Science of the Total Environment 408 (2010) 3053–3061

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Science of the Total Environment

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v

Review

Interactions between engineered nanoparticles (ENPs) and plants: Phytotoxicity,

uptake and accumulation
Xingmao Ma a,⁎, Jane Geiser-Lee b, Yang Deng c, Andrei Kolmakov d
a
Department of Civil and Environmental Engineering, Southern Illinois University Carbondale, Carbondale, IL, USA
b
Department of Plant Biology, Southern Illinois University Carbondale, Carbondale, IL, USA
c
Department of Earth and Environmental Studies, Montclair State University, Montclair, New Jersey, 07043, USA
d
Department of Physics, Southern Illinois University Carbondale, Carbondale, IL, USA

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

Article history: The rapid development and potential release of engineered nanoparticles (ENPs) have raised considerable
Received 27 January 2010 concerns due to the unique properties of nanomaterials. An important aspect of the risk assessment of ENPs
Received in revised form 8 March 2010 is to understand the interactions of ENPs with plants, an essential base component of all ecosystems. The
Accepted 19 March 2010
impact of ENPs on plant varies, depending on the composition, concentration, size and other important
physical chemical properties of ENPs and plant species. Both enhancive and inhibitive effects of ENPs on
Keywords:
Engineered nanoparticles (ENPs)
plant growth at different developmental stages have been documented. ENPs could be potentially taken up
Phytotoxicity by plant roots and transported to shoots through vascular systems depending upon the composition, shape,
Uptake and transport size of ENPs and plant anatomy. Despite the insights gained through many previous studies, many questions
Plasmodesmata remain concerning the fate and behavior of ENPs in plant systems such as the role of surface area or surface
activity of ENPs on phytotoxicity, the potential route of entrance to plant vascular tissues and the role of
plant cell walls in internalization of ENPs. This article reviewed the current knowledge on the phytotoxicity
and interactions of ENPs with plants at seedling and cellular levels and discussed the information gap and
some immediate research needs to further our knowledge on this topic.
© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3053
2. Engineered nanoparticles (ENPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3054
3. Phytotoxicity of engineered nanoparticles to plant seedlings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3054
4. Phytotoxicity of engineered nanoparticles to algae and plant cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3056
5. Uptake, translocation and accumulation (UTA) of engineered nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3057
6. Interactions of engineered nanoparticles with plant cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3058
7. Future research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3059
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3060

1. Introduction at least two dimensions between 1 nm and 100 nm (ASCM, 2006;

Nanotechnology is advancing rapidly and could soon become a
trillion-dollar industry (Nel et al., 2006). As a result, release of
substantial amount of engineered nanoparticles (ENPs) into the
environment is inevitable. ENPs are widely accepted as materials with
⁎ Corresponding author. Department of Civil and Environmental Engineering,
Southern Illinois University Carbondale, 1230 Lincoln Drive, Carbondale, IL 62901,
USA. Tel.: + 1 618 453 7774; fax: + 1 618 453 3044.
E-mail address: ma@engr.siu.edu (X. Ma).
SCENIHR, 2007). ENPs of these sizes fall in a transitional zone between
individual molecules and the corresponding bulk materials and
therefore possess unique properties distinctively different from
their molecular and bulk counterparts (Taylor and Walton, 1993).
Examples of the unique properties of ENPs include very large specific
surface area, high surface energy, and quantum confinement. These
unusual properties may result in substantially different environmen-
tal fate and behaviors than their bulk counterparts. An emerging area
of research is now focused on short and medium term studies of the
environmental and ecological impact of released ENPs.

0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2010.03.031
3054 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061

Plants are an essential base component of all ecosystems and play have a stronger chemical activity than bare iron nanoparticles (Lee
a critical role in the fate and transport of ENPs in the environment and Sedlak, 2008; He and Zhao, 2005). nZVIs produced from different
through plant uptake and bioaccumulation (Monica and Cremonini, methods possess somewhat different properties and caution should
2009). Even though scientific investigation on plant uptake and be taken in comparison of experimental results. Nurmi et al. (2005)
accumulation of ENPs is still in its rudimentary stage, some new characterized nZVIs prepared with two widely adopted methods, one
publications have been added to the literature in the past few years by the reduction of goethite with heat and H2 and the other by
yielding new advances in the area of ENP toxicology and uptake by precipitation with borohydride, and found that nano irons have
plants. This review is intended to provide a critical assessment of the different compositions and sizes and consequently result in different
progress taking place on this topic and to shed light on future research reactivities with different environmental contaminants. These varia-
needs. tions complicate the determination and comparison of the toxicity,
fate and impact of nZVIs.
