AN EVALUATION OF THE TOXICITY OF ZINC OXIDE AND TITANIUM DIOXIDE
NANOPARTICLES TO Caenorhabditis elegans
by
Krithika Umakanth
Dr. Joel Meyer, Advisor
May 2011
Masters project submitted in partial fulfillment of the
Requirements for the Master of Environmental Management degree in
The Nicholas School of the Environment of
Duke University
2011
MP Advisor's signature
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ABSTRACT
Nanoparticles are present in a bulk of consumer products and their disposal is unregulated by law.
Consequently nanoparticles have found their way into the environment in a variety of ways. This might
pose a health risk to environmental and human health. This project evaluated the potential for toxicity
of zinc oxide and titanium dioxide nanoparticles (present in sunscreens, fungicides, etc.) to the
nematode Caenorhabditis elegans under controlled laboratory conditions.
Methods: C. elegans were dosed with zinc oxide at concentrations ranging from 200 mg/L to 6.25 mg/L,
and titanium dioxide nanoparticles at concentrations of 150 mg/L to 18 mg/L along with a control (C.
elegans grown in reconstituted hard water). After a 3-day dosing regimen, the endpoints growth
inhibition and lethality were observed and compared to the control.
Results: Titanium dioxide nanoparticles caused growth inhibition at concentrations as low as 18 mg/L
but were not lethal even at the highest concentration tested (150 mg/L). Zinc oxide nanoparticles were
lethal at concentrations 75 mg/L and above but caused growth inhibition from 50 mg/L to 6.25 mg/L.
Cytoviva imaging showed that zinc oxide nanoparticles were present inside the body of the worm.
Uptake was not assessed for TiO2 NP as they were not toxic at low concentrations.
Discussion: Titanium dioxide nanoparticles caused growth inhibition at 18.5 mg/L but are unlikely to be
toxic in the environment where they would be present at significantly lower concentrations. Zinc oxide
nanoparticles caused death at 75 mg/L and above, and inhibited growth below that concentration. Thus,
at environmentally relevant concentrations (6 mg/L and below), zinc oxide nanoparticles have the
potential for growth inhibition which warrants further testing to elucidate mechanism of toxicity. The
results of this study could also be used to design new studies to determine if factors such as pH,
temperature and sunlight affect the toxicity of nanoparticles.
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TABLE OF CONTENTS
ABSTRACT………………………………………………………………………………………………………………………………………………1
TABLE OF CONTENTS ……………………………………………………………………………………………………………………………..2
INTRODUCTION ………………………………………………………………………………………………………………………………………3
OBJECTIVE……………………………………………………………………………………………………………………………………………….4
GENERAL CHARACTERISTICS OF NANOPARTICLES……………………………………………………………………………………5
REASON FOR USING C. ELEGANS……………………………………………………………………………………………………………..7
ROUTES OF EXPOSURE…………………………………………………………………………………………………………………………….8
REVIEW OF PRE-EXISTING LITERATURE………………………………………………………………………………………………….10
METHODS……………………………………………………………………………………………………………………………………………..19
RESULTS ………………………………………………………………………………………………………………………………………………..21
DISCUSSION ………………………………………………………………………………………………………………………………………….27
IMPLICATIONS FOR FUTURE TOXICITY TESTING…………………………………………………………………………………….30
ACKNOWLEDGEMENTS …………………………………………………………………………………………………………………………31
REFERENCES ………………………………………………………………………………………………………………………………………….32
APPENDIX………………………………………………………………………………………………………………………………………………34
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INTRODUCTION
Nanoparticles are defined as particles that are less than 100 nanometers in at least one dimension ().
Recent advances in knowledge and technology have led to the use of nanoparticles in a large number of
consumer products ().Due to their small size, nanoparticles have a large surface area compared to their
volume, which is very useful as it enhances the surface area: volume ratio of nanoparticles, enabling
nanoparticles to find application in a variety of products and services ranging from wrinkle-free clothing
and sunscreens to drug delivery systems used for killing tumor cells in some types of cancer ().Titanium
dioxide and zinc oxide nanoparticles are manufactured in large quantities for use in commercial
products such as anti-microbial agents, paints, dyes, etc.
As nanoparticles are very efficiently and innovatively designed to meet consumer needs, the use of
nanoparticles is burgeoning. Therefore, the volume of nanoparticles being released into the
environment by being accidentally washed off the skin (from cosmetics) or by combustion of fuel and
other ways is increasing. Therefore, there is some concern about nanoparticles as an emerging
contaminant. Metallic and metallic oxide nanoparticles in the environment can be very reactive due to
reaction with water and other chemicals, giving them the potential to be toxic to living organisms in a
number of ways. They are also very mobile, enabling them to move from one system to another which
means that exposure to nanoparticles could occur in a number of ways.
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OBJECTIVE
To address the issue of nanotoxicity, the Center for Environmental Implications of Nanotechnology
(CEINT) was established in 200(). Headquartered at Duke, this group is a collaborative effort between
UNIVERSITIES which works to study nanotoxicity with an interdisciplinary approach. At Duke, Theme 1
teams study the physical and chemical properties of several kinds of industrial nanoparticles and
characterize them. Theme 2 teams study the toxicity of nanoparticles at the cellular and organismal
level, for instance in model organisms such as C. elegans and zebrafish. Theme 3 teams study the
interaction of nanoparticles with the environment at a larger scale in mesocosms. Together, the results
of the research help in determining the nanoparticles can be toxic to the environment.
My research was carried in Dr. Joel Meyer’s lab at the NSOE, Duke University, where I evaluated the
toxicity of ZnO and TiO2 nanoparticles to C. elegans (Theme 2). My objective was to evaluate whether
ZnO nanoparticles and TiO2 nanoparticles were toxic to the microscopic nematode C. elegans.
