NanoSafety

A short overview of knowledge gaps, future aspects and regional actors in the field

Per Gustavsson, Department of Biology, Section of functional zoology, Lund university

Table of contentsIntroduction.................................................................................................................................4

Types of ENPs and applications....................................................................................................6

TiO2...........................................................................................................................................6

ZnO...........................................................................................................................................7

Cu and CuO...............................................................................................................................7

FeO...........................................................................................................................................7

Ag.............................................................................................................................................7

Au.............................................................................................................................................8

SWCNT and MWCNT................................................................................................................8

Fullerenes.................................................................................................................................9

Physicochemical properites determining biological effects.......................................................10

Geometry and particle size.....................................................................................................10

Surface functionalisation........................................................................................................12

Metal contaminants and metal leakage.................................................................................13

Protein and biomolecule binding...........................................................................................14

Aggregation and solubility......................................................................................................15

Summary and research outlooks............................................................................................16

Mechanisms of toxicity..............................................................................................................18

Oxidative stress......................................................................................................................18

Frustrated phagocytosis.........................................................................................................19

Effects of ENPs on reproduction and fetal development...........................................................20

Routes of exposure....................................................................................................................22

Airway exposure.....................................................................................................................22

Dermal exposure....................................................................................................................22

Oral exposure.........................................................................................................................24

Summary and future research needs.....................................................................................25

Human exposure........................................................................................................................26

Workplace emission of ENPs..................................................................................................26

Consumer exposure...............................................................................................................26

Biomarkers of exposure to ENPs............................................................................................27

Summary and future aspects..................................................................................................28

2

Effects of long term exposure to ENPs.......................................................................................29

New technologies for ENP detection..........................................................................................30

Nanosafety and food technology...............................................................................................32

Summary, knowledge gaps and research needs.....................................................................34

Environmental and ecotoxicogical aspects of nanosafety..........................................................35

Adsorption of biological materials to ENPs............................................................................35

Effects of ENPs on plants and crops.......................................................................................37

Food chain transfer of ENPs and biomagnification.................................................................38

Life cycle assessment of ENP..................................................................................................40

Summary and future research needs.....................................................................................41

Actors within nanosafety and related fields...............................................................................42

Sweden...................................................................................................................................42

Lund – Malmö....................................................................................................................42

Stockholm...........................................................................................................................44

Kalmar................................................................................................................................45

Gothenburg........................................................................................................................45

Linköping............................................................................................................................47

Piteå...................................................................................................................................47

Denmark.................................................................................................................................47

Copenhagen university / Nano-Science Center..................................................................47

DTU....................................................................................................................................48

National Research Centre for the Working Environment...................................................49

Aarhus University...............................................................................................................49

Identified knowledge gaps.........................................................................................................51

References.................................................................................................................................53

3

Introduction

The aim of this report is to give an overview of the knowledge and the knowledge gaps

that exist today concerning the safety of engineered nanoparticles (ENP). Nanosafety is

by nature a truly interdisciplinary science which necessitates an understanding of both

physics of particles and the biology of humans, cells, animals and plants. There is no

official definition of what an ENP is, but it is generally accepted that an ENP is a man-

made particle having one dimension in the size range of 1-100 nm.

Why is there a need to study ENPs and the effect of ENPs on living organisms? All life

on earth has been exposed to nanosized particles some time during evolution, and still

is. Nanosized particles can be formed under natural conditions such as during

combustion, such as forest fires and volcanic eruptions (Oberdorster et al., 2005) and

animals have developed defence mechanism to deal with such exposure. The human

skin is relatively impermeable to particle penetration, the mucociliary elevator

continuously clears particulate matter from our airways and that which cannot be

cleared this way is engulfed and digested by macrophages. As ENPs are designed for a

specific purpose and pose new structures that life on earth has not been exposed to

during evolution, there is a possibility that these particles might pose a larger threat than

unintentional anthropogenic nanosized particles. Risk is a function of exposure and

hazard and when the exposure increases, so does the risk. As the use of nanotechnology

and ENPs are estimated to increase dramatically in the coming decade, so will the

exposure and also the risk. If the exposure cannot be reduced, the hazard could be an

important target. Reducing the hazard by designing ENPs which are less toxic could be

a solution. However, when assessing risk, it is of great importance to make as correct as

possible estimations on the exposure. Early animal studies of carbon nanotube exposure

used very high levels of carbon nanotubes as compared to levels which could be

expected in an occupational setting (Lam et al., 2004). In order for nanotoxicological

data to be credible, experimental levels of ENP exposure must be in parity with that

found in a real environment.

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In 2005, the Environmental Defence Fund and DuPont teamed up to form the Nano

Risk Framework, in order to investigate the risks of nanotechnology. Initiatives such as

these could benefit both society and commercial interests (PEN 19, 2010). The study of

ENPs originates from studies on ultra-fine particles (Oberdorster et al., 2005) and using

the knowledge of the toxicology of other particles, e.g. asbestos and graphite, could be

beneficial for the novel field of nanosafety. After reviewing the literature on carbon

nanotube safety and toxicity, it seems that the study of ultra-fine particles has been

forgotten by some researchers. It has been suggested that it would take decades to make

complete risk assessments on nanomaterials, as compared to that of regular chemicals.

The many different forms of nanomaterials is one key factor. In order not to slow down

the development of nanomaterials nor allow hazardous nanomaterials to reach the

market, new strategies for risk assessement have been suggested (Grieger et al., 2010).

The general public’s knowledge on nanotechnology and nanosafety is limited. Two

surveys conducted on laymen in Switzerland, where the perceived risks and benefits of

nanotechnology in food technology was examined showed that people perceived

nanotechnology in food packaging as less problematic than nanotechnology in food.

The trust of the interviewees and the naturalness of food was important factor that

influenced their acceptance of nanotechnology in food technology (Siegrist et al., 2007;

Siegrist et al., 2008). Research activities within nanosafety could in an extended

perspective impact society by providing the general public with unbiased information

on the safety of nanotechnologies and allow laymen to make informed decisions.

5

Types of ENPs and applications

The possible applications of ENPs are immense and equally diverse. Carbon nanotubes

can be used to enforce polymeric materials such as plastics and are used in Li-ion

batteries to improve their capacity. ZnO nanoparticles are used in sunscreens, TiO2

nanoparticles are used in paints, cosmetics and sunscreens among other things. The use

of ZnO and TiO2 in sunscreens is nothing new, both substances have been used as a part

of formulations since the 1990’s. An inventory of commercially available products

which contain some sort of nanosized components can be found at

http://www.nanotechproject.org/inventories/ a webpage maintained by the Woodrow

Wilson International Center for Scholars Project on Emerging Nanotechnologies. The

Project on Emerging Nanotechnologies regularly publishes reports on the status of

nanotechnology and society. The most recent report published in November 2010 is

called “Voluntary initiatives regulation and nanotechnology oversight: Charting a Path”

(Fiorino, 2010).

Chen et al. (2010)(Chen et al., 2010) recently showed in an experiment conducted at

NIOSH facilities, that a commercial spray can product containing TiO2 produced

respirable nanoparticles. The intended usage of the spray was cleaning and sanitizing

bathrooms. The nanoparticle fraction in the spray was 170 µg/m3 which was equivalent

to1.2x105 particles/cm3 with a mean particle diameter of 75 nm. The researchers

estimated the dose to 0.075 µg TiO2 per m2 alveolar epithelium and minute. Exposure

levels for rats were determined to 0.03 µg. This was the first report to ever show that a

commerical product can create respirable nanoparticles and would serve as a basis for

further toxicological studies on respirable TiO2 particles.

TiO2

Titanium dioxide (TiO2) nanoparticles is one of the most common type of nanoparticles

produced on an industrial scale and used in a large number of products and applications.

TiO2 occurs in two different chrystalline forms known as rutile or anatase. TiO2

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nanoparticles are used in sunscreens and cosmetics, used as pigments in paints, as dirt

repellants on windows and in household applications and can be added as a filler in

concrete (Lee et al., 2009). When used in sun screens the TiO2 nanoparticles are usually

coated in order to stabilize them and to prevent particle aggregation.

ZnOZinc oxide nanoparticles are used in sunscreens and cosmetics added to rubber in order

to improve material properties and used as a pigment in paints. The use of ZnO

nanoparticles in sunscreens and cosmetics was banned by the European parliament in

2009 (European Parliament and Council Regulation (EC) No 1223/2009) due to

concerns and uncertainties on the toxicity of nanosized ZnO.

Cu and CuOCopper oxide nanoparticles have applications in the construction industry and can be

added to concrete to improve mechanical properties (Lee et al., 2009). Certain

cosmetics used for tanning puropses reportedly contain copper nanoparticles as well as

beverages sold as health supplements (The Project on Emerging Nanotechnologies,

2011).

FeOIron oxide nanoparticles have several potential uses, especially within experimental

biomedicine. Magnetic nanoparticles made from FeO are under investigation by various

companies with intention of being used as contrast agents or cancer treatment.

AgSilver nanoparticles and colloidal silver has been used since the antiquity as a remedy

and as a health booster. The most prominent biological property of silver nanoparticles

is their antimicrobial effects. Recent tecnological development has led to the use of Ag

nanoparticles being used in various household products, such as refrigerators and

washing machines, to reduce bacterial growth but also in toothpaste and textiles (Project

on emerging nanotechnologies; ENRHES 2009).

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AuSolid gold and larger gold particles are biologically inert. When downsizing particles on

the nanoscale, different effects occur. A great number of mechanistic studies on ENPs

have been carried out using gold nanoparticles. Effects on macrophage uptake have

been done using spherical and rod shaped Au ENPs, as well as studies on alveolar

translocation of Au ENPs of different sizes.