2. Engineered nanoparticles (ENPs)
3. Phytotoxicity of engineered nanoparticles to plant seedlings
Most commonly encountered ENPs in the environment fall into
one of the five following categories: carbonaceous nanoparticles, ENPs closely interact with their surrounding environment and
metal oxides, quantum dots, zero-valent metals and nanopolymers, plants are an essential base component of all ecosystems. As a result,
with new products gradually being added to the list. Due to limited ENPs will inevitably interact with plants and these interactions such
publications on the toxicological effects of dendrimers, this review as uptake and accumulation in plant biomass will greatly affect their
will focus on the toxicological effect and interactions of the rest four fate and transport in the environment, Fig. 1. ENPs could also adhere
categories with plants. A brief introduction of ENPs of each category to plant roots and exert physical or chemical toxicity on plants.
will be provided in this section and detailed information on ENPs Increasing numbers of publications have emerged recently
including their chemistry, environmental behavior and cytotoxicity is concerning the interactions of ENPs with plants (Battke et al., 2009;
referred to elsewhere (Christian et al., 2008; You and Reid, 2008). Lin and Xing, 2007; Lin et al., 2009). Most of these studies are focused
Carbonaceous nanoparticles are the most abundant ENPs and on the potential toxicity of ENPs to plants and both positive and
consist primarily of fullerenes and nanotubes. Fullerenes are enclosed negative or inconsequential effects have been reported.
cage-like structures comprised of twelve 5-member carbon and A recent work indicated that MWCNTs at the concentration range
unspecified 6-member rings in defect-free form. Even though an of 10–40 mg/L dramatically enhanced the seed germination and
icosahedrally symmetrical structure (nC60) is the most commonly growth of tomato plants (Khodakovskaya et al., 2009), Fig. 2. The
encountered fullerene, both smaller fullerene such as C28 and C36 and researchers hypothesized that the positive effect of MWCNTs arose
very large spherical fullerene conformations have been identified and from the capability of CNTs to penetrate seed coat and therefore
characterized (Ugarte, 1992; Wang et al., 2001). Carbon nanotubes
(CNTs) can be defined as carbon whiskers. A carbon nanotube is a
honeycomb lattice rolled on to itself, with diameter of the order of
nanometers and length of up to several micrometers. An excellent
review of carbonaceous nanomaterials and their applications in the
environmental field is now available in literature (Mauter and
Elimelech, 2008).
Metal oxide nanoparticles, represented by titanium dioxide (TiO2)
and zinc oxide (ZnO), are of great technological importance in the
field of heterogeneous catalysis for catalytic support of a wide variety
of metals (Biener et al., 2005). Metal oxide nanoparticles also find
extensive applications in sunscreen industry due to their ultraviolet
blocking ability and visible transparency of nanoparticulate forms
(Klaine et al., 2008). Quantum dots (QDs) are artificial “droplets” of
charge that can contain anything from a single electron to several
thousand electrons (Kouwenhoven and Marcus, 1998). QDs demon-
strated many similar quantum phenomena as in real atoms and nuclei
and therefore are frequently used as models of different atoms by
simply changing its size and shape (Kouwenhoven and Marcus, 1998).
QDs generally possess a reactive core which controls optical
properties and an inert shell to protect the core from oxidation
(Dabbousi et al., 1997).
Silver nanoparticles (AgNPs) are by far the most prevalent metallic
nanoparticles in consumer products due to their antimicrobial activity
(Klaine et al., 2008) while nanoscale zero-valent iron (nZVI) is the
most popular metallic nanoparticles in environmental remediation
applications (Zhang, 2003). Due to the high specific surface area
(∼24–35 m2/g) and more reactive surface sites, nZVI shows extremely
high chemical reductive reactivity in the absence of oxygen and high
oxidative reactivity in the presence of oxygen. The nZVIs-based
technology has been used to remove diverse contaminants through
reductive dechlorination or oxidation, such as chlorinated organics
(Wang and Zhang, 1997; Liu et al., 2005), heavy metals (Ponder et al., Fig. 1. An overview and general principle of uptake, transport and accumulation of
nanoparticulate matter by plants. Nanoparticulate matters in a natural environment are
2000; Kanel et al., 2006) and other inorganics (Choe et al., 2000). Of absorbed by primary roots (A2) or lateral roots (A1 and then B). These nanoparticles are
note, bimetallic nZVI, consisting of elemental iron and another more then transported from root (C) through stem (D and I) to leaf (E, F, G, H). Nanoparticles
stable metal element (e.g. Ni, Pd and Ag), have been demonstrated to could also be adsorbed on the surface of roots.
 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061
Fig. 2. Effects of MWCNTs on seed germination and plant development. A. Phenotype of
tomato seeds incubated three days with and without CNTs on MS medium and B.
phenotype of tomato seedlings exposed to CNTs at different concentrations.