I used the ecologically relevant endpoints of growth and survival to assess whether exposure to ZnO
nanoparticles and TiO2 nanoparticles had adverse health impacts on C. elegans. I used TiO2 NP
concentrations ranging from 150 mg/L or part per million (ppm) to 18.5 ppm. I used ZnO NP ranging
from 200 ppm to 6 ppm. These concentrations are high enough to detect any potential toxicity if NP
accidentally spill or accumulate but also range to low values that are closer to environmentally relevant
levels that might occur as a result of disposal.
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GENERAL CHARACTERISTICS OF NANOPARTICLES
The general characteristics of nanoparticles are listed in the table below:
General characteristics of nanoparticles
Nanoparticles are less than 100 nm in at least one dimension
Nanoparticles have several sources:
• Natural sources: Produced by the combustion of organic compounds (fires),
volcanic eruptions
• Anthropogenic: Engineered nanoparticles (ZnO nanoparticles in sunscreens, Ag
nanoparticles in wrinkle-free clothing), combustion of fuels, etc.
Nanoparticles have high surface area: volume ratio compared to larger sized
particles
Nanoparticles tend to aggregate in solution
Nanoparticles are highly mobile
Engineered nanoparticles are manufactured in a wide range of sizes and forms. Most nanoparticles
present in commercial products have a core and a coating. The core is composed of the metal or metal
oxide while the coating is usually a substance such as gum arabic, citrate, PVP, silicones, silicone oxides,
etc. The coatings serve to stabilize the usually reactive core or modify its reactivity depending on the
purpose for which the nanoparticles is intended.
nanoparticles can enter the environment by being released into the atmosphere, water bodies or soil.
The transport of nanoparticles in these systems is complicated and not well-understood (Lin et al 2010).
The fate of nanoparticles in the environment is determined by the transformation of the nanoparticles
(affected by properties of the nanoparticles and its interactions) and organismal interactions (Lin et al
2010). The physical and chemical properties of nanoparticles affect their bioavailability to biota. Some
nanoparticles are inert but can be made reactive due to interaction with chemicals in the environment
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or changes in pH and light. For instance, nanoparticles can undergo oxidation in the environment
depending on conditions such as sunlight and become capable of causing generation of free radical
species that can damage the health of biota (Lin et al 2010). nanoparticles can also undergo dissolution,
photodegradation and change in the coating due to interaction with the environment. The fate of
nanoparticles is also affected by biota. Living organisms can uptake nanoparticles, transform them intracellularly or extra-cellularly, degrade them or translocate nanoparticles from one place to another (Lin et
al 2010).
The partition coefficient of nanoparticles is a measure of whether they will pass into body tissues or stay
in inorganic media. The partition coefficient of nanoparticles is difficult to determine by the fact that
nanoparticles aggregate in solution and the partition coefficient is the altered.
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REASON FOR USING C Elegans
C. elegans is a nematode that lives in the soil and in the interstitial water between soil particles and can
be easily cultured within the laboratory in aquatic medium (Leung et al. 2008). Its genome has been fully
sequenced and documented. It has a life span of about 5 days. C. elegans feeds on bacteria and fungi in
the soil. It is translucent and easily observed under the microscope and by a variety of visualization
techniques (fluorescence, etc.). C. elegans reproduces quickly and in large numbers allowing for
relatively high throughput toxicological screening assays. Due to this short life cycle and high
reproductive capacity along with the ability to store stocks frozen in liquid nitrogen long-term,
maintenance and culture of C. elegans are straightforward. In the lab, C. elegans is fed the slow growing
uracil auxotroph OP50 strain of E. coli that is also easy to maintain and quantify by measuring the optical
density (OD) of the bacteria solution at 570 nm. For these reasons, C. elegans is ideal as a model
organism in which to study the toxicity of nanoparticles.
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ROUTES OF EXPOSURE
The risk assessment of the toxicity of any compound needs to consider the component of exposure.
Exposure affects whether or not a compound poses a risk to the environment and/or human health. For
instance, exposure via inhalation of ethidium bromide does not cause toxicity while exposure via dermal
contact does cause toxicity. The exposure is also affected by the physical and chemical properties of the
compound under study. Tetrachloroethylene (a solvent for dry-cleaning) is volatile and thus the
inhalation route of exposure is more relevant than dermal contact though tetrachloroethylene is toxic
through dermal contact too.
While considering nanoparticles as emerging contaminants, we must consider the ways in which these
nanoparticles are released into the environment and the potential for contact with living organisms.
Fortunately, there have been no spills or contaminations by nanoparticles on a large scale yet. Though
this makes it difficult to assess the impacts of nanoparticles on a larger scale than the laboratory or the
mesocosms, we can assume for practical purposes that a release of nanoparticles in the environment is
most likely to occur incidentally or accidentally rather than as a large volume of pure nanoparticles
(analogous to an oil spill).
In particular, the nanoparticles under study here (Zinc oxide and titanium dioxide nanoparticles) are
present in consumer products such as sunscreens and sunblock. A potential route of exposure of the
environment to these nanoparticles is if the nanoparticles present in sunscreens, etc. wash off the
consumer’s skin and enter the aquatic ecosystem. If the consumer uses the sunscreen in a swimming
pool which undergoes water treatment, the nanoparticles are still bioavailable as most water
purification systems cannot currently target and remove nanoparticles. Nanoparticles could accumulate,
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especially in a closed system like a pond or lake and alter the chemical balance of the lake which in turn
affects the ability of the aquatic organisms to lead a healthy life.
C. elegans, the test organism in this study, is a nematode that lives in the soil. Nanoparticles that enter
the water in a pond could deposit in the soil of the banks. Due to the inherent tendency of nanoparticles
to clump together, it is difficult to use an octanol-water partition test to determine whether
nanoparticles prefer to stay in the water or in organic matter (Crane et al 2008) as clumps of
nanoparticles do not partition in the same way as mono-dispersed nanoparticles. Thus, C. elegans could
come into contact with these nanoparticles in the following ways:

Dermal contact: The nanoparticles in the soil could cause toxicity to C. elegans by contact with
the skin. They could potentially enter the body of the worm through the skin and cause damage
internally or cause damage to the skin and thus affect the health of the worm.

