This presentation is based on our review paper ‘Engineered nanoparticles in the soil and their potential implications to microbial activity’, Geoderma, 2012, 173-174, 19-27 (http://dx.doi.org/10.1016/j.geoderma.2011.12.018)

1. Engineered nanoparticles in the soil and their potential implications to
microbial activity
R. Dinesh
M. Anandaraj
V. Srinivasan
S. Hamza
Indian Institute of Spices Research
(Indian Council of Agricultural Research)
Marikunnu PO., Calicut-673012
Kerala State, India
This presentation is based on our review paper ‘Engineered nanoparticles in the soil and their potential implications to
microbial activity’, Geoderma, 2012, 173-174, 19-27 (http://www.sciencedirect.com/science/article/pii/S0016706111003661)
(http://dx.doi.org/10.1016/j.geoderma.2011.12.018)

2. Nanotechnology
The U.S. National Nanotechnology Initiative (NNI) has defined nanotechnology as
thescience, engineering, and technology conducted at the
nanoscale, which is about 1 to 100 nanometers (nm).
Nanoscience and nanotechnology are the study and application of extremely small
things and can be used across all the other science fields, such as chemistry,
biology, physics, materials science, and engineering. Nanotechnology is not just a
new field of science and engineering, but a new way of looking at and studying at
the nanoscale where unique phenomena enable novel applications
(www.nano.gov; accessed on 16 February 2012).
A joint report by the British Royal Society and the Royal Academy of Engineering
similarly defined nanotechnology as “the
design, characterization,
production, and application of structures, devices and systems
by controlling shape and size at nanometer scale.

3. The schematic figure
(a) depicts a logarithmic-length scale
showing the size of a classical
nanomaterial (C60 fullerene) compared
with various biological components
(adapted from 17). Particles of various
sizes are drawn to scale.
(b) Rat macrophage cells with
internalized rope-like bundles of
single-walled carbon nanotubes
(SWCNT). For comparison,
mitochondria are marked with arrows.
Human macrophages are up to two
times larger than their rat
counterparts.
(c) Human lung carcinoma cells with
evidence of internalization of iron
oxide nanoparticles of ∼20 nm in
diameter.
Source: Shvedova et al. (2010)
3

4. [Source: terressentials.com]
4

5. Types of NPs
Nanoparticle: A sub-classification of ultrafine particles with lengths in
two or three dimensions greater than 1 nanometer (nm) and smaller than about
100 nm, and which may or may not exhibit size-related intensive properties.
Natural nanoparticles: Particles with one or more dimensions at the
nanoscale originating from natural processes, e.g. soil colloids.
Incidental nanoparticles: Nanoparticles formed as a by-product of man-
made or natural processes, e.g. welding, milling, grinding or combustion.
Engineered nanoparticles (less frequently also “manufactured
nanoparticles”): Nanoparticles manufactured to have specific properties or a
specific composition.

6. Types of ENPs
• Fullerenes (grouping Buckminster fullerenes, CNTs, nanocones etc.)
• Metal ENPs (e.g. elemental Ag, Au, Fe)
• Oxides (or binary compounds when including carbides, nitrides etc.). E.g. TiO2, Fe oxides.
[Source: Norwegian Pollution Control Authority. 2008]
• Complex compounds (alloys, composites, nanofluids etc., consisting of two or more
elements) e.g. Cobalt-zinc iron oxide.
• Quantum dots (or q-dots) are binary or complex compounds often coated with a polymer.
They are usually regarded apart due to unique use and composition. Q-dots are ENPs that
exhibit size-dependent electronic or optical properties due to quantum confinement. E.g.
cadmium-selenide (CdSe) which has light emission peaks that varies according to particle
size; green for 3 nm diameter particles, red for 5 nm, etc. Used in electronics/experimental
biology/medicine.
• Organic polymers (dendrimers, polystyrene, etc.)
Animations courtesy Dr S. Dr. Maruyama; http://www.photon.t.u-tokyo.ac.jp/~maruyama/agallery/agallery.html

