Will Lush

Environmental Bio-Detection Products Inc (Biotoxicity.com).

WHY AREN’T WE TESTING FOR GENOTOXINS

IN OUR ENVIRONMENT?

Will LushV. President of EBPI (Environmental Bio-Detection Products Inc.)10 Years in management role with EBPIOversaw and key contributor of the development of:

Ames ExpressTM StrainsMod-ISO KitEBPI’s Educational Line of Test Kits

Will Lush has presented at conferences and universities internationallyfocusing on the importance of genetic effects of emerging contaminates.

Education:Biology Systems EngineeringInternational Trade

wlush@biotoxicity.com905.826.8378

Conferenza• Introduction to EBPI

• Application of EBPI Product Line

• Technical Presentations• Toxi-ChromoTest Kit

• SOS-ChromoTest Kit

• UMU-ChromoTest Kit

• Ames Test Kits• Muta-ChromoPlate Kit

• Mod-ISO Kit

• Ames-ISO Kit

• Ames Express Strains

EBPI – Environmental Bio-detection Products Inc.

• Specialized Canadian biotechnology company based out of Mississauga Ontario

• “Measuring the Health of our Environment”

• Develop, Manufacture, Distribute testing products (kits) to assess various toxic endpoints and bacterial contamination

• Mainly biological based assays

• Focused on Research and Development

What is Toxicity

Toxicity is the degree to which a substance can damage an

organism’s capability to function in its environment.

Acute toxicity – destroys its ability to function (kills it)

Chronic toxicity – impairs its ability to function (reduces its competitiveness)

What is Genotoxicity

Genotoxicity describes the property of chemical agents that

damages the genetic information of an organism.

Effect is most commonly chronic but can be acutely toxic

What is Mutagenicity

Mutagenicity refers to the induction of permanent transmissible

changes in sequence of the cells genetic material. These changes

(mutations) may involve a single gene or a block of genes. Common

mutation types include:

• Frameshift Mutations

• Base-Pair Mutations

Consequences of Responses to Genetic Effects

High fidelity genetic transcription is critical ! Organisms have evolved many repair mechanisms to maintain genome integrity.

If serious genetic damage cannot be repaired, cellular death is often triggered

If genetic damage is not repaired then “disease” can be the consequence

Often genetic diseases such as cancer take many years to manifest through genetic errors

New Chemicals and Pharmaceuticals

Regulated industry requiring testing for mutagenicity and genotoxicity to limit Human and environmental exposure to genotoxins and mutagens

Applications to areas beyond new chemicals and drugs

1) Infrastructure Researcha) Drinking waterb) Waste water

Water Contaminants

Research in Canada

University Of Toronto: Drinking Water Research Chair.

University of Windsor Wastewater Effluent

Western UniversityDrinking water and wastewater

Drinking Water Sampling Locations in North America

Courtesy of Drinking Water ResearchGroup University of Toronto

Results of Drinking water testingMicrotox Test

0

20

40

60

80

100

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

O3/C

l2

LP

UV

LP

UV

/Cl2

MP

UV

MP

UV

/Cl2

AO

P

AO

P/C

l2

EC

20 1

5 m

in (

%)

Edmonton Peterborough WaterlooSmiths Falls Truro BalaCamrose Tampa 1 Tampa 2

0

20

40

60

80

100

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

O3/C

l2

LP

UV

LP

UV

/Cl2

MP

UV

MP

UV

/Cl2

AO

P

AO

P/C

l2

EC

20 1

5 m

in (

%)

Edmonton Peterborough WaterlooSmiths Falls Truro BalaCamrose Tampa 1 Tampa 2

0

20

40

60

80

100

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

O3/C

l2

LP

UV

LP

UV

/Cl2

MP

UV

MP

UV

/Cl2

AO

P

AO

P/C

l2

EC

20 1

5 m

in (

%)

