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TiO2 Nanoparticles Increase the Toxicity of Roundup

WeatherMax® in Exposed Frog Tadpoles

Christina Fridgen1, Marguerite Xenopoulos2, Chris Metcalfe3Trent University, Peterborough, ON, CanadaTrent University, Peterborough, ON, CanadaTrent University, Peterborough, ON, CanadaTrent University, Peterborough, ON, CanadaTrent’s Integrated Nanomaterials Investigations Group

1. Environmental and Life Sciences Graduate Program

2. Department of Biology

3. Environmental and Resource Studies

Manufactred nanoparticles (MNs)

Cellular interaction

• brakes, binds, and activates cell membranes

• cellular uptake of MNs through gills and intestines for potential distribution throughout the body.

• taken in through processes of diffusion, endocytosis and adhesion

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• with 100% bioavailability intravenously administered nTiO2 accumulated in lung, spleen, liver, kidney

• Only liver retained nTiO2

particles on day 28

Distribution through organs

Direct and indirect toxicity?

• MNs are useful in medicines for increased drug delivery

• MNs can increase the bioavailability and bioaccumulation of metals (arsenic and cadmium) to fish (Zhang et al, 2007; Sun et al, 2009)

• Presence of MNs can change the toxicity of xenobiotics to aquatic organisms (Baun et al. 2008).

• Interaction with common pesticides and natural factors (UV, NOM)?

Influence of titanium dioxide nanoparticles on speciation and bioavailability of arseniteSun et al., 2009 Environmental Pollution, Volume 157, Issue 4, Pages 1165-1170Hongwen Sun, Xuezhi Zhang, Zhiyan Zhang, Yongsheng Chen, John C. Crittenden

Aquatic Model Organisms

• Ranids– Northern Leopard Frog (Rana pipiens)

• fast time to metamorphosis (~2-3 months)

– Bullfrog (R. catesbeiana) • slow metamorphosis time (2-3 years)• large tissue samples

• Gene Chips

• Vertebrate Development– Growth (SVL, mass)– Differentiation (Metamorphosis)– Organogenesis

Nano Titanium Dioxide(nTiO2)

As a photocatalyst under ultraviolet light. The strong

oxidative potential of the positive holes oxidizes water to create hydroxyl radicals. It can also oxidize oxygen

or organic materials directly.

•sterilizing, deodorizing, anti-fouling,hydrolysis catalyst.

•chemical solar cell, converting sun to energy.

wastewater remediation – (the environmental

‘white night’)detoxification occurs under ambient conditions

very slowly, direct UV light exposure increases the rate of reaction.

nTiO2 (NA) = Nanoamour Product (10nm)

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Questions

• Will exposures of leopard frog (Rana pipiens) and bullfrog (R. catesbeiana) tadpoles to titanium dioxide 10nm nanoparticles (nTiO2) produced an acute toxic response?

• Will long term exposures to nTiO2 particles accumulate in organs and cause tissue damage?

• Will co-exposures of MNs influence the acute toxicity of the herbicide, Roundup WeatherMax®.

• 4mg of powder (10nm) / 40ml MilleQ water

• sonicate for 1.5 min at 20kHz/300 Watts/L

centrifuge – 4X40ml

- 10 min

- RPM???

nTiO2 (NA) Preparation

nTiO2 QuantificationSize analysis Concentration

DLS - 91nm (± 41)

SEMUV Absorption: λmax

360nm33 mg/L (± 0.5 mg/L)

33% of nominal –particles lost due to

centrifugation

nTiO2 (NA)96hr Acute Toxicity

• R. pipiens tadpoles, 2008

• no mortality was observed (1 to 250 mg/L - nominal)

• tadpoles filtered suspended nTiO2 out of

the water column

– concentrate it in their intestines before defecating it in pellet form.

nTiO2 (NA)Chronic toxicity - Histology

• Over a more chronic exposure (21d), we found no signs of tissue injury in bullfrog tadpoles

exposed to nTiO2 (concentration 0.1 to 1 mg/L)

– Gross morphology observed – no significant pathologies

– Mast cell number counted – no significant increase

*No presence of goblet cells at such an early stage in Bullfrogs even at day 150 days old.