Ingestion exposure: C. elegans feed on bacteria and fungi dwelling in the soil. They could ingest
nanoparticles accidentally while eating bacteria/fungi. Once inside the body, nanoparticles could
potentially cause toxicity to C. elegans.

Inhalation exposure: C elegans could breathe in air containing nano-sized particles during
normal respiration.
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REVIEW OF PRE-EXISTING LITERATURE ON NANOPARTICLES TOXICITY
Nanoparticles have been present in the air we breathe and in the environment for a long time as a result
of forest fires, volcanic eruptions, etc. However, recently engineered nanoparticles are being used in a
wide variety of commercial applications due to the unique properties of nanoparticles and the ease of
engineering them to meet specific needs. Nanoparticles are released into the environment at a greater
rate now than before. Thus, studying the impacts of nanoparticles on the health of living organisms is
essential in understanding the potential of nanoparticles to cause toxicity.
There are several studies being conducted on nanoparticle toxicity but because it is a nascent subject,
toxicity studies are still revealing new potential mechanisms of toxicity of engineered nanoparticles. The
toxicity of nanoparticles depends, like other toxicants, on a number of factors. An analysis of the existing
studies on nanotoxicity reveals that these factors influence toxicity of nanoparticles:

Physical and chemical properties of nanoparticles

Solvent/medium in which nanoparticles are present

Dose

Route of exposure
1. Physical and chemical properties of nanoparticles:
Nanoparticles are very small in size and hence have high surface to volume ratio. This lends
them the useful property of having high surface reactivity. The stability of nanoparticles is a
function of the size of nanoparticles. Therefore, nanoparticles are coated with substances such
as gum Arabic, etc. to stabilize the nanoparticles (below 25 nm in size). Often, the coating causes
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toxicity in organisms. High surface reactivity implies that nanoparticles would react with
substances they come in contact with more easily than their larger sized counterparts. However,
nanoparticles also tend to aggregate in solution. This complicates toxicity testing as the
nanoparticles are no longer nano-sized but are larger aggregates. The aggregation is influenced
by the pH, temperature, nature of the nanoparticles and the solvent and some other factors.
Aggregation occurs to a larger extent at higher concentrations of nanoparticles than at lower
concentrations. However, it has been recognized that aggregation of nanoparticles in lab
conditions during toxicity testing might provide a better picture of toxicity of nanoparticles in
the real environment (Crane et al 2008).
Nanoparticle composition and structure vary according to the manufacturer. TiO2 and ZnO
nanoparticles are present in a range of shapes and sizes with different coatings. The shape and
size of the nanoparticles also affects the way nanoparticles react with other material and
systems and therefore influences toxicity. The coatings can also cause toxicity due to their
nature.
TiO2 nanoparticles reflect sunlight which is the reason for their use in sunscreens but can absorb
70% of incident UV radiation (Dunford et al). In the presence of light they have been known to
catalyze DNA damage to human cells in vitro. Thus, environmental or biotic transformations of
nanoparticles can affect the toxicity of nanoparticles.
2. Nature of solvent/medium:
The medium into which nanoparticles are released in the environment and the medium in which
they are tested in the laboratory affect the toxicity of nanoparticles. For instance, a suspension
of nanoparticles in seawater differs in toxicity from a suspension of nanoparticles in freshwater.
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3. Dose:
The dose of the nanoparticles administered in the laboratory tests or the amount of
nanoparticles available for reaction with living organisms in the environment affects the toxicity
of the nanoparticles. Some nanoparticles may be toxic at small doses, while some may be toxic
only at very high doses.
4. Route of exposure:
The route of exposure is also important as nanoparticles could be toxic when organisms are
exposed to them via one route of exposure but not another. For instance, exposure via dermal
contact only could not be toxic as nanoparticles might not be capable of entering the body of
the organism (Nohynek et al 2007). The same nanoparticles could be toxic if ingested (Trouiller
et al 2009) because it might be capable of causing oxidative stress or physical damage in the gut
or by entering the other body tissues from the gut.
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TITANIUM DIOXIDE NANOPARTICLES:
General Characteristics:
Titanium dioxide nanoparticles are manufactured and used in a large number of commercial
applications and products. Titanium dioxide nanoparticles are present in sunscreens, sunblock lotions,
anti-bacterial substances, white pigments for paints, paper, plastics, and printing inks, etc. TiO2
nanoparticles reflect UV rays and are used as opacifying agents and colorants in over-the-counter
sunscreens and a number of other cosmetics (Crane et al 2008). Titanium dioxide nanoparticles are
manufactured in a variety of shapes and sizes and with different coatings such as citrate and acetate.
Titanium dioxide is generally inert and does not dissociate into ions in solution like some other metallic
nanoparticles.
TiO2 nanoparticles toxicity studies show conflicting results: some show that TiO2 nanoparticles are toxic
to cells and organisms whereas other studies show that they are not. The reason for the difference in
the results might be due to the fact that there are differences in TiO2 nanoparticles types and their
aggregation, different testing media and conditions of pH, temperature, sunlight, etc.
Though usually inert, when exposed to UV radiation certain forms of TiO2 nanoparticles become reactive
due to the formation of reactive oxygen species by a phenomenon called surface plasmon resonance
(Brennan et al USEPA) and are capable of causing oxidative stress and DNA damage. Phototoxicity of
TiO2 might be influenced by the aggregation of nanoparticles and the size of the resulting aggregates as
the surface properties are modified in the aggregated phase. This has implications for its phototoxicity,
which is especially relevant in shallow ponds and lakes or in soil where TiO2 nanoparticles are exposed to
sunlight.