Images are adapted and modified with permission from Khodakovskaya et al., 2009, ACS
Nano, copyright 2009 American Chemical Society.
promote water uptake. Water uptake in seed germination is critical
because mature seeds are relatively dry and need a substantial
amount of water to initiate cellular metabolism and growth. The
measured water moisture content of seeds and the detection of CNTs
inside seeds supported the hypothesis; however, the specific
penetration mechanisms through the coat and the enhancement of
water uptake by CNTs were not reported. TiO2 nanoparticles (2.5 g–
40 g/kg soil) were shown to improve the growth of spinach by
enhancing photosynthesis and nitrogen-fixation capability in leaves
and roots respectively (Yang et al., 2007). A latest work confirmed
these findings showing that TiO2 could promote the energy utilization
and conversion efficiency in D1/D2/cyt b559 complex (Su et al., 2009).
Similarly, a mixture of SiO2 and TiO2 nanoparticles at low concentra-
tions increased nitrate reductase activity in the rhizosphere of
soybean and consequently expedited soybean germination and
growth (Lu et al., 2002). SiO2 nanoparticles also enhanced the growth
of Changbai larch (Larix olgensis) and the enhancement effect
increased with concentration up to 500 mg/L (Lin et al., 2004).
A few studies also indicated inconsequential effects of ENPs on
plants. For instance, a study with willow tree cuttings indicated that
TiO2 nanoparticles have limited effects on their growth in terms of
water usage and transpiration (Seeger et al., 2009). In a sand column
study, aluminum nanoparticles did not demonstrate any toxic effects
on kidney bean (Phaseolus vulgaris) and rye grass (Lolium perrene) at
concentrations up to 17 mg/L (Doshi et al., 2008). Al2O3 nanoparticles
up to 4000 mg/L did not have any detectable effects on root
elongation and development of Arabidopsis even though slight
inhibition of seed germination was detected (Lee et al., 2010).
3055
However, most studies with ENPs indicated certain degree of
phytotoxicity, especially at high concentrations. SWCNTs significantly
affected root elongation of tomato, cabbage, carrot and lettuce but
promoted the growth of onion and cucumber in 24 to 48 h (Canas et
al., 2008). Tomato (Lycopersicon esculentum) showed the highest
degree of sensitivity to SWCNTs among the six species tested. The
researchers showed that functionalized CNTs demonstrated different
toxic behaviors but generally were less toxic than non-functionalized
CNTs. However, how CNTs were functionalized was not specified.
Nevertheless, this work highlighted the importance of surface
properties of CNTs in determining the phytotoxicity of CNTs. Metallic
oxide nanoparticles (e.g. ZnO) were shown to be inhibitive at different
developmental stages of plants such as seed germination and root
elongation (Lin and Xing, 2007; Yang and Watts, 2005). In terms of
metallic nanoparticles (MNPs), copper nanoparticles were shown to
be toxic to two crop species, mung bean (Phaseolus radiatus) and
wheat (Triticum aestivum), as demonstrated by the reduced seedling
growth rate (Lee et al., 2008). Mung bean is more sensitive than wheat
and the authors attributed this phenomenon to differences in root
anatomy and architecture; mung bean is a dicot with one large
primary root and several smaller lateral roots developing from this
primary root while wheat is a monocot with numerous small roots
without a primary root. Our recent lab work indicated that very low
concentrations of AgNPs (b1 ppm) could be toxic to seedlings of thale
cress (Arabidopsis thaliana), another dicot with root structures like
mung bean. AgNPs of 20 nm to 80 nm clearly stunted the growth and
their phytotoxicity is concentration and particle size dependent, Fig. 3.
The root tip (cap and columella) were observed to turn light brown
when primary roots were exposed to AgNPs. The brown tip was
attributed to the adsorption of AgNPs either itself or in conjunction
with cell wall materials or secondary metabolites produced by root
tips. Exact mechanisms are yet to be elucidated. Determination of
phytotoxicity of metallic nanoparticles and their oxides is complex
due to the potential dissolution of metallic ions from these
nanoparticles and the potential toxicity and uptake of metallic ions.
A recent study on the phytotoxicity of ZnO nanoparticles to rhygrass
(Lolium perenne) indicated that phytotoxicity cannot be explained by
the dissolution of ZnO nanoparticles from bulk materials alone (Lin
and Xing, 2008). Similarly, the phytotoxicity of ZnO nanoparticles to
Arabidopsis was much stronger than solutions containing same
concentration of soluble Zn (Lee et al., 2010). A separate study on
the phytotoxicity of five different nanoparticles (MWCNTs, Ag, Cu,
ZnO and Si) on an agricultural plant, zucchini (Cucurbita pepo),
supported the conclusion that dissolution from bulk materials alone
cannot account for the observed phytotoxicity of ENPs (Stampoulis et
al., 2009). Even though bulk materials of Ag and Cu resulted in
Fig. 3. Phenotype of Arabidopsis thaliana exposed to A. Hoagland solutions, B. 340 μg/L
20 nm AgNPs and C. 340 μg/L 40 nm AgNPs. Phenotype was imaged under a light
microscope (Leica S6D).