7. Properties of ENPs
• ENPs have different optical, electrical, magnetic, chemical and mechanical properties from
their bulk counterparts are that in this size-range (between 1-100 nm) quantum
effects start to predominate and the surface-area-to-volume ratio (sa/vol)
becomes very large.
• The sa/vol of most materials increases gradually as their particles become smaller, which
results in increased adsorption of the surrounding atoms and changes their
properties and behavior.
• Materials reduced to the nano-scale can suddenly show very different properties,
compared to what they exhibit on the macro-scale, which enables unique applications.
• For example, opaque substances become transparent (copper); stable materials become
combustible (aluminum); inert materials become catalysts (platinum); insulators become
conductors (silicon); solids turn into liquids at room temperature (gold)
Source: Hristozov and Malsch (2009)

8. Source: http://www.deakin.edu.au/itri/images/nanoparticles2.bmp

9. Research in nanotechnology has resulted in applications across a wide range of
areas like medical and pharmaceutical sectors, the development of new materials,
personal care products, to applications in agriculture and food. Today, nanoscale
materials find use in a variety of different areas.
According to the Project on Emerging Nanotechnologies (PEN) over 1,300
manufacturer-identified, nanotechnology-enabled products have entered
the commercial marketplace around the world and If the current trend
continues, the number of products could reach 3,400 by 2020.
(http://www.nanotechproject.org/inventories/consumer; accessed on 30 January
2012)
Because of the potential of this technology there has been a worldwide increase in
investment in nanotechnology research and development (Guzman et al., 2006).
The production of engineered nanoparticles (ENPs) was estimated to be
2000 tons in 2004 and is expected to increase to 58,000 tons in 2011-
2020 (Maynard, 2006).
9

10. ENPs and microorganisms
They may have an impact on soil microorganisms via
(1) a direct effect (toxicity)
(2) changes in the bioavailability of toxins or nutrients
(3) indirect effects resulting from their interaction with natural organic compounds and
(4) interaction with toxic organic compounds which would amplify or alleviate their toxicity
While toxicity mechanisms have not yet been completely elucidated for most ENPs, possible
mechanisms include:
• disruption of membranes or membrane potential
• oxidation of proteins
• genotoxicity
• interruption of energy transduction
• formation of reactive oxygen species (ROS) and
• release of toxic constituents
[Source: Simonet and Valcárcel (2009); Klaine et al. (2008)]
This is a magnification of E. coli exposed to a low concentration (10 mg/L) of titanium dioxide nanoparticles.
Cells with compromised membranes are stained red.
[http://esciencenews.com/articles/2009/03/26/nanoparticles.cosmeticspersonal.care.products.may.have.adve
rse.environmental.effects]

11. Fig. Possible mechanisms of nanomaterials toxicity to bacteria
Source: Klaine et al. (2008)
11

12. Source: Lellouche et al. (2009)
Scanning electron microscope analysis of normal and
MgF2·Nps treated cells. (a) E. coli and (b) S.aureus untreated
cells after overnight growth; (c) E. coli and (d) S. aureus treated
with MgF2·Nps (1 mg/ml).
Transmitted electron microscopy of MgF2·Nps treated and untreated
cells. TEM micrographs of E. coli and S. aureus thin sections:
Untreated E. coli (a,b) and S. aureus (c,d); MgF2·Nps (1 mg/ml)
treated E. coli (e,f) and S. aureus (g,h). Arrows indicate MgF2 12
nanoparticles.