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Toxicity of TiO2 nanoparticles in other species
Toxicity studies of TiO2 have been conducted in several organisms such as microorganisms, C. elegans,
aquatic organisms such as zebrafish and rainbow trout as well as in mammals like rats.
TiO2 nanoparticles are toxic to bacteria and fungi. Studies have shown that TiO2 nanoparticles are toxic
to zebrafish and cause growth inhibition and damage to their liver and gills (Chen et al 2010). TiO2
nanoparticles were found in gill, heart and brain tissue in the zebrafish that were sacrificed after the
study, indicating that the toxicity is due to the small-sized TiO2 nanoparticles that were able to penetrate
the body tissues of the zebrafish.
Studies in rainbow trout showed that TiO2 nanoparticles suspensions caused respiratory distress and
oxidative stress but were not hemolytic or ionoregulatory toxicant (Federici et al 2007). Studies in
rodents have found that TiO2 nanoparticles can be genotoxic. A study in mice showed that exposure to
TiO2 nanoparticles via ingestion of drinking water containing different nanoparticles concentrations
induced 8-hydroxy-2′-deoxyguanosine, γ-H2AX foci, micronuclei, and DNA deletions and a moderate
inflammatory response in a dose-dependent manner (Trouiller et al 2009) and the clastogenic property
of TiO2 DNA was observed at 500mg/kg.
As mentioned earlier, phototoxicity of certain engineered TiO2 nanoparticles is gaining attention in the
scientific community. TiO2 exposed to UV radiation becomes an efficient semiconductor due to a
phenomenon called surface plasmon resonance and generates reactive oxygen species such as hydroxyl
radicals on reacting with hydroxide ions in water (Brennan et al). Few studies have investigated the
phototoxicity of TiO2 nanoparticles.
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A study by L’Or´eal, a cosmetic company manufacturing products that contain ZnO and TiO2
nanoparticles conducted a study which showed that these nanoparticles are not toxic to humans
(Nohynek et al 2007). TiO2 nanoparticles present in sunscreens usually have silicones or silicon oxides as
coatings which exacerbate apoptosis of cultured human skin cells in vitro compared to plain TiO2
(Rampaul et al 2007). Another study showed that anatase nano TiO2 exhibited photoactivated toxicity to
medaka larvae at concentrations as low as 2.42 mg/L (LC50) under solar simulated radiation (SSR)
approximating 25-30% of daylight (Brennan et al). Thus, phototoxic forms of TiO2 nanoparticles
deposited in shallow water bodies or on the surface of the soil has potential to cause toxicity to
organisms exposed to it, especially under condition of brighter sunlight.
Toxicity of TiO2 nanoparticles in C. elegans
C. elegans has been used as model for studying organismal level toxicity of several toxicants including
nanoparticles. C. elegans could be exposed to TiO2 nanoparticles if they enter the soil from nearby
water bodies into which they are accidentally released. Exposure to TiO2 in C. elegans would be via
ingestion and dermal contact.
A study has shown that titanium dioxide nanoparticles are toxic to C. elegans under certain
experimental conditions by causing a decrease in growth, survival rate and fertility potentials, probably
by affecting the expression of the cyp35a2 gene (Roh et al 2010). The same study also found that
smaller-sized nanoparticles (7 nm) are more toxic to C. elegans than larger-sized nanoparticles (20 nm).
The mechanism of the toxicity is not fully known and needs to be explored with further toxicity testing.
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ZINC OXIDE NANOPARTICLES:
General Characteristics:
ZnO nanoparticles are white in color and have low water solubility (upto 5 mg/L). ZnO nanoparticles are
used in anti-bacterial applications, in sunscreens and as part of drug delivery systems in cancer therapy.
ZnO nanoparticles are engineered in a variety of shapes and sizes with different coating. ZnO
nanoparticles rapidly dissolve in water to yield Zn2+ ions. The free ionic form of metals is usually
considered to be the most toxic to biota. Thus, it is unclear if the toxicity of ZnO nanoparticles is a result
of the toxicity caused by the ionic Zn2+ species released by dissolution of ZnO nanoparticles or by the
ZnO nanoparticles as a function of their small size and large surface area (Ma et al 2009) because several
experiments have yielded different answers to this question (Poynton et al 2010).
Toxicity of nanoparticles in general is affected by the rate of release of ions in solution (seawater,
freshwater, etc.). ZnO nanoparticles availability and toxicity were shown to be a function of the release
of the Zn2+ ions from the solution in which they are dispersed, rather than the sizes of the ZnO
nanoparticles (Zhang et al 2010).
Very few studies have focused on ZnO nanoparticles phototoxicity. ZnO nanoparticles proved not to be
more cytotoxic when irradiated with UV light than when not in a study conducted by Diambeck et al.
ZnO nanoparticles also proved to be only slightly more genotoxic when irradiated with UV rays in an in
vitro study (Dufour et al).
Toxicity of ZnO nanoparticles in other species:
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The mechanism of zinc oxide nanoparticles toxicity has been attributed sometimes to the production of
reactive oxygen species (ROS). Other studies exhibit effects that range from acute effects such as growth
impairment to chronic effects such as impaired reproductive capability.
The route of exposure of ZnO nanoparticles is also important in determining the toxicity of these
nanoparticles to organisms. For instance, E. coli exhibited no reduction in viability when exposed to high
concentrations (>40 mg/L) of ZnO nanoparticles, but were inhibited in growth when the same ZnO
nanoparticles were sprayed as an aerosol to a biofilm of the E. coli (Wu et al 2010). The toxicity in this
case was also a function of the Zn2+ ionic species dissolved in the media rather than the ZnO
nanoparticles themselves.
In humans, in vivo ZnO nanoparticles remained in the stratum. Additionally, the outermost layers of the
stratum corneum have a good turnover rate, implying that the ZnO nanoparticles are unlikely to result in
safety concerns to humans.