SWCNT and MWCNTSingle walled carbon nanotubes are made up of graphene layers folded into a tube. They

are commonly 0.8-1.2 nm in diameter and up to several micrometeres long, depending

on the mode of synthesis. Multi wall carbon nanotubes are made up of multiple layers

of graphene sheets folded in to tubes which are held to ether by van der Waals forces.

The external diameter can vary from a few nm up to 150 nm and they can be up to

several micrometers long (Gustavsson et al., 2011). Both the surface of SWCNT and

MWCNTs are inherently hydrophobic, causing the tubes to form

aggregates/agglomerats. An organism exposed to pristine carbon nanotubes will

therefore most likely encounter bundles or aggregates of tubes instead of single tubes.

Most research in the past, as well as more recent research has been focused on how to

make carbon nanotubes water soluble and monodispersed. Addition of hydroxy- or

carboxyl groups to the surface generally makes carbon nanotubes hydrophilic and thus

water soluble. Carbon nanotubes can be modified with other functional groups in order

to modify the properties of the tubes. Examples of such are addition of chelators or

poly(ethylene glycol) (PEG) to bind metal ions or to decrease the half-life of carbon

nanotubes intended for biomedical applications. Applications of carbon nanotubes

include uses in composite materials in order to make the material stronger or electrically

conductive, applications in electronics such as electrodes in lithium ion batteries and are

also being investigated for applications in drug delivery (Gustavsson et al., 2011).

8

FullerenesFullerenes is an allotrope of carbon which form a three-dimensional 60 carbon atom

structure. The applications of fullerenes are within biomedicine and drug delivery, as

reinforcers of polymer matrices and fullerenes functionalised with metals could function

as catalysts (ENRHES 2009).

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Physicochemical properites determining biological effects

Various physicochemical properties of ENPs have been suggested to influence their

biological effects and toxicity. A short description and examples of these are given

below and is intended as an overview only.

Geometry and particle sizeHigh aspect ration nanoparticles (HARN) have been suggested to cause frustrated

phagocytosis by macrophages and also to be retained in the pleural stomata. This is due

to the fact that such particles are too long and stiff to be efficiently phagocytosed by

macrophages (Donaldson et al., 2010). Parallells can be drawn to asbestos and silica

particles which cause similar effects. This type of geometry can cause ENPs to have a

longer biopersistency in the body, increasing the risk of negative effects on cells and

organs.

The geometry and size may also have implications for tissue biodistribution as well as

elimination and excretion of ENPs. Carbon nanotube show some characteristics of

HARNs, but have been shown to under certain circumstances be excreted through the

kidneys. Multiwalled CNTs which were made water soluble by surface

functionalization could be excreted through the kidneys and into the urine if the CNTs

were well separated and aligned longitudinal to the filtration slits in the kidney

glomeruli (Lacerda et al., 2008a; Lacerda et al., 2008b). PEG-ylated gold ENPs shaped

either as spheres or rods were tested for their uptake into macrophages in vitro and in

the organs of mice in vivo and assayed for their protein binding capacity. Macrophages

were found to take up the spherical ENPs almost four-fold as compared to the rod

shaped ENPs. The absence of serum proteins greatly influenced the uptake leading to a

five-fold greater uptake of spherical gold ENPs. The spherical particles also bound BSA

to a much higher degree as compared to the rod-shaped particles. As for uptake into the

organs of mice both spherical and rod-shaped ENPS were taken up and accumulated in

liver and spleen, while only the rod-shaped ENPs were significantly taken up in heart,

lung and kidneys. The rod-shaped form of gold nanoparticles had a longer blood

10

circulation time than their spherical counterpart. (Arnida et al., 2011). Similar effect of

length and shape were observed when four different kinds of MWCNTs were injected in

the abdominal cavity of mice. Two types of the MWCNTs were short and flexible and

formed smaller entangled aggregates, while the other two types were longer and more

rigid and less tangled. After introduction to the abdominal cavity the response of the

mesothelial tissue which lines the abdomen was investigated. It was found that the

thickness of the tissue was much larger for those animals which had been treated with

the longer and more rigid MWCNTs. The animals treated with the longer MWCNTs

also exhibited granulomas while the other two groups did not (Poland et al., 2008). The

authors of the report suggested that the shape and size of the MWCNTs were the reason

for their non-degradability by phagocytising cells and suggested that certain types of

MWCNTs could exhibit asbestos-like effects in biological systems.

Yet another study on the effect of ENP shape examined the effect of length on TiO2

particles. Nanospheres (60-200 nm diameter), short nanobelts (60-300 nm diameter,

0.8-4 µm long) and long nanobelts (60-300 nm diameter, 15-30 µm long) were

compared for their effects on mice after airway exposure and on macrophages. First, all

of the three studied materials induced reactive oxygen species by alveolar macrophages

in comparable levels. The nanospheres and the short nanobelts were taken up into

lysosomes, while the longer nanobelts were unable to elicit functional lysosomes and

were observed free in the cytoplasm. Cytotoxicity was only seen for the longer

nanobelts at higer concentrations and only the long type of nanobelts induced cytokine

production. It would seem that changing the shape of a material could confer different

biological response (Hamilton et al., 2009).

Two gold nanoparticles of different sizes, one 1.4 nm and one 18 nm in diameter, were

studied for their biodistribtion in rats after intratracheal instillation and intravenous

injection. The smaller particle is known to be cytotoxic, while the larger particle is

rather intert. Interestingly, the particles showed very different biodistributions. After

intratracheal instillation, most of the 18 nm sized particles remained in the lungs, while

a part of the smaller 1.4 nm sized particles were able to translocate from the lung and

into the blood, liver, kidneys, skin and urine (Semmler-Behnke et al., 2008). These

11

results further show that the behaviour of ENPs is dependent on size and is not easily

predictable.

The size of a particle can not only affect its toxicity or protein binding, but also their

clearance and translocation from the airways. Henning et al. (Henning et al., 2010)

investigated the speed of mucociliary clearence of ENPs with different sizes and

chemical compostitions. The experiments were conducted in an in vitro model using

chicken embryo tracheas. It turned out that polystyrene particles ranging from 50 nm to

6000 nm were all transported with a similar speed, while particles made from various

combinations of poly(lactic-co-glycolic acid) (PLGA) showed a high variety of speeds.

The speeds were dependant on the chemical compostion of the ENPs.

Surface functionalisationAs important as the small size of nanoparticles is the functionalization of the surface. In

order for nanoparticles to be compatible with various applications they often require

surface functionalisations (Hirsch and Vostrowsky, 2005). As an example on how

surface functionalization can affect biological response, Table 1 summarizes some

functionalizations of carbon nanotubes and their effect on the circulatory half live. The

carbon nanotubes used in the studies differ in length, diameter and aggregation state.

However, it can be seen that certain modifications, especially PEG-ylation, has dramatic

effects on the half-life of carbon nanotubes in plasma. A short half-life in plasma does

not necessarily indicate that the nanotubes are eliminated and excreted, rather it

indicates the rate of absorption by internal organs.

12

Table 1. The effect of surface modification/functionalization of carbon nanotubes

(CNTs) on the circulation half-life in plasma. Adapted from Gustavsson et al. (2011).

Type of

CNT

Surface modification Half-

time

Administration Species Reference

MWCNT - 3 h i.v. Mouse Lacerda et al. 2008a

MWCNT glucosamine 5.5 h i.p. Mouse Guo et al. 2007

SWCNT Hydroxyl 50 min i.v. Mouse Wang et al. 2008

SWCNT Pluronics F108 1 h i.v. Rabbit Cherukuri et al. 2006

SWCNT DTPA 3-3.5 h i.v. Mouse Singh et al. 2006

SWCNT chitosan 3-4 h i.v. Mouse Kang et al. 2009

SWCNT PEG 15 h i.v. Mouse Liu et al. 2008

SWCNT PEG 15.4 h i.v. Mouse Yang et al. 2008

Metal contaminants and metal leakageDuring the production of carbon nanotubes by the CVD method, metals are used as

catalysts. The resulting carbon nanotubes therefore usually contain various levels of

metal contaminants. The most common metals are Fe, Co, Mn, Ni. Other forms of

carbon than CNTs are also usually found after synthesis such as organic carbon species.

The CNTs are purified to a certain level depending on their application, e.g. electronics

or medicals. Some authors contribute toxicity of CNTs to the presence of metal

contaminants (Kagan et al., 2006). Such contaminants could potentially cause oxidative

stress through the production of reactive oxygen species.

Another physicochemical property of ENPs suggested to cause toxicity is leakage of

metal ions from metal ENPs. A recent review on the subject suggest that a majority of

the toxic effects of metal nanoparticles can be attributed to their level chemical stability,

since stable particles show little cytotoxicity as compared to those who are more easily

dissolved for instance (Auffan et al., 2009).

13

As an example the release of copper ions from two kinds of copper oxide nanoparticles

were examined in cell culture. One of the particles was made from copper oxide and

soluble under intracellular conditions, and the other made from copper and coated with

carbon. The particles had similar hydrodynamic diametres but slightly different surface

area per gram. The copper particles which were stabilized by a carbon layer were less

toxic than the soluble copper particles. The difference was found to be due to

dissolution of copper from the particles and treatment with copper ions at similar levels

as that released from instable particles gave a similar toxicological profile (Studer et al.,

2010).