3056 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061
reduced biomass compared to controls, plants exposed to ENPs
demonstrated a higher degree of reduction, indicating that at least
part of the toxicity is from elemental MNPs. Phytotoxic phenotypes in
seedlings caused by ENPs include stunt growth, reduced biomass and
root cap deformity. The morphology changes of rye grass roots caused
by high concentrations of ZnO nanoparticles were demonstrated by
Lin and Xing (2008) and are shown in Fig. 4. It can be seen that root
tips shrank and epidermal and cortical cells were highly collapsed in
the presence of high concentrations of ZnO.
It needs to be cautioned that the inhibition of plant growth may
not derive directly from chemical phytotoxicity of nanoparticles.
Instead, toxicity may result from the physical interactions between
nanoparticles and plant cell transport pathways, i.e. by inhibiting
apoplastic trafficking by blockage of the intercellular spaces in the cell
wall or cell wall pores, or the symplastic connections between cells
through blockage of the nano-sized plasmodesmata. A latest work
showed that the inhibition of leaf growth and transpiration of maize
seedlings (Zea mays L.) by bentonite and TiO2 nanoparticles is
primarily due to the reduction of hydraulic conductivities (Asli and
Neumann, 2009). A further examination showed that diameter of
Fig. 4. Longitudinal sections of ryegrass primary root tips observed under light
microscope, demonstrating the deformity of morphology of root tips by ZnO
nanoparticles or ions. A. Controls, B. primary root tips exposed to 1000 mg/L ZnO
nanoparticles and C. primary root tips exposed to 1000 mg/L Zn2+. rc: rootcap; ep:
epidermis, ct: cortex and vs: vascular cylinder.
Images are adapted with permission from Lin and Xing, 2008, Environ. Sci. Technol,
copyright 2008 American Chemical Society.
maize root cell wall pores was reduced from 6.6 nm to 3.0 nm by
pretreatment of nanoparticles. Another factor which needs to be
considered in phytotoxicity study is the solvent effect. The duration of
nanoparticles in solution is very short without stabilizers and most
commercial products contain certain stabilizers. The synergetic effects
of ENPs and stabilizers should be taken into consideration in
phytotoxicity studies. Barrena et al. (2009) indicated that three
metallic nanoparticles demonstrated low to zero toxic effects on two
vegetable plants, lettuce and cucumber and the observed positive or
negative effects could be primarily attributed to the presence of
stabilizers. For most nanoparticles, relatively high concentrations are
needed to cause observable toxicity on plants and the toxicity
threshold is species dependent (Lin and Xing, 2007; Lee et al., 2008).
Several latest research papers have also indicated that particle size
and specific surface area are more appropriate indicators of
phytotoxicity than nominal concentrations of nanoparticles (Barrena
et al., 2009). Our laboratory studies on the phytotoxicity of silver
nanoparticles (AgNPs) on thale cress seedlings appear to support the
hypothesis that surface area is a better indicator of phytotoxicity than
nominal concentration (data not published). Yang and Watts (2005)
also indicated that surface characteristics are important for phyto-
toxicity of nanoparticles. By coating alumina nanoparticles with
phenanthrane, the authors found that phytotoxicity of alumina
nanoparticles to seedlings of five different species was reduced, as
indicated by root elongation. Fourier Transform Infrared Spectroscopy
(FTIR) was employed to evaluate the surface characteristics and the
result revealed that alumina hydroxyl radicals disappeared from the
surface and the authors concluded that hydroxyl radicals contributed
to phytotoxicity of alumina nanoparticles. This conclusion was proved
by the reduced phytotoxicity of alumina nanoparticles after the
addition of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO).
Studies dedicated to investigate the effect of other physiochemical
properties of ENPs on phytotoxicity such as particle shape and surface
charge are not currently available.
Taken together, the apparent differences on the toxicity of
nanoparticles to plants may arise from properties of nanoparticles,
plant species and ages, exposure time and concentrations. Research
has been conducted to highlight the importance of preparation
methods because different sample preparations resulted in different
size distributions and toxicological properties (Nurmi et al., 2005;
Lovern and Klaper, 2006). As mentioned above, nZVI prepared with
various methods demonstrate highly different properties. Other
researchers who studied the toxicity of fullerene dispersions to
several aquatic species reached the same conclusion that preparation
methods should be considered in comparison of phytotoxicity results
(Oberdorster et al., 2006; Zhu et al., 2006). An equally important
parameter in the phytotoxicity study is the selection of phytotoxicity
indicator. Seed germination and root elongation are two standard
indictors of phytotoxicity suggested by U.S. Environmental Protection
Agency, yet several research has indicated the insensitivity of seed
germination for nanoparticles (Stampoulis et al., 2009). Plant biomass
and chlorophyll levels have been shown to be more sensitive in
several studies and more work is probably needed to standardize
phytotoxicity studies of nanomaterials.