CONTROL nTiO2 1ppm - 21 daysTad

po

le A

bd

om

en

4x

Inte

stin

es 4

0x

nTiO2 particles?

Ep

ithe

lial C

ells

3

nTiO2 (NA) 1ppm - 21 days

Liver 40x Intestine 40x

CONTROL nTiO2 1ppm - 21 days

Filte

r Ap

para

tus

Gill T

uffs

Howe (Fridgen) et al., 2004. ET&C. 23(8) 1928–1938, 2004

nTiO2 influenced the acute toxicity of the

herbicide, Roundup WeatherMax® (RW)

• The presence of nTiO2

increased the 96hr toxicity of Roundup WeatherMax® at 1.8

(mg FAE/L)

Trials were variable (consistent according to brood)

0.6 1.8 3.6 6.0 0.6 1.8 3.6 6.0 0.6 1.8 3.6 6.0

1010

60%63.3%36.6%

• Furthermore, tadpole weight was significantly (P< 0.05) decreased by Roundup WeatherMax®only in the presence of nTiO2.

Roundup 3ppm Roundup 3ppm and TiO2 1ppm Roundup 3ppm and TiO2 10ppm

•This decrease in mass was correlated with an increase in tail damage, indicating increased tadpole stress.

0.6 1.8 0.6 1.8 0.6 1.8

nTiO2 influenced the acute toxicity of the herbicide, Roundup WeatherMax® (RW)

RW 1.8 mg FAE/L alone RW 1.8 mg FAE/L + nTiO2 1ppm RW 1.8 mg FAE/L + nTiO2 1ppm

Vive Nano Products

Nano Silver, nAg (-)• Anti-bacterial, anti-viral and anti-

fungal applications

• []1.5g/L; size (TEM): 2-10nm

• ICP – 27.74% AG

• UV Absorption: λmax 420nm

• pH: 6.8, colour: amber

SEM (<10nm)

Nano Titanium Dioxide, nTiO2 (-)

• []1.5g/L; size (TEM): 3-8nm

• ICP – 27.86% Ti

• UV Absorption: λmax ??

• pH: 9.10, colour: clear

LOGO HERE!

‘Our main focus is on the development of [water dispersible nanomaterials] for the catalyst and crop protection industries’.

(capped with carboxy groups (-) polyacrylate sodium)

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SPECIES COMPARISON 96hr LC50s

Silver ion

yet to be analyzed!!

• nTiO2 (NA) >35mg/L

• nTiO2 (- VN) >35mg/L

• nAg (- VN) BF- 3.8 mg/L; GF- 10.1 mg/L

• RW BF- 3.05 mg FAE/L; GF- 4.55 mg FAE/L

nTiO2 (-) VN

nAg (-) VNRoundup WeatherMax

0.3 1.2 2.1 3 3.9

0.3 1.2 2.1 3 3.9

0.3 1.2 2.1 3 3.9

Chronic Toxicity??• Evidence of the mechanism of this

contaminant interaction.

• Animals preserved for:

– Tissue histology and TEM (filtering apparatus, gills, intestines, and liver)

– Bioaccumulation• Tissue contaminant loads of nAg,

nTiO2 and glyphosate (ICP-MS)

– Gene expression (C.Heibing, UVic)• Gene chip vs. targeted gene groups

Enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticlesZhang et al., 2007. Chemosphere, Volume 67, Issue 1, Pages 160-166

Conclusion

When conducting risk assessment we must

remember the influence that MNs may have

directly and indirectly by interacting with existing

contaminants

Enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticlesZhang et al., 2007. Chemosphere, Volume 67, Issue 1, Pages 160-166

Acknowledgements

Thanks to everyone in our TINI research group at

Trent University

And to our funders