13. Antimicrobial activity of carbon based ENPs
• C60 was harmful or has neutral biological consequences.
• C60 aggregates inhibited Escherichia coli and Bacillus subtilis.
• Fullerene water suspensions (FWS) exhibited relatively strong antibacterial activity and were more toxic to
B. subtilis.
• FWS exerts ROS-independent oxidative stress in bacteria, with evidence of protein oxidation, changes in
cell membrane potential, and interruption of cellular respiration.
• Fullerenols (C60 (OH) 12, C60 (OH) 36.8H2O, and C60 (OH) 44.8H2O) have been found to be toxic to six
kinds of bacteria and two kinds of fungi.
• Carbon based ENPs like CNTs have been found to inactivate E. coli, Staphylococcus epidermis, beneficial
soil microbes like P. aeruginosa, B. subtilis as well as diverse microbial communities of river and waste
water effluent.
The toxicity of C60 has been attributed to its ability to bind and deform the DNA stands,
thereby interfering with DNA repair mechanisms.
Source: Lyon et al. (2005) , Jia et al. (2005); Fortner et al. (2005); Lyon et al. (2008); Aoshima et al. (2009); Kang et al.
(2009); Zhou et al. (2005)

14. Antimicrobial activity of metal and metal oxide ENPs
• Microbial toxicity has been reported for metal NPs, like elemental Ag, Au, Fe; oxides like TiO2,
Fe-oxides, Co-Zn-Fe oxide etc. These NPs raise serious environmental concerns because of
their unique dissolution properties and electronic charges, in addition to their small sizes and
large surface-to-mass ratios (Wang et al., 2010).
• Ag NP is toxic to E. coli and Staphylococcus aureus and B. subtilis.
• Even Ag NP biosynthesized by fungi showed potent activity against fungal and bacterial
strains like Aspergillus niger, Staphylococcus sp., Bacillus sp. and E. coli.
• E. coli and S. aureus and Pseudomonas putida were inhibited by ENPs of Ag, CuO and ZnO.
[Source: Rai et al. (2009); Suresh et al. (2010); Jaidev and Narasimha (2010); Jones et al. (2008); Gajjar
et al. (2009) ]
Zn NP
ZnO NP

15. • Even water suspensions of nanosized titanium dioxide (TiO2), silicon
dioxide (SiO2), and zinc oxide (ZnO) were found to be harmful to
varying degrees, with antibacterial activity increasing with particle
concentration. Antibacterial activity generally increased from SiO2 to
TiO2 to ZnO, and B. subtilis was most susceptible to their effects.
• Electrospraying of NPs of NiO, CuO, or ZnO (20 nm, 20 μg, in 10 min)
reduced the total number of living E. coli by more than 88%, 77% and
71%, respectively.
• ZnO, Al2O3 and TiO2 NPs were toxic to the nematode Caenorhabditis
elegans inhibiting growth especially the reproductive capability.
• Likewise, oxides of Zn, Cu and Ti NPs have been reported to be toxic to
the microalgae Pseudokirchneriella subcapitata.
• Exposure of earthworm (Eisenia fetida) to ZnO NPs enhanced mortality
with increasing concentrations of NPs (Li et al., 2011)
Source: Adams et al. (2006); Aruoja et al. (2009); Wang et. al. (2010)
15

16. Jason Unrine’s UK research team mixed earthworms into artificial soil tainted with gold
nanoparticles. After 28 days, Unrine’s team detected gold nanoparticles throughout the
earthworm’s bodies, with the highest concentrations in their gut. Some of the exposed
worms produced 90 percent fewer offspring. This study can serve as a model for how
organisms take up other kinds of nanoparticles. 16

17. Mechanisms of toxicity of metal ENPs
• Pitting of the cell wall, dissipation of the proton motive
force, and finally cell death (Choi et al., 2008).
• Ag NP would also bind with bacterial DNA, and this
might compromise the DNAs replication fidelity (Rai et
al., 2009; Yang et al., 2009).
• These metal oxide NPs may act as „Trojan-Horses‟,
entering cells and releasing ions intracellularly (Limbach
et al., 2007).
FE-SEM images of captured E. coli
using anti-E. coli antibody
functionalized magnetic
nanoparticles 4 (a), (b), (c), and
(d) four different images at
different places of the sample.
[Source: Rastogi et al. (2011)]
Figure 1. left panel: Silica NPs; right panel: E. coli cells with intrenalized Si NPs
[source: http://www.egr.msu.edu/~hashsham/group/project_Yang.shtml]