Protein and biomolecule bindingAs a particle becomes smaller and smaller, it will eventually end up in the size range of

proteins and other biomolecules, that is the size will be on the nanoscale. This will lead

to a different ability of the nanoparticle to interact with biomolecules as compared to a

partilce of the bulk material (Lynch and Dawson, 2008). Such interactions with plasma

proteins in the blood could lead to the exposure of novel epitopes that are normally

encrypted inside the proteins. Many nanoparticles have in common that they can bind

apolipoproteins,The binding of apolipoproteins could be a reason for translocation of

nanoparticles into cells (Lynch and Dawson, 2008). Deng et al. (2009) observed that

TiO2, SiO2 and ZnO ENPs could bind human plasma proteins, with different binding

profiles for each type of ENP. In addition TiO2 ENPs with different shapes such as

spheres, rods and tubes also bound plasma proteins in different manners. Generally, the

spherical TiO2 bound more proteins than the TiO2 rods and tubes did (Deng et al.,

2009). Binding of complement factors by carbon nanotubes from plasma has been

suggested as a possible mechanism whereby nanotubes induce biological effects

(Salvador-Morales et al., 2006; Hamad et al., 2008; Moghimi and Hunter, 2010)..

Apparently few other proteins selectively bind to carbon nanotubes and include

fibrinogen and apolipoproteins. ENPs made from metal oxides have been shown to

interact with lung surfactant proteins (Schelh and Hohlfeld 2009; Schulze et al. 2010).

The function of lung surfactants is to prevent the alveoli from collapsing by reducing

the surface tension in the alveolar fluid. Inhalation is believed to be a major exposure

route for ENPs and the interaction with lung surfactants could influence the biological

14

activity of ENPs (Schelh and Hohlfeld 2009). In one study seven different metal oxide

ENPs were compared, as well as carbon black. Large differences were seen in the

protein binding patterns between all ENPs, also when the particles were made from the

same bulk material (Schulze et al. 2010). If ENPs are able to enter into cells they could

potentially interact with enzyme function and disurb cellular homeostatis. Examples of

ENP interaction with enzymes include acetylcholinesterase and butyrylcholinesterase.

These two enzymes are important for neuronal signaling and control of skeletal muscle

function. Two studies have shown that different ENPs including copper ENPs and

carbon nanotubes, were able to adsorb and inhibit the enzymatic activity of both

enzymes. ENPs of Al and AlO3 and SiO were much less potent in adsorption and

inhibition (Wang et al., 2010b; Wang et al., 2009).. The authors suggested that the

release of metal ions from ENPs could affect enzyme function as well.

Carbon nanotubes commonly have a filamentous like structure, much like the structure

of DNA strands and actin filaments. It has been observed that single walled carbon

nanotubes, which are the thinnest type of carbon nanotubes, are able to cause bundling

of actin filaments and affect cell division negatively. (Holt et al. 2010).

Aggregation and solubilityA key factor determining the toxicity of nanoparticles is their bioavailability, which in

turn is governed by the ability of the nanoparticles to aggregate and to dissolve in water.

Carbon nanotubes for instance, are inherently hydrophobic which mean that they form

aggregates upon contact with water. Functionalization of carbon nanotubes is done in

order to examine toxic effects of single particles different dispersant agents are often

used in cell culture experiments, e.g. detergents as Tween-80. Pauhlun (2010) presented

a study on long-term effect of MWCNT exposure in rats. The results from the study

were interpreted as giving the agglomeration state of the nanotubes more influence on

their toxicity as compared to that of size and shape of individual tubes (Pauluhn, 2010).

Structure-activity relationships

Recent progress has been made in the determination of structure-activity relationship of

engineered nanoparticles. Application of structure-activity relationship methodologies

15

in nanosafety is still in its cradle, as compared to other research areas such as

pharmaceutical development or chemometrics. The activity is not necessarily due to the

physical structure of the ENP, it could also include chemical composition, charge,

aggregation state or levels of contaminants. Adapting and implementing these tools in

nanosafety could help to clarify what properties makes an ENP toxic to organisms. In

principle such relationships could be calculated from dose-response effects reported in

litterature, but there is not enough data sets with enough quality to allow such

comparisons (Puzyn et al., 2009). Fourches et al. (2010) used datasets from previous

studies and constructed a QSAR model which had an external prediction of 73% and an

R2 of 0.72 for regression modelling (Fourches et al., 2010). Models like these could be

beneficial for the design of ENPs which have desired properties without toxic effects.

Bello et al. (2009) did not construct any QSARs, but did investigate the potential of

various ENPs in causing biological oxidative damage, based on the materials ferric

reducing ability of serum in an in vitro assay. When examining the data they found that

93% of the oxidative damage in serum could be explained by a mathematical model

which took into account the combination of two factors: specific surface area and the

level of transition metal content (Bello et al., 2009).

Lanone et al. (2009) examined the effects of 24 nanoparticles on two human cell lines

using two cytotoxicity screening methods (MTT and Neutral Red). The most toxic

nanoparticles according to their experiments were copper and zinc nanoparticles, while

titanium and cerium particles were less toxic. No correlation between size or specific

surface are and the toxocity of the particles was found (Lanone et al., 2009).

Summary and research outlooksLooking at the examples described in the previous section, it is clear that there is not a

single uniting factor that explains the toxicological properties of ENPs. Size, shape,

chemical compostion or surface functionalization - neither of these factors alone can

account for ENP toxicity. Carbon comes in many allotropes which all have different

properties. A nanosized fragment of carbon is not likely to have the same biological

properties as a fullerene or a single-walled carbon nanotubes. Taking carbon nanotubes

16

as an example, it is getting more plausible that these ENPs should be regarded as

distinct chemical entities. A future research niche could be systematic data-mining of

structure-activity or cause-and-effect relationships of ENPs, in order to find a useful

predictor of biological activity. According to the literature, few studies on the subject of

”nano-QSAR” exist (Sayes and Ivanov, 2010)thus warranting further research in this

area.

17

Mechanisms of toxicity

This chapter aims to give a brief overview on the mechanisms by which ENPs cause

toxic effects. The exact mechanism are not clearly understood, and neither is the

knowledge on which step occurs first. However, parallels can be drawn to the effects of

ultrafine particles (Oberdorster et al., 2005).

Oxidative stressThe most commonly accepted theory on the toxic mechanisms of ENPs is about

oxidative stress. Oxidative stress occurs when there is an imbalance between the

antioxidant system inside the cell and the levels of reactive oxygen species (ROS). The

antioxidants inside the cell quench the free radicals under normal conditions to keep the

cellular homeostasis. However, when depleted of antioxidants or exposed to a substance

or particle that can induce ROS, the cell will be affected. The ROS can interact with

proteins and DNA which causes protein malfunction or DNA damage. This in turn

causes cellular processes to dysfunction and leads to cytotoxicity. The ROS not only

cause direct effects on the cellular machinery, but can also induce the expression of pro-

inflammatory proteins. Pro-inflammatory proteins cause the cells of the immune system

to relocate from the circulation to the site where pro-inflammatory proteins are released.

This is prominent for alveolar macrophages which are exposed to ROS-inducing

particles and is a process that is believed to be involved in the response to inhaled

carbon nanotubes (Mocan et al., 2010).

The cytotoxicity of carbon nanotubes is commonly considered to be due to the

formation of ROS, which cause oxidative stress and cellular malfunction. The reason for

the formation of ROS is but metal contaminants from the production process have been

suggested as a plausible cause. The fibre like structure has also been suggested to

participate in the toxic response of macrophages, with analogies to asbestos and quartz

frequently made (Donaldson et al., 2010).

18

Frustrated phagocytosisParticles which have an aerodynamic diameter small enough to allow them to deposit in

the alveoli will eventually encounter alveolar macrophages. The function of the

macrophages is to clear particles from the lungs by chemical breakdown followed by

clearance via the mucociliary elevator in the airways. If an instilled particle is too stiff

and too long, the macrophages will be unable to engulf the particle totally, leading to

the phenomena known as frustrated phagocytosis (Brown et al., 2007).A macrophage

undergoing frustrated phagocytosis will produce pro-inflammatory cytokines which

lead to recruitment of other immune cells such as neutrophils. These recruited immune

cells can in turn potentially cause oxidative stress in the tissue.

An additional toxic effect of carbon nanotubes is that of macrophage functional

impairment. It has been showed in vitro and in vivo that exposure of macrophages to

carbon nanotubes can affect their phagocytic activity. In vitro exposure of macrophages

to SWCNTs can effectively impair their engulfment of apoptotic cells, which under

normal conditions is an important function of macrophages (Witasp et al., 2009).In vivo

airway exposure of mice to SWCNT followed the bacteria Listeria monocytogenes led

to an attenuated macrophage phagocytosis of bacteria. This in turn led to a greater

pulmonary inflammation and less clearance of bacteria in the lungs (Shvedova et al.,

2008). These effects on macrophages indicate that ENP exposure can cause negative

effects on other biological functions, without being directly cytotoxic on their own. It

has been shown that MWCNTs can cause physical damage to the cell membrane of

macrophages. Apart from causing cytotoxic effects in the cells, the carbon nanotubes

were found to have adsorbed numerous proteins including the macrophage receptor

MARCO. Experiments indicated that binding to MARCO could cause effects on the cell

membrane, eventually leading to its disruption (Hirano et al., 2008).

19

Effects of ENPs on reproduction and fetal development

The knowledge on the effects of ENPs on reproduction is limited. A major task is to

determine relevant doses for exposure in animal models. The following section

summarizes some results from animal studies using various ENPs, including carbon

nanotubes and TiO2.