Toxicity of ZnO nanoparticles to C. elegans:
ZnO nanoparticles toxicity could be a function of the ionic Zn2+ released into solution as a result of
dissolution of ZnO nanoparticles in the solvent. A study compared the toxicity of ZnO nanoparticles and
ZnCl2 solution to C. elegans by studying lethality, growth inhibition and reproductive ability as endpoints
(Ma et al 2009). The results of the study showed that there was no significant difference between the
LC50 of the ZnO nanoparticles solution and the ZnCl2 solution, indicating that the toxicity of ZnO
nanoparticles could be due to the release of ionic Zn2+ and the resulting effects of the Zn2+ ions.
Alternatively, ZnO nanoparticles could undergo intracellular biotransformation and produce endproducts similar to those produced by biotransformation of ZnCl2 particles, accounting for the similarity
in their toxicity to C. elegans. The ZnO nanoparticles induced expression of fluorescently tagged mtl2
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gene, indicating that the metallothionine gene (mtl) could have been activated to transform the ZnO
nanoparticles into a form that caused toxicity. Both these observations suggest that toxicity of ZnO
nanoparticles is similar to toxicity of Zn2+ ions which are related to activation of the mtl gene. However,
the concentration of ZnO nanoparticles used was high (325–1,625 mg/L) which interferes with applying
these results to the environment but provides a good basis for understanding the mechanism of toxicity
of ZnO nanoparticles to C. elegans.
Yet another study of zinc oxide nanoparticles toxicity in C. elegans showed that ZnO nanoparticles had a
significantly lower LC50 than bulk ZnO particles, indicating that ZnO nanoparticles were more toxic than
their larger sized counterparts (Wang et al 2008).
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MATERIALS AND METHODS
The methodology involved three main steps:
1. Nanoparticle solution preparation
2. Toxicity testing
3. CytoViva imaging
Part 1: NANOPARTICLES PREPARATION
The nanoparticles used for testing were obtained from Sigma Aldrich in the form of a powder and
analyzed by the lab at Duke University. The shape of both the nanoparticles (ZnO and TiO2) was spherical
and the average particle sizes were 20 nm and 15 nm respectively. The nanoparticles had no coating. To
make the dosing solution, the nanoparticles were dissolved in RO (reverse osmosis) water and then
subjected to sonication to ensure dissolution and dispersion of the nanoparticles. Periodically during
sonication, the nanoparticle solution was analyzed using dispersive light scattering. This technique
characterizes the size distribution of the nanoparticles in the solution. In this way, I ensured that the test
solution was composed mainly of dispersed nanoparticles of the appropriate size range (<150 nm in
hydrodynamic size) rather than big clumps of aggregated nanoparticles. After sonication for 40 min, the
nanoparticle solution had the appropriate size distribution of nanoparticles. The average hydrodynamic
diameter of the TiO2 nanoparticles was approximately 120 nm and for ZnO nanoparticles the
hydrodynamic diameter was 120 nm approximately after being dissolved in RO water. For dosing the
worms, nanoparticles dilutions were prepared in reconstituted hard water (EPA water/low chloride
media). This was chosen due to the low ionic strength (total hardness of 1 20- 1 80 mg/L as CaCO3)
compared to K+ or K media and it more closely reflects the ionic strength of water in the environment.
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Part 2: TOXICITY SCREENING
The methodology that I used was a modified version of the protocol developed by the Meyer laboratory
at Duke University from a high throughput, automated assay described by the Freedman laboratory at
NIEHS, with advice from Windy Boyd (Freedman laboratory) [1].
General Approach:
Age-synchronized stage 1 larvae (L1s) of C. elegans were exposed in liquid culture (reconstituted hard
water) to the nanomaterial of interest (ZnO and TiO2) along with killed uvra as food, and daily
observations were made microscopically to quantify survival and growth. This was an acute toxicity test.
For a detailed record of the methods, please refer to the Appendix.
PART 3: CYTOVIVA® IMAGING
After I identified whether the nanoparticles had any adverse impacts on C. elegans in the toxicity tests, I
used imaging techniques to find out if the nanoparticles entered the worm and to determine their
location inside the worm as it was important to investigate whether the toxicity was due to uptake of
the nanoparticles. I dosed the worms with a sub-lethal concentration of ZnO nanoparticles and used a
control to build up a library of wavelengths and compare the two samples. With the help of Cytoviva®
technology developed by CytoViva® Inc which is a hyperspectral imaging technology that provides very
detailed pictures of the subject I took several pictures of worms dosed with ZnO nanoparticles at
resolutions as low as 25 nm x 25 nm resolution. The nanoparticles are visible in dark field microscopy
because of the glow emitted by the light they scatter. Clumps of nanoparticles appear brighter whereas
mono-dispersed nanoparticles appear as faint and small glowing spots.
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RESULTS
The statistical analysis used to compare if there are significant differences in length between worms
treated with control and different concentrations of nanoparticles was the Kruskal-Wallis test and the
Mann-Whitney U Test. These two tests are non-parametric tests similar to the ANOVA. These tests were
used because they accounts for non-normal distributions in identical populations of differing sizes such
as the ones in this project.
TiO2 NANOPARTICLES:
Below are presented the results of the Kruskal-Wallis and Mann Whitney U tests for analyzing the
lengths (on day 3) of the worms treated with each concentration of TiO2 nanoparticle solution tested
against the control. The results show that the mean ranks of the worms treated with different
nanoparticle concentrations is not the same i.e. the populations are not identical (p < 0.0001). The
results of the Mann-Whitney U test show that even the lowest concentration of TiO2 nanoparticles
tested caused growth inhibition (there was significant difference in lengths compared to the control; p <
0.05). Thus, we can conclude that exposure to titanium dioxide nanoparticles at 18.5 ppm and above
used caused growth inhibition in C. elegans.