18. Schematic diagram of colloidal Ag nanoparticles interaction on captured E. coli cell with NPs 4, over the period
of time and observed biomolecules [Source: Rastogi et al. (2011)]
18

19. Figure 2: Interaction of amorphous TiO2 nanoparticles with
MDCK (Madin Darby canine kidney) culture cells. I h
incubation.
A – nanoparticles fill folds and invaginations of a cell;
B - E – direct contact of the nanoparticles with cell plasma
membrane;
F – invagination of plasma membrane containing
nanoparticles;
G, H – nanoparticles in “coated pits”, clathrin particles are
visible on the pit cytoplasmic surface;
I, J – nanoparticles in endosomes. Arrows show empty
“coated pits”. Ultrathin sections. Transmission electron
microscopy.
Source: Ryabchikova, E; Mazurkova, N; Shikina, N; Ismagilov, Z; “The Crystalline Forms Of Titanium Dioxide
Nanoparticles Affect Their Interactions With Individual Cells”, JMedCBR 8, 27 October 2010
[http://www.jmedcbr.org/issue_0801/Ryabchikova/Ryabchikova_Nano_10_2010.html]
19

20. Effect on soil microorganisms
• Plant growth promoting rhizobacteria (PGPR) like P. aeruginosa, P. putida, P. fluorescens,
B. subtilis and soil N cycle bacteria viz., nitrifying bacteria and denitrifying bacteria have
shown varying degrees of inhibition when exposed to ENPs in pure culture
conditions or aqueous suspensions (Mishra and Kumar, 2009).
• Metal oxide NPs of Cu (80 to 160 nm) showed antibacterial activity against plant growth
promoting Klebsiella pneumoniae, P. aeruginosa, Salmonella paratyphi and Shigella strains
(Mahapatra et al., 2008).
• Iron and copper based NPs are presumed to react with peroxides present in the
environment generating free radicals known to be highly toxic to microorganisms like P.
aeruginosa (Saliba et al., 2006).

21. Bright-field micrographs of Anabaena CPB4337 exposed to increasing concentrations of ceria nanoparticles.
Rodea-Palomares et al. Toxicol. Sci. 2011;119:135-145
(A) Control Anabaena filaments.
(B and C) Anabaena filaments exposed to 1 mg/l N10 for 48 h and 80 mg/l N10 for 72 h.
(D, E, and F) Anabaena filaments exposed to 0.1 mg/l N25 for 72 h, 1 mg/l N25 for 24 h, and 80 mg/l for 24 h.
(G and H) Anabaena filaments exposed to 0.01 mg/l N50 for 48 h and 50 mg/l N50 for 48 h.
(I, J, and K) Anabaena filaments exposed to 1 mg/l N60 for 24 h, 50 mg/l N60 for 72 h, and 80 mg/l N60 for 24 h. Bars, 20 μm.
P.S. Anabaena is a genus of filamentous cyanobacteria that exists as plankton. It is known for its nitrogen fixing
abilities in submerged / paddy soils, and they form symbiotic relationships with certain plants, like Azolla)

22. Published literature on the effects of ENPs on soil microorganisms
ENP Effects Source
Carbon containing No inhibition in the activity of dehydrogenase and activities of enzymes Tong et al.
fullerenes/ CNTs involved in N (urease), P (acid-phosphatase) and C (β-glucosidase) cycles in (2007)
the soil. A slight shift in bacterial DNA, indicating a minor change in the
community structure measured using PCR-DGGE. (Incubation study)
Number of fast-growing bacteria decreased by three-to four folds Johansen et
immediately after incorporation of the C60 and protozoan number decreased al. (2007)
only slightly in the beginning of the experiment. A slight shift in bacterial DNA,
indicating a minor change in the community structure measured using PCR-
DGGE. (Incubation study)
No significant effect on the anaerobic community of biosolids from Nyberg et al.
anaerobic wastewater treatment sludge over an exposure period of a few (2008)
months. No change in methanogenesis and no evidence of substantial
microbial community shifts due to treatment with C60. (Microcosm study)
Multi-walled CNT significantly inhibited the activities of 1,4-β- Chung et al.
glucosidase, cellobiohydrolase, xylosidase, 1,4-β-N-acetylglucosaminidase, (2011)
phosphatase and microbial biomass-C and -N in soils. (Incubation study)
Source: Dinesh et al. (2012)