Repeated administration of carbon nanotubes to rats by intra-scrotal injections led to a

reversible damage of the testis, but no effect on fertility or on health effects of offspring

(Bai et al., 2010). Studies on developmental effects of carbon nanotubes on zebra fish

embryos have been done. Low concentrations of MWCNTs caused offspring defects,

while cell death, embryonal death and hatching delay were observed for higher

concentrations (Asharani et al., 2008). In similar studies using concentrations within the

same span no effects on embryonal development were seen, however, the fertility of the

second generation of zebra fish was compromised by an unknown mechanism (Cheng et

al., 2009; Cheng et al., 2007). The passage of ENPs from the mother to the fetus has

been poorly studied. Some ex vivo studies on human placentas have been carried out. In

one of the studies, human placentas were perfused with fluorescently labeled

polystyrene beads. The diameter of the beads ranged from 50 to 500 nm. Particles with

sizes from 50 up to 250 nm were taken up and were able to cross the placental barrier,

however the viability of the placenta was not affected (Wick et al., 2010). In another

study human placentas were perfused ex vivo with nanosized PEG-ylated gold particles

with a diameter of 10-30 nm. The experiments did not reveal any transfer of gold

nanoparticles from the maternal circulation to the fetus (Myllynen et al., 2008). The

effect of TiO2 nanoparticles on the development of pups was investigated by

subcutaneous injection of TiO2 on pregnant mice. The TiO2 particles used were 20-100

nm in their pure form, but after dissolution into saline solution containing the detergent

Tween-80 the majority of the particles were aggregated. The aggregates could be

divided into either a 27 nm category or a 2,429 nm sized one. The exposure to these

TiO2 particles resulted in an increase in dopamine (DA) and its metabolites in pups from

mothers who were injected with TiO2 (Takahashi et al., 2010). Results like these

20

indicate that certain kinds of ENPs could influence the development of the nervous

system, but it should be remembered that further results are needed and the likelihood of

the exposure level be determined. Subcutaneous injection of 0.1 mg of TiO2

nanoparticles (anatase, 25-70 nm in diameter) on pregnant mice led to significant effects

on their offspring and prenatal-transfer of nanoparticles from the mothers to their pups.

The male offspring were found to have TiO2 nanoparticles in their testis and their brains

and also a reduced sperm production. Cells with apoptotic biomarkers were seen in the

olfactory bulb and brain (Takeda et al., 2009). It would thus seem that the effects of

ENPs are dependent on material composition as well as size and agglomeration in their

abilities to cause effects.

21

Routes of exposure

Airway exposureThe effects on human airway exposure to ENPs are difficult to assess, as there is limited

data available. On the other hand, there is much literature available on exposure to ultra-

fine particles originating from combustion sources.

Nickel is a metal which has been associated with negative health effects upon exposure.

Addition of nickel nanoparticles significantly increases the compressive strength of

concrete (Guskos et al., 2010). Thus it is plausible that humans will be exposed to

nickel ENPs in the future. One case study on nickel nanoparticles exist. Post-mortem

analysis was done 16 years after the time of decease on a male worker who died by

adult respiratory distress syndrome in 1994 after exposure to an aerosol of nickel

nanoparticles. Electron microscopic evaluation of lung tissue revealed <25 nm in

diameter nickel nanoparticles in lung macrophages and his urine contained high levels

of nickel. There was also tubular necrosis seen in the kidneys. The results indicate that

the nanoparticles were able to translocate from the airways into the circulation.

Assessment of the occupational hygiene showed that the concentration of particles

where the subject was working was 382 mg/m3 and that the subject would have inhaled

approximately 1 g particles during a 90 min operation. The subject was reported to

having removed his protective face mask during operation (Phillips et al., 2010).

Dermal exposureThe effects of ENPs on skin, both human and in animal models are unclear. ENPs, such

as TiO2 and ZnO particles, are used in cosmetics and in sun screen. Thus dermal

exposure to certain ENPs is very likely to occur, but the extent of particle penetration

seems judging from available literature to be dependent on particle type, size and

exposure scenario. One of the most probable scenarios is exposure by sunscreens or

cosmetics.

22

A literature survey by Newman et al. (2009) examined the effects of TiO2 and ZnO

sunscreens as reported in 15 peer-reviewed investigations (Newman et al., 2009).

Experiments were carried out on human or pigs skin in vitro. For TiO2, none of the

investigations showed any significant particle translocation below the stratum corneum,

which is the outermost skin layer also known as the horny layer. ZnO nanoparticles

were found to penetrate a little, at levels less than 1% of the applied dose. Combinations

of TiO2 and ZnO did not penetrate significantly either.

Contrasting results have been seen when nude mice and pigs were topically exposed to

TiO2 particles ranging from 4 to 90 nm in size. Repeated exposure of pigs for 30 days

showed that both 4 nm (hydrophobic anatase) and 60 nm (hydrophobic rutile) TiO2

nanoparticles were able to cross the stratum corneum and recovered in the lower

epidermis. A 60 day repeated exposure of nude mice to TiO2 nanoparticles resulted in

uptake of titanium in major internal organs, including brain. Pathological changes were

also observed in organs, especially the skin and the liver which showed indications of

oxidative stress (Wu et al., 2009).

An Australian research team investigated whether or not zinc or zinc oxide could be

released from a sunscreen preparation after dermal application. The researchers

prepared two formulations, one containing 19 nm sized ZnO particles and one

containing >100 nm sized ZnO particles. Both particle types contained radioactive 68Zn

as a tracer. The subjects were then exposed to natural sunlight and were free to conduct

activities during the day. After dermal exposure it was found that the majority of the Zn

was not absorbed by the skin, as determined by measuring radioactivity in urine and

blood. A higher proportion of Zn was seen in females exposed to the 19 nm sized

particles, as compared to males receiving the same treatment and as compared to both

genders treated with >100 nm sized particles. The absorbed dose was 0.001% of the

dose applied, which is very low, but still indicative that metal ions can be absorbed. The

authors were not able to determine if the absorbed Zn was taken up as free Zn ions or as

ZnO particles (Gulson et al., 2010).

23

One study on human dermal exposure to carbon nanotubes was made in 2001(Huczko

and Lange, 2001). In this study, 40 volunteers were subjected to a patch that had been

soaked in a carbon soot containing a high proportion of CNTs. The results from this

study were negative, with no visible irritation or skin changes. The main critique of this

study is that the authors did not quantify the amount of CNTs in the soot, which makes

comparison with other studies impossible. CNTs are known to induce reactive oxygen

species and inflammation in animal models of skin irritation. In the human study no

investigations were made on skin thickness, inflammatory markers or on oxidative

stress.

Quantum dots have been assayed for their ability to cross pig skin. In one study

researchers found that QD621, a nail-shaped cadmium/selenide core quantum dot with a

cadmium sulfide shell coated with PEG, was unable to cross isolated pig skin but

induced cell death in cultured human keratinocytes. This could mean that if quantum

dots cross injured skin, they could possibly exert toxic effects on humans (Zhang et al.,

2008). The likelihood of skin exposure to quantum dots is however rather limited as

compared to TiO2 nanoparticles for instance where exposure is intentional.

Oral exposureThe effects of oral exposure of ENPs have not been well studied. Most articles within

this area are related to drug delivery and few have examined the acute oral toxicity of

ENPs. Yang et al. (2010) synthesised ultra-short SWCNTs and delivered them orally to

mice in order to investigate their therapeutic potential for treatment of Alzheimers

disease (Yang et al., 2010). They found that the ultra-short SWCNTs could translocate

from the gastrointestinal tract to the blood stream and be taken up into neurons in the

brain. Similar studies were carried out by Kolosnjaj-Tabi et al. (2010) where mice

where fed oral doses up to 1000 mg/kg b.w. without exhibiting any adverse effects

(Kolosnjaj-Tabi et al., 2010). In contrast to this, Folkmann et al. (2009) found oxidative

damage to DNA in liver and lungs after a single oral dose of 0.064 mg/kg b.w. of

SWCNTs (Folkmann et al., 2009).

24

TiO2 ENPs have been thoroughly studied in dermal exposure experiments but not to the

same extent after oral delivery. Wang et al. investigated the uptake of 25 and 80 nm

sized TiO2 following treatment with 5 g/kg in mice and found translocation to major

organs two weeks after exposure (Wang et al., 2007). Liver inflammation and increased

serum levels of biomarkers for cardiac injury were also observed. A reservation to the

relevance of the study was raised by Johnston et al., as the dose of 5 g/kg b.w. is

extremely high and would not likely reflect that of any human exposure to nanosized

TiO2 (Johnston et al., 2009). Repeated oral dosage during 14 days of silver

nanoparticles on the size range of 22-71 nm at a dose of 1 mg/kg b.w. to mice led to

translocation of silver nanoparticles to brain, lung, liver, kidney and other organs.

Larger particles bearing a size of 323 nm did not translocate from the gut to organs

under investigation. Silver nanoparticles with a diameter of 42 nm at repeated doses

from 0.25 -1 mg /kg b.w. during 28 days led to negative effects on kidney and liver in

mice (Park et al. 2010). A similar study for 28 days using Sprague-Dawley rats given

an oral dose of 30, 300 or 1000 mg/kg b.w. of 60 nm sized silver nanoparticles indicated

in contrast that doses over 300 mg/kg b.w. resulted in slight liver damage (Kim et al.,

2008).

Summary and future research needsThere is a debate going on as whether or not ENPs are able to cross the dermal barrier.

As it is unlikely that the majority of ENPs would be able to cross the barrier due to their

size, it is possible that small effects of ENP exposure in the short term could have larger

impact in the long term. The importance of conducting repeated exposure experiments

should be stressed, simply giving a high single dose (bolus dose) does not mimic effects

of long term chronic or subchronic exposure. Secondly, crossing of particles may not be

the most important toxic impact; release of metal ions or contaminants which can

migrate across the dermal barrier may have equally important effects. Local reactions in

the skin such as oxidative stress could potentially also have effects, e.g. carbon

nanotube treatment has been shown to cause oxidative stress in cultured skin (Murray et

al., 2009). Such investigations are however scarce.