4. Phytotoxicity of engineered nanoparticles to algae and
plant cells
ENPs are not only toxic to plant seedlings, but also harmful to plant
cells and algae. Using AgNPs and root tip cells of onion (Allium cepa),
researchers demonstrated that AgNPs could disrupt cell division
process causing chromatin bridge, stickiness and cell disintegration
(Kumari et al., 2010) Pseudokirchneriella subcapitata, single-celled
microalgae, have been frequently used in nanotoxicity studies. The
toxicity of CeO2 nanoparticles to P. subcapitata have been observed,
yet the mechanisms could not attribute to either of the mechanisms
 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061
mentioned above for plant seedlings because no evidence showed
that CeO2 nanoparticles adsorbed to the cell surface nor was taken up
by P. subcapitata (Hoecke et al., 2009). In this study, the authors
noticed that CeO2 nanoparticles clustered around P. subcapitata cells
and suggested that localized interactions of CeO2 nanoparticles with
the algal cells may cause toxicity because of local aggregation
resulting in local nutrient depletion and shading.
As indicated earlier, surface area and surface characteristics play
an important role in the phytotoxicity of nanoparticles to seedlings. A
study specifically designed to test the hypothesis that nanotoxicity is
related to specific surface area instead of mass concentrations
supported this statement because the toxicity threshold of suspen-
sions of SiO2 nanoparticles with the diameters of 12.5 nm and
27.0 nm to P. subcapitata is essentially the same when nanoparticles
are normalized to their surface area (Hoecke et al., 2008). In a separate
study, Hoecke et al. found that the toxicity of CeO2 nanoparticles to P.
subcapitata can be related to the surface area as well. Up to this point,
most evidence on phytotoxicity appeared to support the hypothesis
that surface area is a more important factor on phytotoxicity of
nanoparticles to plants at both seedling and cellular levels. However,
evidence to reject this hypothesis has also been reported in other
studies using mammalian cells. For instance, Warheit et al. found that
toxicity of nanoparticles is not solely determined by the surface area
of nanoparticles and suggested that surface activity of nanoparticles
as a dominant factor in determining the toxicity of nanoparticles
(Warheit et al., 2006, 2007). A toxicity study conducted with AgNPs
and nitrifying bacteria demonstrated that reactive oxygen species
(ROS) may play a role in the toxicity of nanoparticles, but ROS is not
the only factor controlling the toxicity of silver nanoparticles (Choi
and Hu, 2008). Phytotoxicity studies on both seedling and cellular
levels adding ROS in the investigation scope are needed.
In addition to the surface characteristics, the role of dissolution in
the toxicity of metallic nanoparticles (both elemental and oxides) to
plant cells is under intense investigation. Franklin et al. noticed that
the toxicity of ZnO nanoparticles, bulk ZnO and ZnCl2 demonstrated
comparative toxicity to P. subcapitata and the toxicity of nanoparti-
culate and bulk ZnO is solely due to the dissolved Zn (Franklin et al.,
2007). In a separate study of the phytotoxicity of AgNPs to algal cells,
AgNPs seemed to be more toxic than Ag+ to Chamydomonas
reinhardtii, mobile single-celled algae (Navarro et al., 2008a,b).
Using a strong silver ligand (cysteine) to bind silver, the authors
found that toxic effects of silver ions or AgNPs can be completely
eliminated, indicating that the toxicity of AgNPs was associated with
the Ag+. The authors concluded that AgNPs are a source of Ag+ and
the rapid accumulation of Ag+ by algal cells was the underlying
mechanism of toxicity of AgNPs, which is in contrast with the
conclusions drawn from seedling studies. In this study, photosynthe-
sis efficiency was used to determine toxicity effect. A recent study
concerning the uptake of SWCNTs by a commonly used cell culture,
tobacco Bright Yellow (BY-2) cells showed that SWCNTs were not
toxic to the cell based on their morphology, metabolic rate and
membrane integrity (Liu et al., 2009). However the applied SWCNTs
are considerably large and are mainly composed of CNT bundles with
an average size of 500 nm.
5. Uptake, translocation and accumulation (UTA) of engineered
nanoparticles
The importance of uptake and accumulation of ENPs by plants is
increasingly recognized by researchers and some scientific studies
have been published recently. The first report was published by Zhu et
al. (2008). It unambiguously showed for the first time that iron oxide
nanoparticles (Fe3O4) was taken up by pumpkin (Cucurbita maxima)
roots and translocated through the plant tissues. A mass balance was
conducted at the end of the experiment and the study showed that
about 45.5% of fed nanoparticles were accumulated in roots and
3057
approximately 0.6% of the nanoparticles were detected in leaves.