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Figure 1: Length of C. elegans dosed with TiO2 nanoparticles
Table 1: Kruskal Wallis Test Results for TiO2 nanoparticles
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Table 2: Mann Whitney U test Results for TiO2 nanoparticles
Other Observations
The worms dosed with high concentrations had trouble moving presumably because of the deposition of
nanoparticles aggregates on the outer body membranes of the worms and the large amount of
aggregates in the dosing solution. The worms at lower concentrations and in the control were fastermoving and more vigorous.
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ZnO NANOPARTICLES
DOSE-RESPONSE:
Dose-response tests for ZnO nanoparticles yielded the following results. ZnO nanoparticles were lethal
to C. elegans at concentrations as low as 75 ppm. Lower than 75 ppm, ZnO nanoparticles caused growth
inhibition in C. elegans at concentrations as low as 6.25 ppm. The lengths of the worms were analyzed
using the Kruskal-Wallis test, the results of which are presented in the table and figure below. The
difference in lengths between the dosed samples and control was analyzed using the non-parametric
Mann-Whitney U test.
Figure 2: Lengths of C. elegans dosed with ZnO nanoparticles
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Table 3: Kruskal-Wallis Test Results for ZnO nanoparticles
Table 4: Mann-Whitney U test results for ZnO nanoparticles
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CYTOVIVA® RESULTS:
As the toxicity test revealed that ZnO nanoparticles caused death of the worms at 75 ppm and above,
and growth inhibition at low concentrations (6 ppm) which might be environmentally relevant, I
performed Cytoviva® imaging to check if the toxicity was due to uptake of the ZnO nanoparticles by the
worms. Uptake of nanoparticles could result in toxicity if the nanoparticles are able to enter the internal
tissues of the worm by crossing the gut or skin cell barriers. Once inside a tissue, nanoparticles can cause
generation of reactive oxygen species (ROS), disrupt the cell membrane or otherwise interfere with
regular molecular and cellular functioning (Carlson et al 2008).
I performed Cytoviva® imaging for C. elegans at 50 ppm of ZnO NP. This revealed that the ZnO
nanoparticles were present on the exterior of the worm’s body, deposited in large amounts on its skin.
The nanoparticles were mostly present as clumps but there were some dispersed nanoparticles, too.
Nanoparticles were also found lining the gut of the worms and some had crossed the digestive lining
and were found inside the body of the worm. The ZnO nanoparticles were in the gut probably as a result
of accidental ingestion of ZnO nanoparticles along with the UVRA (food).
The fact that ZnO nanoparticles are capable of crossing the gut and entering the body is important as it
indicates the potential for generation of ROS after nanoparticles enter the tissues inside the body. This is
a result of their small size.
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DISCUSSION
Titanium dioxide and zinc oxide nanoparticles are high-volume production and use nanoparticles. They
are used as opacifying agents and colorants in cosmetics, anti-fungal or anti-microbial agents, and in a
number of other products. Though approved by the FDA as safe for use on human skin and in some food
colorants, nanoparticles could be toxic to invertebrates and small vertebrates.
Nanoparticles are considered an emerging contaminant because they are not targeted by wastewater
treatment systems, have the potential to be toxic to biological systems and are released into the
environment without regulation. Evaluation of the environmental toxicity of nanoparticles is not
straightforward. Nanoparticles are small in size and have a large surface area: volume ratio; they tend to
clump together and form aggregates in solution or in air. The physical and chemical properties of the
aggregates and their effects on living organisms they come into contact with might be different from
those of the mono-dispersed nanoparticles. The effects also vary depending on the type of nanoparticle
and the coating. The adverse effects of nanoparticles might be due to reaction with internal organs,
interference with normal body functioning, or mechanical stress such as that caused to the body
membranes.
Nanoparticle toxicity studies show differing results. Previous studies have shown that nanoparticles can
cause toxicity to living organisms at high concentrations. A general trend is that while nanoparticles are
toxic to cells at small concentrations in most cell culture studies and to microbes, nanoparticles are not
as toxic unless at higher concentrations when multicellular, eukaryotes with body membranes are
exposed to them. Thus, the body membranes of organisms seemingly play a large role in protecting
organisms from nanoparticles. Once nanoparticles enter organisms’ bodies, some nanoparticles have
shown the capability to cross into body tissue from the gut or from the respiratory organs after
27
exposure via ingestion or inhalation respectively. Simpler or lower-level organisms seem more
susceptible to some nanoparticles as nanoparticles can interfere with the normal functioning of their
bodies and have a consequently larger adverse impact on them than inside the bodies of more complex
organisms.
In addition, a comparison of ZnO nanoparticles toxicity in vitro and in vivo showed that toxicity testing in
vitro is not representative of toxicity in vivo. ZnO nanoparticles were more toxic when instilled
intratracheally or via inhalation to mice than when exposed to lung cell cultures (Warheit et al 2009).
Thus, the results of nanoparticle toxicity tests are conflicting and not easy to integrate to understand the
bigger picture of nanoparticle toxicity just by themselves.
Another problem with nanoparticle toxicity testing is that tests are not identical to exposures that would
occur in the environment but are approximations of environmentally relevant exposures. Though the
results of the toxicity tests in this project showed that the two nanoparticles are toxic to C. elegans,
there are some considerations to keep in mind while interpreting these results.
An important point is that the concentrations I used in the test are higher than are likely to be present in
the environment. For instance, TiO2 or ZnO nanoparticles that are released into a stream would get
highly diluted and be present at concentrations lower than 6 ppm unless they accumulate over a period
of time. Another consideration is that lab conditions are very controlled in terms of temperature, pH
and composition of the medium unlike the heterogeneous and dynamic nature of the environment.