25

Human exposure

There is an immense gap in the knowledge of how ENPs affect human health. There are

three major routes of exposure and uptake of ENPs. The first is inhalation, the second is

by ingestion and the third is dermal uptake. A fourth, but less explored exposure route is

uptake by the olfactory nerve (Oberdorster et al., 2005). Inhalation can be considered

the major route of exposure, as ENPs are likely to become airborne. Ingestion of ENPs

could occur if food is contaminated e.g from food packaging containing ENPs or by

swallowing of ENP containing mucus from the mucociliary elevator. Dermal exposure

is not likely to be a major route of exposure, as the stratum corneum of the epidermis is

considered and effective diffusion barrier to particulate material. The sweat glands

could however pose a port of entry in certain cases (Crosera et al., 2009).

Workplace emission of ENPsStudies on the exposure of workers towards ENPs have been reported in a few cases.

Measurements on carbon nanotubes exposure levels have been reported both for

SWCNT and MWCNTs, both at manufacturing sites and in laboratories were carbon

nanotubes were handled. Maynard et al. (2004) did some of the first measurements on

workplace exposure to carbon nanotubes. They examined exposure to SWCNTs at four

different production sites by personal sampling for 30 min. The exposure was found to

be between 0.7 and 53µg/m3. Analysis of particle morphology showed a compact

structure. Dermal exposure was assayed by examination of protective gloves used by

the workers. Approximately 0.2-6 mg was deposited per glove (Maynard et al., 2004).

Determining dermal exposure is important as it allows for an extrapolation to

experimental dermal exposure in animal models or in cell culture.

Consumer exposureLittle attention has been put to quantify the consumer exposure to ENPs. The studies

that do exist have examined effects of sun screens, looked at release of silver from silver

nanoparticle treated garments and utensils and in one case the release of aerosolised

TiO2 from a bathroom cleaning agent. Release of silver from fabrics has been examined

following washing of ENP endowed fabrics. This was done by first examining the

26

dissolution of silver ENPs used for fabric treatment, followed by actual washing of the

fabrics. It was found that the released silver varied from 1.3 to 35% of the original silver

in the fabric and was dependent on the type of binder system (Geranio et al., 2009).

Chen et al (2010) showed in an experiment conducted at NIOSH facilities, that a

commercial spray can product containing TiO2 produced respirable nanoparticles (Chen

et al., 2010). The intended usage of the spray was cleaning and sanitizing bathrooms.

The nanoparticle fraction was 170 µg/m3, equivalent to1.2×105 particles/cm3, with a

mean particle diameter of 75 nm. The researchers estimated the dose to 0.075 µg TiO2

per m2 alveolar epithelium and minute. Levels for rats were determined to 0.03 µg. This

was one of the first reports to show that a commercial product can create respirable

nanoparticles and would serve as a basis for further toxicological studies on respirable

TiO2 particles. Similar studies on Ag-nanoparticles released from consumer spray

products were done by Hagendorfer et al. who compared pump spray and gas spray

formulations in an experimental setup using a scanning mobility particle sizer in

combination with TEM analysis. The pump spray did not cause any measurable release

of Ag nanoparticles, while the gas driven spray caused significant release of Ag

nanoparticles (Hagendorfer et al., 2010). Thus, different formulations in consumer

products can cause different exposures.

In order to better assess any risk associated with exposure to ENPs, the release of ENPs

from composites, fabrics, paints etc should be determined. For consumer products, such

as fabrics, very little is known. Kulthong et al. examined the presence, release and

antibacterial effects of commercial and laboratory prepared nanosilver containing

fabrics. The authors found that some fabrics claiming to contain nanosilver, contained

no silver at all. The efficacy of fabrics to exert antibacterial effects varied from 0% to

more than 99%. Using an artificial sweat preparation, as to mimic a real exposure

situation, the release of silver was determined to vary from 0 mg/kg to 322 mg/kg of

fabric, depending on pH and fabric (Kulthong et al., 2010). Friends of the Earth issued a

consumer report in 2009 entitled “Manufactured Nanomaterials and Sunscreens: Top

Reasons for Precaution”, where the concern for nanosized particles in consumer

products was raised. However the references made in the report on nanoparticle

translocation across the skin did not concern TiO2 or ZnO nanoparticles, but rather

quantum dots. This is an example which shows the problem in comparing biological

27

effects of nanosized particles without taking into account the differences in particle

physicochemical properties (Friends of the Earth, 2009).

Biomarkers of exposure to ENPsIn order to assess if clinical signs of a patient are related to ENP exposure, the

development of biomarker assays are of high relevance for nanosafety. Few studies

have been conducted in this area at the moment. Higashisaka et al. examined the effects

of commercial silica ENPs with diameters ranging from 30 to 1000 nm and one

functionalized silica ENP bearing either carboxyl and amino groups. (Higashisaka et al.,

2011). Particles were intravenously injected in mice and made a proteomic profiling of

the plasma proteins. This resulted in significantly higher plasma levels of haptoglobulin

for mice treated with 30 or 70 nm particles, while not for those treated with larger

diameter particles. The levels were significantly higher up to 3 days after injection. The

proteins c-reactive protein (CRP) and serum amyloid A (SAA) also increased in a

similar manner. In contrast, he functionalized particles did not elicit a significant

increase of haptoglobulin. Further, 30 nm particles which were intranasally instilled in

mice led to significant increases in the plasma levels in all three proteins, while the

larger 70 nm particles did not cause any effect. These results indicate that a combination

of these three putative markers could be used for assessing exposure, but it should be

borne in mind that other diseases and afflictions could cause similar increases.

Expressed in renal carcinoma (ERC)/mesothelin is a protein that can be used as

biomarker for mesotheliomas in humans. Sakamoto et al recently showed that it can also

be used as a biomarker for mesothelial proliferative lesions in rats after intrascrotal

injection of multi-walled carbon nanotubes (Sakamoto et al., 2010).

Summary and future aspectsIt is clear that exposure to ENPs can occur in the working environment, but that

protective clothing and devices are capable of limiting the personal exposure. Different

operations during work will evidently lead to an extremely high exposure, such as

packing and transfer of ENPs.

28

29

Effects of long term exposure to ENPsAs could be expected, long term data on ENP exposure is scarce. Following a single

intraperitoneal injection of carbon nanotubes, rats were studied for a period of two

years. The aim of the study was to investigate the carcinogenicity of carbon nanotubes,

e.g. if they can cause mesothelioma. The results from the study were negative but not

conclusive as the length of the tubes might not have been sufficiently long in order to

qualify for the fibre definition and was not comparable to that of cancerogenous

asbestos fibres (Muller et al., 2009). Only two studies on repeated long-term pulmonary

exposure to carbon nanotubes exist so far, one by Ma-Hock et al. (Ma-Hock et al.,

2009) and one by Pauhlun (Pauluhn, 2010). Both studies investigated repeated exposure

for MWCNTs on rats during a 90 day period. Both studies found inflammatory cells in

the lungs and different degrees of lung fibrosis. However, neither of the studies found

any systemic effects nor any signs of mesothelioma in the airways. Ma-Hock et al. (Ma-

Hock et al., 2009) used well dispersed MWCNTs, while the MWCNTs used by Pauhlun

(Pauluhn, 2010) were never characterised as individual tubes but rather found as

agglomerates both during aerosolisation and after recovery from the lungs of the

experimental animals. Pauhlun (Pauluhn, 2010) concluded that the toxicity was not due

to the fibre-like characteristics of single MWCNTs but instead due to particle

overloading. The lowest concentration that gave a biological response was that of Ma-

Hock et al. where as little as 0.2-0.3 mg/kg b.w. caused an inflammatory response (Ma-

Hock et al., 2009)

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New technologies for ENP detection

A major difficulty in nanotoxicology and nanosafety is to detect and image

nanoparticles in biological matrices. A biological matrix may be a tissue from an animal

or a plant, a sample of earth or a food specimen. Detection of nanoparticles is usually

done by using electron microscopic techniques, such as TEM or SEM. Fluorescence

microscopy, e.g. confocal microscopy, can be employed if the particles can be excited

using an appropriate wavelength of light. Quantum dots can be detected using

fluorescence microscopy and have found wide applications in bioimaging, either as free

particles or used to label antibodies and other biomolecules. Other nanoparticles can be

detected by labelling with a suitable fluorophore. One drawback of such labelling is that

the addition of a fluorophore may influence the biological activity of a nanoparticle. The

same is true if a nanoparticle is functionalized with a chelating molecule and then

labelled with a radioactive isotope, e.g. In111 (Singh et al., 2006), which may cause the

particle to behave differently from a pristine nanoparticle. Carbon nanotubes have been

detected in animals by radioactive labelling, by TEM and by elemental analysis. As

carbon nanotubes quite often contain detectable amounts of trace metals such as Ni and

Co, the presence of these metals in tissues are indicative of the presence of carbon

nanotubes. Using a combination of both TEM and elemental analysis (STEM-EDX) can

allow more precise detection and validation of ENP presence in a biological matrix, e.g.

carbon nanotubes instilled in a rat lung (Elgrabli et al., 2008).

Another issue is to characterize interactions between nanoparticles and biomolecules in

situ, that is directly in the tissue or biological matrix under investigation without any

isolation steps. Techniques for doing this include soft x-ray microscopy and related

synchrotron radiation techniques (Wang et al., 2010a). Pascolo et al (2011) used

synchrotron soft x-ray imaging to study asbestos in a sample of human lung tissue

(Pascolo et al., 2011). The method allowed the authors to chemically dissect the

asbestos bodies found in the samples and they found that levels of silicon were highest

in the asbestos particles, while the shell of the fibre and the closest surrounding tissue

were higher in Fe, Mg and O. Based on these findings and other experiments, they

31

could conclude that both Mg and Fe were part of the mechanisms which lead to

formation of asbestos bodies. Although the particles under investigation were not ENPs

and not nanosized, the methodology describe could be implemented when investigating

tissue reaction to ENPs and the mechanisms of how different ENPs induce toxicity on

an elemental level.