23. Published literature on the effects of ENPs on soil microorganisms
ENP Effects Source
Metal and Effect of Ag-NP on soil dehydrogenase activity was severe and bacterial Murata et al.
metal colony growth was inhibited at levels between 0.1 and 0.5 mg Ag kg-1 soil. (2005)
oxide (Incubation study)
ENPs
Ag-NP inhibited soil denitrifying bacteria when Ag was added to soils in Throbäck et.
amounts ranging from 0.003 to 100 mg kg-1 dry weight. (Incubation study) al.(2007)
Soil respiration studies show that there were no statistical differences Doshi et al. (2008)
between the time and sizes of peaks in CO2 production and the total
mineralization of glucose due to addition of nano-Al. (Column study using
silica-sand mixture)
The influence of Si-, Pd-, Au- and Cu-NPs on microbial communities was Shah & Belozerova
insignificant. (Microcosm study) (2009)
Ag-NP did not influence microbial biomass-N, enzyme activities, soil pH Hänsch and
and organic C. Microbial biomass was significantly decreased while basal Emmerling (2010)
respiration and metabolic quotient was increased with increasing Ag-NP
application rate. (Incubation study)
(Source: Dinesh et al. (2012)

24. Published literature on the effects of ENPs on soil microorganisms
ENP Effects Source
Metal and TiO2- and ZnO-NPs reduced both microbial biomass and bacterial Ge et al. (2011)
metal oxide diversity and composition indicating that nanoparticulate metal oxides
ENPs may measurably and negatively impact soil bacterial communities.
(Microcosm study)
TiO2- and ZnO-NPs significantly inhibited soil protease, catalase, and Du et al. (2011)
peroxidase activities; urease activity was unaffected. (Field study)
Zn- and ZnO-NPs inhibited the activities of dehydrogenase, β - Kim et al. (2011)
glucosidase and acid phosphatase in soils. (Pot study)
Ag-, Cu- and Si-NPs impacted arctic soil bacterial community; Ag-NPs Kumar et al.
were highly toxic to a plant beneficial bacterium, Bradyrhizobium (2011)
canariense. (Incubation study)
(Source: Dinesh et al. (2012)

25. Fig. Key processes in soil relating to transformation and potential risk from
manufactured nano particles (MNPs; Source: Klaine et al. (2008)
1. Dissolution
2. Sorption/aggregation
3. Plant bioaccumulation
4. Invertebrate accumulation and toxicity
5. Microbial toxicity
6. Direct particle uptake/toxicity
7. Particle migration
MNPs
6
2
3
4
1 Dissolved
pool
7 5

26. Conclusions
• The anti-microbial activity of metal NPs to soil microbial communities holds great
significance.
• Little information is available on how metal ENPs act in the soil matrices
especially their adsorption to clay minerals, organic fractions, toxic substances,
organic pollutants etc.
• More information is needed on interaction of ENPs with soil components and more
quantitative assessments of aggregation/ dispersion, adsorption/
desorption, precipitation/ dissolution, decomposition, and mobility of
ENPs in the soil environment is essential.
• Overall, it is apparent from the studies done in vitro that ENPs pose a potential
hazard to microorganisms.
• Studies done by incubating soils with ENPs, microcosm studies and pot
experiments suggests that in most cases ENPs inhibit soil microbial activity.