When a different plant species, lima bean (Phaseolus limensis), was
tested by the same researchers however, uptake and transport of iron
oxide nanoparticles were not observed. Lin et al. investigated the
uptake and translocation of carbon nanomaterials by rice plants
(Oryza sativa) and they found that fullerene C70 could be easily taken
up by roots and transported to shoots (Lin et al., 2009). Their study
also demonstrated that C70 could be potentially transported
downward from leaves to roots through phloem if C70 entered into
plants through plant leaves. Similar results were not observed for
MWCNTs by the same researchers even when the concentration of
MWCNTs was 800 mg/L. This discrepancy could be due to the
relatively large size of MWCNTs than fullerenes. With a two-photon
excitation microscope, researchers demonstrated that MWCNTs
primarily adsorbed to root surface as individual and aggregated
CNTs even though one or both ends can pierce through root cap cell
walls, Fig. 5 (Wild and Jones, 2009). This unique interaction facilitates
the penetration of organic contaminants into cell cytoplasm but
extensive internalization of ∼ 100 nm diameter CNTs was not
observed. Other researchers who investigated the uptake of ZnO
nanoparticles by ryegrass (L. perenne) noticed no upward transloca-
tion of ZnO nanoparticles from roots to shoots (Lin and Xing, 2008).
ZnO nanoparticles primarily adhered to root surface and individual
nanoparticles were also observed in the apoplast and protoplast
spaces in root endodermis and stele.
For metallic nanoparticles, Cu nanoparticles (CuNPs) could be
taken up and accumulated in the biomass of bean and wheat plants,
and a responsive relationship existed between the bioaccumulated
CuNPs in plant tissues and in growth media (Lee et al., 2008). The
researchers also presented a linear relationship that higher concen-
trations of CuNPs in the growth media resulted in higher uptake and
accumulation of CuNPs in plant tissues and that the aggregation state
of CuNPs in plant tissues is also related to the concentrations of
nanoparticles in growth media. However, this study was carried out in
agar media; it is unknown how this result resembles the uptake and
bioaccumulation of CuNPs in liquid medium and soil. In our laboratory
studies, AgNPs colloids were used and the preliminary data indicated
that AgNPs as large as 40 nm can be taken up by the roots of A. thaliana
and transported to the shoots even though the majority of AgNPs are
adhered to the root cap, Fig. 6. The roots were collected from seedlings
fed with 680 μg/L of 40 nm AgNPs for four weeks and were imaged
under a confocal/multiphoton microscope. AgNPs were observed to
accumulate in an unexpected tissue, columella, in the roots. Exact
reason for the accumulation of AgNPs in columella is still unknown.
Due to the natural dissolution process from metallic nanoparticles and
the capabilities of some plants to reduce metal ions to elemental NPs
inside plant tissues (Harris and Bali, 2008), accumulated MNPs in
plants could come from two sources. No attempt has been made to
distinguish the sources of NPs in plant tissues in the presence of ENPs.
A recently published work showed that uptake and transport of
nanocrystals from roots to leaves within a few days in moth orchid
(Phalaeonpsis spp.) and A. thaliana when watered with an aqueous
colloidal solution of NaYF4:Yb,Er nanocrystals (Hischemoller et al.,
2009). NaYF4:Yb,Er nanocrystals are upconversion fluorescence
nanocrystals which emit visible light under near infrared light. Orchid
roots were soaked in the NaYF4:Yb,Er colloidal solution for different
time and NaYF4:Yb,Er nanoparticles in plant tissues were visualized
using a confocal laser scanning microscope. The images clearly
demonstrated the route of the penetration of the nanocrystals into
plant tissues, from velamen radicum (a special epidermis occurring
only in aerial roots) to passage cells in about ten minutes and finally
reaching vascular tissues in days (Hischemoller et al., 2009). This
work is probably the first study to show the uptake kinetics and to
illustrate the potential penetration routes of nanoparticles into plant
tissues. Whether this observation is true to subterranean roots is
unknown. Even though progresses have been made, the investigation
3058 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061

Fig. 5. A. Unstained MWCNTs at the surface of a living root, B. piercing of epidermal cellular structure by MWCNTs. Both ends of the MWCNTs penetrated through the cell wall and the
middle of MWCNTs is at the root surface, and C. 3D reconstruction of multiple xy TPEM sections showing the adsorption of MWCNTs at the root surface and piercing of MWCNTs
through the epidermal cell walls of several cells.
Images are adapted with permission from John and Wilds, 2009, Environ. Sci. Technol., copyright 2009 American Chemical Society.