32

Nanosafety and food technology

There has been an increased interest for applications of nanotechnology in the food

industry in recent years. The applications of nanotechnology, i.e. nanoparticles, in food

technology include food safety such as packaging, sensors for detection of pathogens or

food spoilage, encapsulation of flavours or nutrients in nutraceutical applications, in

novel additives and during food production (Bouwmeester et al., 2009; Das et al., 2009;

Restuccia et al., 2010). The use of nanoparticles in the food industry is clearly not only

an issue for occupational hygiene, but also an issue for consumer safety.

Using nanoparticles in food applications has been judged to pose a likely risk for human

exposure (Bouwmeester et al., 2009). In order to assess such risks for exposure, there is

a need to develop techniques to find and characterize ENPs in food. Technologies for

assessing nanoparticle contents in food include electron microscopy and x-ray

microscopy used in conjunction with separation techniques such as field-flow

fractionation and chromatography. These techniques are described with in-depth detail

by Blasco and Picó (Blasco and Pico, 2011) which also includes an extensive list of

products on the market which allegedly incorporates nanoparticle technology.

Food applications of ENPs are also an important part of life cycle analysis for ENPs as

nanotechnology does evidently come into many of the different steps in food

productions. In agriculture ENPs can have applications on water and soil cleaning. In

food processing nano-coatings and sieves can be used to prevent biofouling and to filter

bacteria. Applications in packaging serve to prevent bacterial growth by nanostructured

coatings for instance (Bouwmeester et al., 2009). Examples of coatings are silicate

ENPs, composites, Ag and ZnO ENPs. Nanoparticles could also be used to improve

bioavailability of substances of nutritional value, in a similar way to that of nanoparticle

based drug delivery (Bouwmeester et al., 2009). Examples of ENPs used in or

suggested for use in food applications are metal/metal-oxide ENPs are Ag, ZnO, Cu and

TiO2, as well as nanosized lipid and polymer based delivery systems. The two latter

types of nanoparticles are intended to degrade upon consumption and release factors

33

with nutritional benefits. Ag-nanoparticles have been incorporated into foams and used

as filters to reduce bacteria (Jain and Pradeep, 2005).

The toxicological effects of nanoparticles employed in food technology are not well

characterized. The genotoxicity of chitosan-poly(methyl methacrylate) (PMMA)

composite nanoparticles for use in food packaging films in order to improve food

preservation was examined by de Lima et al (De Lima et al., 2010). Chitosan is

accepted for use as an additive in food in some countries, including Japan, and has

applications in the pharmaceutical industry (Illum, 1998). The particles used in the

study were 60, 82 and 111 nm in diameter and concentrations ranged from 1.8, 18 and

180 mg/ml. No DNA damage was seen, but 82 and 111 nm particles reduced the mitotic

index at the highest concentration. The smallest particles, with a diameter of 60 nm did

not cause any significant effects on gentoxicity. These results suggest that if chitosan-

composite nanoparticles are released from food packaging, the genotoxic effects are

limited (De Lima et al., 2010) However, studies such as these should be complemented

with animal experiments.

Chorianopoulos et al. applied nanosized TiO2 particles to metal and glass surfaces as a

means of photosterilization. They found that Listeria monocytogenes biofilms were

significantly reduced on surfaces that had been coated with TiO2 nanoparticles and then

subjected to UV-A radiation. The authors proposed that such coatings could be used to

improve food safety with an antimicrobial perspective (Chorianopoulos et al., 2011).

The potential release of nanoparticles from such coatings should be studied from the

nanosafety perspective, since release of TiO2 nanoparticles could potentially influence

human health. The biological effects of TiO2 nanoparticles after oral intake are very

limited and the studies that exist have mainly shown negative effects on internal organs

(e.g. liver and kidneys) at doses of 1 or 5 g/kg body weight in rats and mice

respectively (Bu et al., 2010; Wang et al., 2007) Treatment with lower doses such as

0.16 and 0.4 g/kg bodyweight led to increases in serum biomarkers of liver injury and

cytotoxicity markers (Bu et al., 2010).

34

Other issues in food nanosafety that need to be addressed include whether or not ENPs

used in food production and processing could carry unintended biomolecules with them

on their passage from the gut lumen into the body (Das et al., 2009).

Summary, knowledge gaps and research needsAccording to Bouwmeester et al. (Bouwmeester et al., 2009) important issues in food

nanosafety include development of analytical tools for detection of ENPs, studies on

oral uptake and behaviour of ENPs related to food production and identification of

products on the market which may contain ENPs as well as setting up policies and

regulations on this matter. As a consequence of the lack of knowledge, the European

Union issued two regulations in 2004 and 2009 which address some of the problems of

food nanosafety and food packaging specifically (Restuccia et al., 2010). Not all

nanoparticles or nanosized materials used in food technology are associated with

possible toxic risks. Nanoparticles made from the maize protein zein have found

applications in food packaging and as carriers of flavouring and dietary supplements

(Srinivas et al., 2010).

35

Environmental and ecotoxicogical aspects of nanosafety

The majority of research within nanotoxicology is focused on cytotoxicity and animal

studies. The impact of nanomaterials and specifically engineered nanoparticles on the

environment and populations needs to be assessed with similar effort. The antimicrobial

effects of certain nanoparticles, e.g. silver ENPs, which are desirable when applied to

kitchenware, utensils and to clothes may have totally undesirable effects on microbes in

an ecosystem. The propagation and accumulation of ENPs through food chains/food

webs are of an environmental concern. The effect on plants is largely unknown, a Web

of Science search on “plant uptake of nanomaterials gives only 50 hits.

Modelling of the possible adverse effects of three different kinds of nanoparticles was

made by Muelller and Nowack (2008). They made risk assessments based on two

scenarios one realistic and one high-exposure worst case scenario. Predictions were

made for Ag NP, TiO2 NP and carbon nanotubes (both SWCNTs and MWCNTs), based

on litterature findings of production, use and flows of nanoparticles. They found that the

risk quotient, that is the predicted environmental concentration in relation to the

predicted no effect concentrations, for Ag NP and carbon nanotubes was less than one

which suggest that there would be no reason to expect any adverse effects from these

particles. On the other hand, values for TiO2 NP indicated that the effects of such

particles on the environment should be further studied. As this is only a model, the

results must be interpreted with caution. This particular model was based on

circumstances specific for Switzerland. This meant that flows from sewage treatment

plants to landfills and soil was excluded, as waste sludge application to soil in

Switzerland is forbidden and the sludge is incinerated.

Adsorption of biological materials to ENPsJust as the adsorption of proteins to ENPs in animals or cell culture forms a protein

corona which determines their effects, ENPs in the ecosystem will also associate with

other materials. Carbon nanotubes are considered a reactive carbonaceous material and

concerns have been raised on their impact on both humans and environment. Studies

36

were made on oxidized carbon nanotubes which had been stored in fresh water for 2.5-7

years and in PBS or natural organic matter (NOM) for up to 120 days. The purpose of

this treatment was to investigate how environmental parameters could affect toxicity. It

was found that storage in fresh water did not affect the toxicity of the nanotubes, they

were still as toxic as freshly pepared CNTs. In contrast, when nanotubes were stored in

PBS or NOM, the toxicity was reduced to control levels (Panessa-Warren et al., 2009).

Investigations like these provide useful information on how to reduce biological impact

of ENPs.

In a meta-analysis of available literature, Kahru and Dubourguier investigated the LC50

and EC50 (L(E)C50) values reported for ENPs in the available literature. The ENPs

included the traditional spectra e.g. C60 fullerenes, carbon nanotubes, TiO2 and Ag.

They found 77 reported values and arranged the nanoparticles under investigation

according to their median L(E)C50 on relevant species in representative food chains.

From their analysis made on this basis they concluded that the most harmful ENPs

would be nanosized Ag and ZnO with L(E)C50 less than 0.1 mg/l, followed by C60,

CuO, SWCNT, MWCNT and TiO2 in that order of decreasing harmfulness. Nanosized

Ag and ZnO were considered as “extremely toxic” (Kahru and Dubourguier, 2010)

This type of meta-analysis is useful as it provides a basis for assessment of possible

ecological impact and ecotoxicity of different ENPs. However, it should be noted that

merely using mass as a dose-metric when comparing the toxicity of different ENPs can

give a false impression of the relative toxicity of particles. Much indicates that it is not

that simple (Warheit, 2010).

In order to handle the impact of ENPs on society and the environment in the near future,

nanowaste management must be considered since waste is thought be an important

source of environmental contamination and dissipation of ENPs (Musee, 2011). The

environmental impact of nanowaste does not necessarily implicate that the ENP cause a

direct effect on organisms and populations, there is also a possibility that the ENPs

carry with them other pollutants that cause biological effects in an unexpected manner

as compared to that of the pollutant only.

37

It has been shown that polycyclic aromatic hydrocarbons (PAH) can be absorbed by

ENPs. Yang et al. (Yang et al., 2006) showed that SWCNTs, MWCNTs and fullerenes

could adsorb PAHs including pyrene and naphtalene. Carbon nanotubes were potent

adsorbers of PAHs which could lead to environmental effects not only depending on the

inherent toxicity of the carbon nanotubes but also due to adsorbed pollutants. The same

authors presented data in a subsequent publication in 2007, where they showed that

adsorbed PAH could desorb from carbon nanotubes and fullerenes in water. The authors

suggested that this could lead to the release of PAHs should an organism be exposed to

carbon nanomaterials carrying PAH pollutants (Yang and Xing, 2007). The adsorption

of substances such as pollutants to carbon nanomaterials does not necessarily have to be

a negative event, and has been proposed to be something that could be exploited in

order to remove pollutants from aqueous environments or from gas phase (Ren et al.,

2010). The most prominent carbon nanomaterials described as having adsorbent

properties are carbon nanotubes, which according to the review are able to bind divalent

heavy metals including Cd and Pb and aromatic compounds.