Nanoparticles released into water bodies might be altered in chemical nature by reacting with chemicals
in the water, pH of the water, etc. For instance, TiO2 nanoparticles generate reactive oxygen species
when exposed to sunlight and hydroxide ions thereby altering their toxicity.
28
In addition, this test used sonication to disperse the nanoparticles but the nanoparticles aggregated in
the test solution after a while. This is similar to what would happen to nanoparticles in the environment
i.e. they would tend to aggregate rather than be mono-dispersed. Though there are limitations in
directly extending the results of lab tests to real life, toxicity tests can help to identify whether
compounds have the potential to be toxic to living organisms.
Nanoparticles have a lot of important uses including application in cancer therapy and more recently as
substances that can promote the mineralization and subsequent inactivation of organic contaminants in
polluted water (Dionysiou, EPA). Thus, it is not feasible or even wise to stop the use of nanoparticles in
products. However, toxicity tests such as this project contained can inform regulatory agencies such as
the US EPA and decision makers about policies regarding the use and disposal of nanoparticles. This can
help prevent environmental damage and can save the cost of remediation in case we fail to regulate
nanoparticle disposal properly.
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IMPLICATIONS FOR FUTURE TOXICITY TESTING
Studying toxicity in the lab can help establish the basis for future studies that could elucidate the
mechanism of toxicity of the nanoparticles. Further testing can be done by using worm strains deficient
in one or more genes such as metallothionine to determine if the toxicity of the metallic nanoparticles is
due to the interference of normal functioning of that gene. Testing under different conditions of pH and
temperature can be carried out to obtain an idea of what differing environmental conditions could do to
impact toxicity. Phototoxicity is also an aspect that can be explored through co-exposure of the testing
solution and C. elegans to UV or direct sunlight.
Toxicity testing C. elegans can also serve as a model to inform us of toxicity of nanoparticles in other
organisms including vertebrates. This can also help in designing studies in other organisms which could
be exposed to these nanoparticles. Insights into the effect of nanoparticles on C. elegans’ genome can
help in the elucidation of the mechanistic basis of nanoparticles toxicity in eukaryotes.
Nanoparticles that show toxicity in lab tests can show no toxicity in the environment because of
dynamic factors such as pH, other chemicals in the environment and several other variables. Thus, the
results of the tests carried out in lab for this project can be combined with studies in the mesocosms in
Duke Forest to get a more integrated picture of nanoparticle toxicity.
30
ACKNOWLEDGEMENTS
I would like to express my heartfelt gratitude to my advisor Dr. Joel Meyer for his guidance and advice in
making this project a success. I would also like to thank all the members of the Meyer Lab who have
helped me in my research and taught me a lot about toxicological research.
My academic advisor Dr. Jennifer Swenson was instrumental in guiding me to take up this project and
for that I thank her.
I also appreciate the support provided by friends and family during this project.
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REFERENCES
Carlson C, Hussain SM, et al. 2008. Unique cellular interaction of silver nanoparticles: size-dependent
generation of reactive oxygen species. J. Phys. Chem. B, 112 (43): 13608–13619.
Dionysiou, Dionysios D. 2005. Biotemplating of Titanium Dioxide Nanoparticles for the Green Production
of Photochemically Active Catalysts: Synthesis, Characterization, and Photocatalytic Evaluation. EPA
Dunford R, et al. 1997. Chemical oxidation and DNA damage catalysed by inorganic sunscreen
ingredients. FEBS Letters 418: 87-90.
Federici G et al. 2007. Toxicity of titanium dioxide nanoparticles to rainbow trout: Gill injury, oxidative
stress, and other physiological effects. Aquatic Toxicology 84: 415-430.
Wu B, Wang Y, et al. 2010. Comparative eco-toxicities of nano-ZnO particles under aquatic and aerosol
exposure modes. Environ Sci. Technol. 44:1484-1489.
Miao A, Zhang X, et al. 2010. Zinc oxide-engineered nanoparticles: dissolution and toxicity to marine
phytoplankton. Environmental Toxicology and Chemistry 29(12): 2814-2822.
Trouiller B, et al. 2009. Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo
in mice. Cancer Res 69 (22): 8784-8789.
Meyer JN, et al. 2010. Intracellular uptake and associated toxicity of silver nanoparticles in
Caenorhabditis elegans. Aquatic Toxicology 100: 140–150.
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Rampaul A, Parkin IP, Cramer LP, et al. 2007. Damaging and protective properties of inorganic
components of sunscreens applied to cultured human skin cells. Journal of Photochemistry and
Photobiology A: Chemistry 191: 138-148.
Roh JY, Park YK, et al. 2010. Ecotoxicological investigation of CeO2 and TiO2 nanoparticles on the soil
nematode Caenorhabditis elegans using gene expression, growth, fertility, and survival as endpoints.
Environmental Toxicology and Pharmacology 29: 167–172.
Warheit DB, Sayes CM, et al. 2009. Nanoscale and fine zinc oxide particles: can in vitro assays accurately
forecast lung hazards following inhalation exposures? Environ. Sci. Technol. 43: 7939–7945.
Crane M, Handy RD, et al. 2008. Ecotoxicity test methods and environmental hazard assessment for
engineered nanoparticles. Ecotoxicology 17:421–437.
Ma H, Bertsch PM, et al. 2009. Toxicity of manufactured zinc oxide nanoparticles in the nematode
Caenorhabditis elegans. Environmental Toxicology and Chemistry, Vol. 28 (6): 1324–1330.
Leung MCK, et al. 2008. Caenorhabditis elegans: An emerging model in biomedical and environmental
toxicology. Toxicological sciences 106(1): 5–28.
Brennan, AA, Hoheisel, S, Fernandez, JD, Diamond, SA. Phototoxicity of Nano-Scale Titanium Dioxide.
U.S. EPA, Mid-Continent Ecology Division.
Nohynek GJ, Lademann J, et al. 2007. Grey goo on the skin? Nanotechnology, cosmetic and sunscreen
safety. Critical Reviews in Toxicology 37: 251–277.