of plant uptake and accumulation of nanoparticles is still in its infant 6. Interactions of engineered nanoparticles with plant cells
stage. Many hurdles are ahead and many critical questions remain
unanswered and these critical questions will be discussed in the ENPs have to penetrate cell walls and plasma membranes of
Future research needs section. epidermal layers in roots to enter vascular tissues (xylem) in order to

Fig. 6. Uptake and accumulation of 40 nm AgNPs by wall cress (Arabidopsis thaliana). A. Root controls grown in Hoagland solutions, B. the adsorption of AgNPs on primary and lateral
root caps, and C. the uptake and accumulation of AgNPs in plant roots, small fractions of AgNPs can be detected in the vascular tissues. D. Magnified root cap indicating that AgNPs are
primarily accumulated in root cap and some AgNPs are sequestered in columella. The roots were imaged with a confocal/multiphoton microscope.
 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061
be taken up and translocated through stems to leaves. Cell walls,
through which water molecules and other solutes must pass to enter
into roots, are a porous network of polysaccharide fiber matrices. The
pore sizes of plant walls are typically in the range of 3–8 nm, which is
much smaller than many tested ENPs (Carpita and Gibeaut, 1993).
Navarro et al. hypothesized that cell walls typically with the thickness
of about 5 to 20 nm function as natural sieves. ENP aggregates with a
size smaller than the largest pore are expected to pass through and
reach the plasma membrane and the larger particle aggregates will
not enter into plant cells. But the authors also admitted that ENPs may
induce the formation of new and large size pores which allow
the internalization of large ENPs through cell walls (Navarro et al.,
2008a,b). Proseus and Boyer investigated the penetration of Cu
nanoparticles of various sizes into and across algal cell walls using
confocal laser scanning microscopy and found that gold nanoparticles
with a diameter of 10 nm or above could not penetrate through algal
cell walls even under pressured conditions (Proseus and Boyer, 2005).
However, cell internalization of ENPs of different sizes and
compositions has been observed for different plant species (Lin et
al., 2009; Liu et al., 2009). Liu et al. demonstrated that SWCNTs treated
with strong acids can be taken up by BY-2 cells. The authors further
indicated that BY-2 cells can also uptake the SWCNTs–fluorescein
isothiocyanate and SWCNT–DNA conjugates, making SWCNTs a
promising nanotransporter for intact plant cells (Liu et al., 2009).
Torney et al. used protoplasts in their studies to demonstrate the
internalization of mesoporous silica nanoparticles and the potential
delivery of DNA and other chemicals with silica nanoparticles to plant
cells (Torney et al., 2007). Other researchers showed that protoplasts
from sycamore-cultured cells can effectively take up 40 nm polysty-
rene nanospheres and 20 nm CdSe/ZnS QDs (Etxeberria et al., 2006).
Protoplasts are plant cells after enzymatic removal of cell wall and
certain surface proteins. Etexberria et al. showed that it is likely that
ENPs pass through plasma membranes of sycamore cells in a similar
manner as they do through mammalian cells via endocytic pathways.
The authors also demonstrated a mechanism of recognition, separa-
tion and redistribution which exists in the early stage of solute
uptake: the 40 nm nano-sphere was stored in the vacuoles of
sycamore cells as most other solutes but the 20 nm QDs remained
in the cytoplasmic membranous compartments after 18 h (Etxeberria
et al., 2006. Whether the different sequestration location is related to
the particle sizes or the composition is unknown and yet will be highly
interesting to unravel. Even though protoplasts can provide invalu-
able information on the internalization mechanism how nanoparticles
enter plant cells, it is questionable that whether protoplasts could
resemble intact plant cells surrounded with cell walls. The employ-
ment of intact cells is still needed to elucidate the mechanisms of
nanoparticles internalization into plant cells, especially after it was
shown that intercellular transport of nanoparticles in cell walls could
occur.
Once micro- and macromolecules enter plant cell walls, the
molecules can be transported through plasmodesmata, the intercel-
lular organelles of 20–50 nm in diameter (Lucas and Lee, 2004;
Heinlein and Epel, 2004). Selective and non-selective pathways
through plasmodesmata are found to transport regulatory proteins
and RNAs in short distances (Kim, 2005). We found AgNPs of 20 nm
taken by plants were mostly in the intercellular spaces could be
transported inside plant cells through plasmodesmata, Fig. 7. Root
samples were analyzed under a transmission electronic microscope
three weeks after exposed to 20 nm AgNPs. Plasmodesmata have been
observed to dilate in different states in leaf epidermal cells when they
were transfected, in a non-invasive way, with green fluorescent
protein (GFP) reporters (Crawford and Zambryski, 2000). At least one
protein, Size Exclusion Limit2, is found involved in the size exclusion
of plasmodesmata (Kobayashi et al., 2007). The size of the largest
molecule that can pass through plasmodesmata defines the size
exclusion limit. In terms of size, Stokes radius has been considered to
3059
Fig. 7. Transmission electron micrographs showing the accumulation of silver
nanoparticles (AgNPs) in plasmodesmata of Arabidopsis thaliana. A. 20 nm AgNPs
(arrows pointing to) in intercellular space, i.e. plasmodesmata. B. Control. Scale bars:
1 μm.
better describe size exclusion limit and is defined as the radius of a
hard sphere in hydration and with shapes that diffuses the same rate
as the molecule. Dashevskaya et al. (2008) showed that anionic GFPs
with negative charges had larger Stoke radius than neutral GFPs of the
same sizes in epidermal cells of Nicotiana benthamiana.