Effects of ENPs on plants and crops The impact of ENPs on plant growth is not well characterized. Future contamination of

plants and crops with ENPs is a scenario with increasing probability, with ENPs

released into the atmosphere or into ground water. Applications of nanomaterials and

nanoparticles in agriculture are being found, e.g. as pesticides or as carriers of

pesticides. A recent review of ENPs and their interactions can be found in Ma et al.

(2010). A study was conducted on deposition and uptake of cerium dioxide (CeO2)

nanoparticles in maize plants. CeO2 nanoparticles were generated using flame spray

pyrolysis, which led to nanoparticles with a mean size of 37 nm and a surface of 110

m2/g. Exposure of maize plants was done by aerosol generation in a controlled

environment or by irrigation of plants with water containing CeO2 nanoparticles. The

CeO2 nanoparticles were deposited onto the leaves of the maize plants, and significantly

more when plants were exposed to light. Significant amounts of CeO2 nanoparticles

were adsorbed to the leaves, as evident after washing the leaves. However, no

translocation of nanoparticles was observed after aerosol exposure. The irrigation

38

experiments indicated no uptake or accumulation of nanoparticles and most of the CeO2

was detected on the surface of the soil. This suggested that the soil functioned as a

filtration mechanism of nanoparticles, and all together the results implicated that plants

were more resilient to nanoparticle exposure and uptake as compared to mammalian

systems (Birbaum et al., 2010). When soybeans exposed to CeO2 and ZnO nanoparticles

(7 nm and 8 nm in size), slightly different results were seen. The effect on seed

germination and growth, as well as uptake of nanoparticles was investigated. Neither of

the particles affected germination but genotoxic effects of CeO2 nanoparticles was

observed. Elemental analysis of plant roots revealed that CeO2 nanoparticles were taken

up by the plants while ZnO nanoparticles were not (Lopez-Moreno et al., 2010).

Tomato seed treated with carbon nanotubes during germination were observed to

exhibit increased rate of germination and plant growth with increasing concentrations of

carbon nanotubes, without any observable negative effects on plant integrity. The

authors of the study suggested that the observed phenomena could be exploited in

agricultural application. The proposed mechanism was that carbon nanotubes were able

to penetrate the seeds and thus increased the uptake of water into the seeds

(Khodakovskaya et al., 2009). Should nanoparticles like as carbon nanotubes find their

way into agricultural and horticultural applications, the risk of human or animal

exposure will surely increase and studying the effects of oral administration of ENPs

will become more urgent.

Food chain transfer of ENPs and biomagnificationJudy et al. (Judy et al., 2011) showed in an experimental set up that gold nanoparticles

in the size range of 5-15 nm were able to accumulate in a terrestrial food chain, from a

primary producer to a primary consumer. Tobacco plants, Nicotiana tabacum, were

seeded and after 4 weeks placed in test tubes with 100 mg/ml of gold nanoparticles.

Hornworms, the larvae of Manduca sexta, were then allowed to grace on plants

previously exposed to gold nanoparticles. Gold could be detected in dried tobacco

leaves and in the gut of the hornworms. Calculations showed that total gold (bulk gold)

accumulated in the hornworms relative to the tobacco leaves and that 10 and 15 nm gold

particle treatments led to higher bulk gold accumulation in the worms. The total number

of gold nanoparticles which accumulated in the hornworms did not depend on the

39

particle size, and the number of particles was significantly higher than in the tobacco

leaves (Judy et al., 2011).Similar transfer has been shown for TiO2 nanoparticles in a

simple food chain too, although bioaccumulation of nanoparticles was not observed.

Daphnia magna were exposed to 0.1 or 1 mg/ml of TiO2 nanoparticles with a mean size

of 21 nm. After exposure the D. magna were transferred to tanks with zebra fish, Danio

rerio which had been trained to eat D. magna. Analysis of the zebra fish and the D.

magna revealed that TiO2 nanoparticles taken up and biomagnified in the D. magna and

transferred to the zebra fish, without biomagnification (Zhu et al., 2010). The uptake of

quantum dots (QD) was studied in an invertebrate food web using bacteria (E. Coli),

ciliates (Tetrahymena pyriformis) and rotifers (Brachionus calyciflorus). The QDs were

ellipsoid, with major axis of 12 nm and a minor axis of 6 nm and had a core made up

from CdSe and a shell of ZnS. Two different surface chemistries were used, one

carboxylated and one biotinylated. Accumulation of QDs in bacteria was not observed,

probably due to the surface chemistry and hydrodynamic diameter of the dots. Ciliates

were found to accumulate QDs with biotinylated QDs being twice as biopersitent as

carboxylated QDs. Transfer of QDs to rotifers which preyed on ciliates was observed,

but without any biomagnification (Holbrook et al., 2008). In another similar simplified

food chain, Pseudomonas aeruginosa bacteria were exposed to CdSe QDs. QDs

accumulated in the bacteria which were then preyed upon by the protozoan

Tetrahymena termophila. Biomagnification of the QDs at a five-fold level were

observed in the protozoan which had ingested bacteria containing QDs. In addition, it

was observed that protozoans had difficulties in digesting QD laden bacteria which

eventually resulted in reduced growth of protozoan. The cellular integrity of the

protozoans remained intact however and the majority of the QDs found inside the

protozoans were intact. The observations of reduced growth indicate that QD could

impact ecosystems at an early producer/consumer level (Werlin et al., 2011).

The biopersistency of ENP is a critical issue when assessing associated risks. Some

ENPs are designed to degrade, e.g. particles intended for drug delivery while for

particles intended for enhancing material properties degradability would not be expected

to be a desired property. Kummerer et al. (Kummerer et al., 2011)investigated the

degradability of various nanoparticles in an aqueous environment. They found that

40

nanocrystals of starch and cellulose were more biodegradable than the corresponding

bulk material, while fullerenes and carbon nanotubes were not biodegradable at all. The

stability of carbon nanotubes was studied by Liu et al (Liu et al., 2010) who found that

surface functionalisation could influence bioperistency. Carbon nanotubes with

carboxyl groups attached to their surface could be degrade over time in simulated

phagolysosomes, while nonfunctionalized tubes were inert to such degradation.

Similarly Kagan et al. showed that carbon nanotubes functionalized with antibody

fragments could be degraded by neutrophiles, a cell type which normally serves

important functions in clearing particulate materials from the lungs (Kagan et al., 2010).

Life cycle assessment of ENPLife cycle assessment or life cycle analysis (LCA) is a technique by which the impact of

a process or product can be assessed. The impact that can be evaluated include

environmental, economical and societal. As an example, the energy expenditure of

methods for producing TiO2 nanoparticles has been examined (Grubb and Bakshi,

2010). A life cycle assessment of socks containing silver nanoparticles suggested that

the user phase of the product, e.g. washing, had a stronger impact than that of the

production phase (Meyer et al., 2011). Healy et al (2008) investigated the environmental

impact of three different carbon nanotube production methods using LCA. When taking

into account both energy expenditure and chemicals employed in the production, the

method which had the least environmental impact under normal production condition

was high-pressure carbon monoxide (HiPco), while if using best case yield conditions

the arc technique had the lowest impact. Köhler et al (2008) investigated two case-

studies where carbon nanotubes were involved. One case was related to the application

of carbon nanotubes in Li-ion batteries, which is a growing area where the use of carbon

nanotubes is expected to grow, and the other related to the application of carbon

nanotubes in textiles. The main purpose of this study was to investigate the points

during a products life cycle where carbon nanotubes could potentially be released. They

found that the knowledge on where release of carbon nanotubes is possible during a

products life cycle is limited and urged further research in the area. For Li-ion batteries,

the nanotubes are part of a matrix which is porous but the matrix is enclosed in casing

which makes release during usage unlikely. For fabrics the nanotubes are bound more

41

strongly to a polymer matrix but wear and tear, such as mechanical stress and exposure

to UV-light could lead to release of nanotubes during usage. The authors suggest that

there is a potential for product and application developers to take into account the

potential release points in the life cycle, especially since there are few products on the

market (which was the case in 2008). Recycling of products is also mentioned as a

possible risk of occupational exposure.

Summary and future research needsThere is evidence that nanoparticles can be transferred from producer to consumer in a

food web/chain, albeit in very simplified models. Data for populations of e.g. mammals

is not available. As the organisms used in these studies, bacteria, protozoa and

hornworms, serve as food for other organism, such as mammals, it is very plausible that

biomagnification on basal levels will lead to detrimental effects on higher levels in a

food chain. The current understanding on the biological effects after consumption of

nanoparticles is limited. All together, results such as this show that further studies on

bioaccumulation/magnification are warranted.

42

Actors within nanosafety and related fields

Sweden

Lund – Malmö

Governmental organisations

Arbetsmiljöverket Malmö

Mats Ryderheim mats.ryderheim@av.se

Lena Lindskog lena.lindskog@av.se

Monica Björk monica.bjork@av.se

Lund institute of technology (LTH)

Dept of Fire Safety Engineering and Systems Safety /LUCRAM

The department of fire safety engineering at LTH are conducting highly competitive

research within risk management. Not currently active within nanosafety at the moment,

but could be of assistance with risk management and policies.

http://www.lucram.lu.se/

Lund University

Biomedial Polymer Technology, Dept of Experimental Medical Science

Researchers Henrik Kempe and Maria Kempe are developing magnetic nanoparticles of

various kinds that could be used for treatment of thrombosis related to implanted stents.

