. Effect of Cerium Oxide Nanoparticles on the

Quality of Rice (Oryza sativa L.) Grains. J. Agric. Food Chem., 2013, 61 (47), 11278–

11285.

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11. Hong, J., Jose R. Peralta-Videa, Cyren Rico, Shivendra Sahi, Marian N. Zappala, Lijuan

Zhao, Jorge L. Gardea-Torresdey. Evidence of Translocation and Physiological Impacts

of Foliar Applied CeO2 Nanoparticles on Cucumber (Cucumis sativus) Plants. Environ.

Sci. Technol., 2014, 48 (8), 4376–4385.

Oral Presentations

1. Hong, J., Gary Banuelos, and Z.-Q. Lin. 2009. Integrating Phytoremediation and

Biofortification: Use of Se-laden Plant Materials for Production of Se-enriched Edible

Mushroom. Proceedings of 6th

International Phytotechnologies Conference. December 1-

4, 2009, St. Louis, Missouri.

2. Hong, J., Z.-Q. Lin. 2010. Selenium Phytoremediation Magement: Development of

Selenium-Biofortified Mushrooms from Plant Waste. 102th Annual Meeting of Illinois

State Academy of Science.April 9-10, 2010, Decatur, Illinois.

3. Hong, J., Rico, C.M., Zhao, L., Peralta-Videa, J.R., and Gardea-Torresdey, J.L. 2013.

Toxicity effects of seven Cu compounds/nanoparticles in lettuce (Lactuca sativa) and

alfalfa (Medicago sativa). 2nd Annual Conference of Sustainable Nanotechnology

Organization, November 3-5, 2013, Santa Barbara, California.

4. Hong, J., Sahi, S., Rico, C.M., Zhao, L., Peralta-Videa, J.R., and Gardea-Torresdey, J.L.

2013. Foliar uptake of CeO2 nanoparticles by cucumber plants (Cucumis Sativus) and the

physiological impacts. SETAC North America 34 th Annual Meeting, November 17-21,

2013, Nashville, Tennessee.

Poster Presentation

1. Hong, J., Shivendra Sahi, Cyren Rico, Lijaun Zhao, Jose R. Peralta-Videa, Jorge L.

Gardea-Torresdey. Translocation and Physiological Impacts of Foliar Application of

CeO2 Nanoparticles on Cucumber Plants (Cucumis Sativus). American Chemical Society

246th National Meeting at Indianapolis, Indiana, from September 8-12, 2013.

Awards and Scholarships

1. Research Grants for Graduate Students (RGGS) award. Southern Illinois University –

Edwardsville, spring 2009.

2. Graduate student travel grant award, 6th International Phytotechnologies Conference.

December 1-4, 2009, St. Louis, Missouri.

3. Graduate student travel grant award, 102th Annual Meeting of Illinois State Academy

of Science. April 9-10, 2010, Decatur, Illinois.

4. The Student Presentation Award at the 102th Annual Meeting of Illinois State Academy

of Science in 2010. Decatur, Illinois.

5. Honorary Citizen of the city of El Paso in Texas. 2011. 6. EPA P3 (People, Prosperity and the Planet) award: Honorable Mention. (A Student

Design Competition for Sustainability)Washington, D.C., April, 2012.

7. Scholarship from Japan Student Services Organization (JASSO) Student Exchange

Support Program.

8. Travel Award from the ACS Division of Chemical Toxicology for American Chemical

Society 246th National Meeting.

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Synergistic Activities

Vice president of UTEP Chinese Students and Scholars Association (CSSA) 2010-2011

Organized the CSSA members to participate the UTEP Food Fair 2010

President of UTEP Chinese Students and Scholars Association (CSSA) 2011-2012

Organized the CSSA members to participate the UTEP Food Fair 2011

Held the Mid-Autumn Day & Welcoming New Students Party

Held the event to celebrate Chinese New Year.

Exchange Student in Nagaoka University of Technology, Japan 09. 2012- 12.2012

Permanent address: 302, Unit 1, West 3 Building, Residential Quarter,

Shanxi Agricultural University,

Taigu, Shanxi, China, 030801

This thesis/dissertation was typed by Jie Hong.

Documents

Uptake Of Copper And Cerium By Alfalfa, Lettuce And