7. Future research needs
Despite the rapid progress in the study of phytotoxicity, uptake
and accumulation of ENPs in the past two years, we are still in the very
early stage of this field and numerous questions with tremendous
scientific or practical importance need to be investigated. In the
phytotoxicity study of nanoparticles, the most urgent need is probably
to build connections between the characteristics of nanoparticles (e.g.
surface area, particle size, surface activity) with phytotoxicity. Equally
important is the need to understand the role of plant species and the
composition of nanoparticles on phytotoxicity of nanoparticles.
Previous studies have clearly demonstrated the different resistivity
of nanoparticles by different plant species, yet how and why different
plant species demonstrate different resistance to nanoparticles
remains unsettled. Does the resistance of plants correlate with the
biomass of roots, or surface of roots? How does the vascular structure
of plants affect the plant's resistance to toxicity? What is the genetic
response of plants and what genes are up-regulated and what genes
are down-regulated in the presence of ENPs?
On the aspect of plant uptake and accumulation, little studies have
been performed to assess the uptake kinetics of ENPs and to
investigate how the composition, particle size and aggregation state
affect the uptake kinetics and the fate and transport of nanoparticles
in plant systems. Information is also lacking on how plant species and
environmental factors will affect the uptake and accumulation of ENPs
by plants. Xylem structure of plants could be an essential parameter
because it has been recognized that xylem structures determine the
speed of water transport (Guthrie, 1989). As a result, plants with
different xylem structures may demonstrate different uptake kinetics
of nanoparticles and studies with plants having different xylem
structures will reveal tremendous information on the impact of plant
species on uptake kinetics. Another important need is to understand
the penetration route of NPs into vascular tissues. Solutes enter into
vascular tissues either apoplastically or symplastically. Preliminary
studies with AgNPs in our study appeared to indicate apoplastical
pathway of entrance to plant tissues and cross membrane through
plasmodesmata, yet more studies are needed to confirm this
hypothetical pathway. Where ENPs are sequestered in plant tissues
3060 X. Ma et al. / Science of the Total Environment 408 (2010) 3053–3061
Fig. 8. Top left: the design of simple environmental cells based on the electron transparent LUXFilm™ membrane; bottom: SEM images of the Arabidopsis thaliana root tissue
immersed in water. The individual AgNPs accumulated in the plant tissue are seen as bright spots.
and are they bioavailable to human beings and wild lives? The answer
to the last question will have considerable implications for food safety
because nanoparticles can be potentially accumulated in edible
portions of plants and crops if they are exposed to ENPs.
From the experimental point of view, the future phytotoxicity and
uptake research will benefit from the in vivo microscopy methods
which allow the quantitative kinetic studies of the uptake and
accumulation. Up to now, most of this kind of research relies on
optical detection of the fluorescent nanoparticles (quantum dots) or
on nanoparticles functionalized with fluorescent markers (Gonzalez-
Melendi et al., 2008). This powerful tool however has several inherent
limitations such as limited resolving power which leads to the
fundamental impediments in observing the uptake, transport and
accumulation dynamics on the cellular level. In addition, the vast
majority of the nanoparticles relevant to phytotoxicity is not
fluorescent and cannot be detected directly by this method. The
common functionalization of the surface of these particles may lead to
alteration of their chemical reactivity and deviate from the real world
behavior.
One of the potential methods capable of addressing these
challenges relies on the recent developments in environmental
scanning (SEM) and transmission (TEM) electron microscopy. The
development of electron transparent but gas/liquid impermeable
membranes opened the possibility to image the living cells in their
natural environment with unprecedented resolution (de Jonge et al.,
2009). Using this approach, in vivo imaging of the individual
nanoparticles and their short and long term dynamics inside the
individual cells and pant tissue become feasible. Fig. 8 shows AgNPs
taken up by thale cress seedlings imaged with this innovative
technology by one of the authors.
Finally, the majority of studies conducted so far on phytotoxicity
and uptake of ENPs by plants were carried out in hydroponic settings.
Without adequate information, hydroponic studies could reveal
considerable information on the interactions of plants and ENPs in
water without the compounding effect of soil. Yet the complicated
nature of environmental media calls for fate and transport studies of
ENPs in more “nature-like” conditions. These research needs are by no
means a complete list in the study of interactions of ENPs with plants,
yet they probably represent some most urgent research needs in this
area as the production of ENPs is expected to grow rapidly.
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