Their experience in particle synthesis and biocompatibility studies could be of

synergetic effects to the NanoSafety cooperation.

http://www.biomedicalpolymers.bmc.lu.se/Index.html

43

MaxLab/ESS

The use of synchrotron radiation in nanotoxicological investigations has been suggested

to be a new and emerging technology. Synchrotron radiation facilities are already

present in Lund and Maxlab IV and ESS will be ready in the future. This technology is

expensive and limited beam line time will be available. Thus, it could be of a strategic

value to realise and assess the potential of this technology for nanosafety on an early

scale. Synchrotron techniques could allow investigations of ENP interaction with

biomolecules in situ in both cells and tissues.

Companies

Genovis

Genovis have developed magnetic nanoparticles, called NIMT FeOdots. The particles

can be used for drug delivery and can be functionalized with a variety of chemical

entities. The particle size is 10 +/- 1 nm. Genovis currently markets their products in

several countries.

www.genovis.com

Camurus

Camurus focuses on drug delivery using FluidCrystal® nanoparticles, for oral, dermal

and injection delivery. The nanoparticles are based on lipid membranes.

www.camurus.com

Entomopharm

Entomopharm is a Danish biotechnology company based at the Bioincubator at

IDEON/BMC. Entomopharm has developed models for drug transport over the blood

brain barrier (BBB) using locusts as an experimental system. This technology could be

useful for screening and modelling how nanoparticles interact with the BBB or with cell

membranes.

www.entomopharm.com

44

SPAGO Imaging AB

Makes contrast agents for MRI detection of tumours by using nanoparticles based on

gadolinium oxide. SPAGO Imaging is based in Lund.

www.spagoimaging.se

Stockholm

Governmental organisations and projects

The Institute of Environmental Medicine

Recruiting three post doc/ PhD students during Autumn 2010, in mathematical

modelling of nanoparticle toxicokinetic behavior.

www.ki.se

NANOMMUNE

An FP7 sponsored cooperation between researchers at KI, UU, KTH in Sweden, with

European partners including EMPA in Switzerland and NIOSH in the US. The focus of

this project, which will finish in 2011, was to elucidate the effects of nanomaterials on

human health with the immunesystem in particular. Specific efforts were put on

material characterization. Coordinated by Prof. Bengt Fadeel at KI.

http://ki.projectcoordinator.net/~NANOMMUNE

The Swedish Defence Research Agency (FOI)

In 2009, FOI received a FORMAS grant in cooperation with Umeå University and

Uppsala University for a project on the health risks on nanoparticles. The focus was on

metal oxide particles. At this time, FOI had been given a total of 8 million SEK in

grants to conduct research within this area.

www.foi.se

45

The Swedish Chemical Inspectorate (Chi / KemI)

www.kemi.se

Companies

Nanologica

Nanologica is a Stockholm based company which specializes in synthesizing

nanoporous materials, which are less than 100 nm in size. The materials have industrial

applications from pharma to photovoltaics. Nanologica participates in the FP7

sponsored EU project NanoSustain. http://www.nanologica.com/index.php

Kalmar

Kalmar University

Within the Department of Chemistry and Biology, the Nanosciences group conducts

research within biology and nanoscience. According to their webpage they work with

nanoparticles.

http://www.kob.hik.se/nanoscience/research/index.php

Gothenburg

University of Gothenburg - Nanoparticles in Interactive Environments

According to the report The Nano Guide 2010 published by Nano Connect Scandinavia,

this research platform was a collaboration between 2006-2010 between the Department

of Physics and Chemistry at the University of Gothenburg. Interesting partners within

this collaboration would be the Atmospheric science group, Gothenburg Atmospheric

Science Centre, Marine Chemistry, Nano Toxicity Göteborg Science Centre for

Molecular Skin Research Centre for Environment and Sustainability.

The work has recently resulted in the FORMAS funded collaboration GU Nanosphere,

from 2010-2015. The effort in this project is to give a total science perspective of the

impact of nanoparticles on humans, the environment and society.

46

GU NanoSphere

http://www.cefos.gu.se/forskning/Samhallets_riskfragor/nanosphere/

Åsa Boholm asa.boholm@cefos.gu.se

Martin Hassellöv martin.hassellov@chem.gu.se

Chalmers Energi och Miljö

http://www.chalmers.se/ee/SV/forskning/forskargrupper/miljosystemanalys/forskning/

projektbeskrivningar/nanorisksv

Björn Sandén bjorn.sanden@chalmers.se

Sverker Molander sverker.molander@chalmers.se

Rickard Arvidsson rickard.arvidsson@chalmers.se

Companies

IMEGO

Gothenburg Uni spinn-off. Manufactures magnetic nanoparticles, as well as other

nanotechnology related materials and devices.

http://www.imego.com/Expertise/Electromagnetic-sensors/Magnetic-nanoparticles/

index.aspx

Linköping

Companies

Exova

Based in Linköping, develops composite materials in cooperation with SAAB among

others and have been reported to use carbon nanotubes.

www.exova.se

47

PiteåCompanies

Swerea SICOMP

A Swedish research institute located in Piteå. Specializes in polymeric fibre composites.

Uses carbon nanotubes in some applications.

http://www.swerea.se/sicomp/

Denmark

Copenhagen university / Nano-Science Center

Centre for Pharmaceutical Nanotechnology and Nanotoxicology

The centre is designated to examine the relationship between nanomaterials and

biological substances by structure-activity assessments.

http://nano.ku.dk/groups/cpnn/ Contact: Moein Moghimi, momo@farma.ku.dk

NanoPhysics

Employes x-ray methods to study semiconducting nanoparticles and nanowires, as well

as medical imaging.

Contact: Robert Feidenhans’l

Nanotoxicology

This group conducts research on the health effects of nanoparticles. Belongs to the Dept

of Environmental Health and is located at the Institute of Public Health.

Contact: Steffen Loft

48

DTU

CINF- Centre for individual nanoparticle functionality

Explore and understand fundamental relations between surface morphology and

reactivity on the nanometre scale.

Contact: Ib Chorkendorff

Nanotechnology and Risk

Studies potential environmental risks of engineered nanomaterials. Uses a laboratory

based approach and one desk-based approach to analyze and suggest solutions to

regulatory and management solutions from nanotechnology. The aim is to promote

sustainable development and reduce any adverse effects.’

Contact: Anders Baun

http://www.env.dtu.dk/English/Research/Research%20Themes/Environmental

%20Chemistry%20and%20Microbiology/Nanotechnology%20,-a-,%20Risk.aspx

DTU-food

http://www.food.dtu.dk/Default.aspx?ID=23670

Ulla Birgitte Vogel ULBVO@food.dtu.dk

Katrin Löschner kals@food.dtu.dk

Niels Hadrup nilha@food.dtu.dk

Erik Huusfeld Larsen ehlar@food.dtu.dk

Current known activities at DTU:

During december 2010, DTU is recruiting one PhD student in ”Life cycle assessment of

Nanomaterials” and one post-doc for risk assessement of nanomaterials.

49

National Research Centre for the Working Environment

http://www.arbejdsmiljoforskning.dk/Aktuel%20forskning/Nanoteknologi.aspx?lang=en

Håkan Wallin hwa@arbejdsmiljoforskning.dk

Keld Alstrup kaj@arbejdsmiljoforskning.dk

Peder Wolkloff (KU) pwo@arbejdsmiljoforskning.dk

Gunnar D. Nielsen gdn@arbejdsmiljoforskning.dk

Aarhus University

INANO

iNANO is the interdisciplinary research center at Aarhus university, Denmark.

Research on nanoethics is being made:

http://inano.au.dk/research/research-areas/nanotoxicology-and-nanoethics/nanoethics/

Research is also made in the area of food safety and nanotechnology:

http://inano.au.dk/research/research-areas/nanofood/

The School of public health at the University of Aarhus and iNANO are working within

a collaboration with Chinese scientists in the FP7 sponsored programme SIDANO. The

project aims to assess the risk of engineered nanotechnology materials. The strategy is

to use in vitro and in vivo methods to look at the toxic response of engineered

nanomaterials, validate in vitro systems, look at molecular pathways involved in the

toxic response to nanomaterials etc.

Companies

LM Windpower

www.lmwindpower.com

LM Windpower produces wind power plants. LM Windpower employs a composite

material known as Hybtonite in some of their construction. Hybtonite is a composite

containing approximately 0.5% carbon nanotubes and is produced by Amroy Europe Oy

in Finland.

50

Identified knowledge gaps

The purpose of this list is to provide a short overview of areas of nanosafety were there

are knowledge gaps that could potentially be exploited for further applications for

funding.

Determination of relevant levels of exposure – for implementation in animal and

cell experiments

Determining the relevant dose metrics of nanosized materials: size, shape,

particle number, surface area, mass etc.

Toxicity of ENPs after having adsorbed other substances,

Bio-persistency – how long will an ENP be present in an ecosystem or in the

body of an animal. Can they decompose?

Aging of ENPs and the effect of time and environmental factors on their toxicity

Bioaccumulation of ENPs

Biotransformation of ENPs – the environmental fate and the effect of

biochemical processes on the shape, size and function of ENP is relatively

unknown

Release of ENPs from composite materials – during processing, machining,

wear or aging of materials.

Toxicity of composite materials containing ENPs

Risk and life cycle analysis of ENPs and ENP containing materials

51

Structure-activity relationships for ENPs

Effects on skin and topic allergy

Recycling and waste handling of ENP and ENP containing materials

Lack of human data – very few case reports on human exposure

Aerosol formation from commercial products containing ENPs

Consumer exposure

Biomarkers for ENP exposure

Finding reliable reference materials when doing toxicity screenings, both

negative and positive controls.

Long term inhalation studies on animals

Synchrotron radiation technology for assessing ENP interactions with biological

systems and materials

Translation of actual exposure levels to an in vitro or in vivo situation /

Translation of in vitro results into in vivo models

Characterization of ENPs after cellular uptake/interactions

Reproductive effects

52

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