27. • This underlines the fact that the effects of ENPs on microbial
community in soils under field conditions is still in its infancy,
the smothering effects of SOM and HA on ENPs are still being
speculated and the bacterial self protection-mechanism on
encountering ENPs in the soil matrix is yet to be extensively
studied.
• Considering that attempts are being made to employ some of these
ENPs as carrier materials for smart delivery of chemical fertilizers and
pesticides to crops, (DeRosa et al., 2010) it is imperative that we set
specific standards for the manufacture, use, and disposal of
ENPs.
• Therefore, conclusive evidences need to be obtained to draw strong
conclusions about the potential toxicity of ENPs to microbial activity
under field conditions and herein lies one of the main challenges in
environmental risk assessment of spreading ENPs.

28. the risks of NPs for
However, before these advantages can come into play,
the environment and crops have to be defined to ensure their
sustainable and beneficial application.
Relevant ecotoxicological information on exposure and effects of NPs as a basis
for a comprehensive risk assessment is needed.
(Source: Bucheli TD- Effects of NANOparticles on beneficial soil MIcrobes and CROPS
(NANOMICROPS);
http://www.nfp64.ch/E/projects/environmental_research/effects_nanoparticles_microbes_crops/Pa
ges/default.aspx; accessed on 28/2/20120)

29. An Engineered Nano Particle Risk Assessment (ENPRA) Approach
ENPRA aims at developing and implementing a novel integrated approach for ENP Risk Assessment.
This approach is based on the Exposure-Dose-Response Paradigm for ENP (see figure below).
• This paradigm states that exposure to ENP of different physico-chemical characteristics via
inhalation, ingestion or dermal exposure is likely to lead to their distribution, beyond the portal-of-
entry organ to other body systems.
• The cumulative dose in a target organ will eventually lead to an adverse response in a dose-
response manner.
The Exposure-Dose-Response paradigm
[Source: http://www.mapfre.com/fundacion/html/revistas/seguridad/n114/articulo1En.html]
29

30. The approach proposed by ENPRA is in line with the grand challenges described by Maynard
et al. (2006). The rationale of ENPRA is summarised graphically in the below figure .
[Source: http://www.mapfre.com/fundacion/html/revistas/seguridad/n114/articulo1En.html; accessed on 29/02/2012]
30

31. Nanotechnology has not yet been proven to be safe for humans or for the environment.
Oxford University and Montreal University linked titanium dioxide and zinc oxide nanoparticles in
sunscreen to causing free radical and DNA damage in skin. And numerous other studies have
found that nanoparticles are easily absorbed by cells, where they cause other untold harm within the
body.
Nanoparticles have been found to cause brain damage in fish and other aquatic species
exposed to them.
ENPs have been found to be toxic to microorganisms.
Possible toxicity mechanisms include
• disruption of membranes or membrane potential
• oxidation of proteins,
• genotoxicity,
• interruption of energy transduction Quantum dots within D. magnia
• formation of reactive oxygen species and [Source: http://cben.rice.edu/ShowInterior.aspx?id=148]
• release of toxic constituents
• structural changes to the microbial cell surface that may eventually lead to cell death.

32. 'The key question therefore is how to benefit from nanotechnologies while
limiting these risks?'
One suggestion is to ‘take into account the ethical, health, environmental and
regulatory considerations linked to nanotechnologies as early on as possible in
the R&D phase, and to encourage dialogue with the public’.
The aim is to ‘spark debate early on among all those concerned so as to avoid
repeating the mistakes made with genetically modified organisms (GMOs)
where the public were reluctant to accept anything mildly related to GMOs’ (Stephen
Schaller, EU funded Nanologue project).
(Source: ‘Dissecting the pros and cons of nanotechnologies‟ at
http://cordis.europa.eu/fetch?CALLER=NEWSLINK_EN_C&RCN=26524&ACTION=D) accessed on 28/2/2012)
Further reading : Dinesh, R., Anandaraj, M., Srinivasan, V., Hamza, S., 2012. Engineered nanoparticles in the soil and
their potential implications to microbial activity. Geoderma 173-174, 19-27.

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