Lin D et al. 2010. Fate and transport of engineered nanomaterials in the environment. J. Environ. Qual.
39: 1896–1908.
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APPENDIX
PART 1: GROWTH ASSAYS
Egg Prep
1.
I applied K-medium to loosen eggs on a 4-day old egg prep plate and rubbed with a bent glass
bar. I poured this solution that contained worms and eggs into a 15-ml centrifuge tube with a sterile
pipette. I repeated the rinse to obtain the maximum number of eggs from the plate.
2.
I filled the tube with K-medium until volume of the liquid in the tube is ~15 ml and centrifuged
at 2000 rpm for 2 min to pellet worms and eggs.
3.
I removed the supernatant and resuspended the pellet in about 10 ml bleach solution and let it
sit for 10 min on the shaker at 20 C. This bleach solution kills the worms and leaves the eggs intact.
4.
I centrifuged at about 2000 rpm for 2 min to pellet and removed the supernatant.
5.
Resuspended pellet (only viable eggs, no viable worms) in 13 ml of K- medium and centrifuged
for 2 min.
6.
Removed supernatant, making sure to leave enough to suspend the eggs in the K+ medium for
the overnight hatch.
UV-Killed OP50 preparation
Instead of using OP50 for this preparation, a UV-sensitive strain (uvra) of E. coli was used to ensure UVkilling.
34
1. Transferred ~6mL of a ~16hr culture of live uvra E. coli in LB-broth to a 150cm Petri dish.
2. Based on the UVC intensity reading, dosed uvra bacteria with at least 1000J/m2
3. Divided the needed time of exposure by 6 and shook up/swirled plate for a few seconds after each
interval to ensure that all bacteria are exposed equally and do not shield each other from the UVC.
4. After dosing, pipetted bacteria into a 15mL conical tube and stored at 4◦C for use. To make sure all
bacteria were killed, I diluted a small volume of dosed bacteria 1:20,000. I then lawned out 50uL of this
diluted sample onto an LB agar plate and incubated at 37C overnight. No colonies were observed
indicating that there were no live bacteria.
Toxicity Test: Setting up Exposure
1.
At Day -1, Egg prepped and placed rinsed eggs in K+ medium overnight in 25 cm2 cell culture
flasks with vented caps to get synchronized L1s (Lewis and Fleming 1995). This ensured that the L1s are
synchronized (at the same stage of development).
2.
Every 24 hrs prepared dosing solutions:
•
Dosing solutions were 500uL total in reconstituted hard water (EPA water) for 24 well plates.
•
Spun down the UV-killed bacteria and removed the supernatant.
•
Repeated the rinse with reconstituted hard water twice and resuspended the bacterial pellet in
600 ul of reconstituted hard water.
•
Pipetted appropriate volumes of this bacterial solution into each well (5ul on day 1, 10ul on day
2, 50 ul on day 3).
35
•
Added nanoparticles from stocks as calculated to achieve target dosing concentrations.
3. At Day 0, spun down synchronized L1s at 2200 x g for 3 minutes in 15 mL centrifuge tubes.
4. Decreased volume of EPA water by aspiration, resuspended worms, then pipetted 2uL onto glass slide
and counted worms to determine worms per uL.
5. Into a 24 well plate, added appropriate volume such that I ended up with ~500 L1 larvae in each well.
6. Incubated at 20ºC (no shaking).
Observations
Recorded every 24 hours
Survival
I observed nematodes in wells to check for viability. If any potential lethality (lack of motion) was
observed, continued with survival assessment; otherwise, proceeded to growth assessment.
1.
Tilted plate and let worms settle in bottom cusp of the wells (allow ~30 minutes for settling)
2.
Using a 1 ml pipette, pipetted out larvae and dosing solution into 15 ml centrifuge tube
3.
Washed each well with EPA water and added to centrifuge tube
4.
Spun at 2200 x g for 2 minutes
5.
Pipetted ~10 nematodes onto a small k-agar dish and gently prodded with platinum wire worm
picker to assess viability. Nonresponsive nematodes were defined as dead.
36
Growth
If some or all of the nematodes were alive, I quantified the growth of the nematodes as follows.
1.
Same as steps 1-4 of survival assay.
2.
Pipetted between 1-5 µl out of the bottom of the vial (at least 20 larvae) onto a slide
3.
Added 2-3 µl of 170 mM NaN3 to immobilize the nematodes
4.
Observed and captured image.
5.
In empty wells of the same 24-well plate,, replenished dosing solutions (every 24hrs to ensure
consistent dosing with nanoparticles and enough OP50 to keep C. elegans well fed)
6.
Spun worms down again (2200 x g for 2 minutes)
7.
Pipetted remaining larvae from the bottom of the tube (in as small a volume as possible [~20uL]
to prevent dilution of the dosing solution) into the proper replenished well in the new 24 well plate.
8.
Used imaging software NIS-Elements to measure and record the captured images of the worm
lengths
37
CYTOVIVA IMAGES
Figure 3:
Control (o ppm ZnO)
Figure 4,3 : ZnO nanoparticles inside C.elegans (50 ppm)
NP are visible as bright spots
38
R SCRIPT
Dataset <read.table("C:/Users/Krithika/Documents/Spring 2011/MP/zno/anar1.csv",
header=TRUE, sep=",", na.strings="NA", dec=".", strip.white=TRUE)
boxplot(Length~Concentration, ylab="Length", xlab="Concentration",
data=Dataset)
Dataset1 <read.table("C:/Users/Krithika/Documents/Spring
tio2/Book2alof.csv",
2011/MP/New
header=TRUE, sep=",", na.strings="NA", dec=".", strip.white=TRUE)
boxplot(Length~Concentration, ylab="Length", xlab="Concentration",
data=Dataset1)
39
nanoparticles/new