Edmonton Peterborough WaterlooSmiths Falls Truro BalaCamrose Tampa 1 Tampa 2

No Significant Change for Most Treatments – Weak Positives

Courtesy of Drinking Water Research Group University of Toronto

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

O3

Cl2

LP

UV

Cl2

MP

UV

Cl2

AO

P

Cl2

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

O3

Cl2

LP

UV

Cl2

MP

UV

Cl2

AO

P

Cl2

Mu

tag

en

icit

y R

ati

o

Edmonton 2.0 mg/LPeterborough 6.0 mg/LMannheim 6.0 mg/LSmiths Falls 6.7 mg/LTruro 7.3 mg/L

95% Significance

Muta ChromoPlateTM TestLow TOC water (<10 mg/L)

Weak Mutagenicity Associatedwith Chlorination

Courtesy of Drinking Water Research Group University of

Toronto

Muta ChromoPlateTM Test High TOC (>10 mg/L)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

NH

2C

l

ClO

2

O3

LP

UV

MP

UV

AO

P

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

O3

Cl2

LP

UV

Cl2

MP

UV

Cl2

AO

P

Cl2

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

95% Significance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Co

ntr

ol

ND

Cl2

O3

Cl2

LP

UV

Cl2

MP

UV

Cl2

AO

P

Cl2

Mu

tag

en

icit

y R

ati

o

Bala 14.1 mg/L

Camrose 17.9 mg/L

Tampa 1 21.8 mg/L

Tampa 2 21.8 mg/L

95% Significance

Significant Mutagenicity Associatedwith Chlorination

Courtesy of Drinking Water Research Group University of Toronto

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25

Total Organic Carbon (mg/L)

Mu

tag

en

icit

y R

ati

oCl2

O3/Cl2

LP/Cl2

MP/Cl2

AOP/Cl2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25

Total Organic Carbon (mg/L)

Mu

tag

en

icit

y R

ati

oCl2

O3/Cl2

LP/Cl2

MP/Cl2

AOP/Cl2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25

Total Organic Carbon (mg/L)

Mu

tag

en

icit

y R

ati

oCl2

O3/Cl2

LP/Cl2

MP/Cl2

AOP/Cl2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25

Total Organic Carbon (mg/L)

Mu

tag

en

icit

y R

ati

oCl2

O3/Cl2

LP/Cl2

MP/Cl2

AOP/Cl2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25

Total Organic Carbon (mg/L)

Mu

tag

en

icit

y R

ati

oCl2

O3/Cl2

LP/Cl2

MP/Cl2

AOP/Cl2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25

Total Organic Carbon (mg/L)

Mu

tag

en

icit

y R

ati

oCl2

O3/Cl2

LP/Cl2

MP/Cl2

AOP/Cl2

Muta-ChromoPlate and TOCPositive correlation with Cl treatments and TOC

Courtesy of Drinking

Water Research

Group University of

Toronto

Disinfection By Products

THM

Nitrosamines

MX

Haloquinones

Bromate

Canary in a Coal Mine

Mutagenicity Assay Data of Wastewater Effluent

99% significance

Why Regulate?

Things are changing in society• Reuse of waste water for drinking water

• Thousands of new chemicals being released into our

environment everyday

• Build up of persistent chemicals in the environment

• Interactions between chemical compounds and

organisms in the environment is largely unknown.• Availability of low cost techniques and technologies to evaluate at a molecular

level are available

• Regulators are recognising them, and starting to RECOMMEND testing

South Africa

UMU-TestISO 13829 (Water Quality Determination of the genotoxicity of water and waste water using the umu-test)

SOS-ChromoTest

Water services provider that purifies 3600 megalitres of water per day.

Services 11 million people in Gauteng and parts of Mpumalanga, North West and Free State Provinces

3000 km of pipelines, to 19 water services authorities

Recharging Aquifers

Municipalities in Florida are requiring genotixicty testing so as to not inadvertently contaminate the aquifer.

Municipality Regulations for Genotoxicty Testing

Genotoxicity Testing Internationally

European Union European legislation includes genotoxicitytesting as part of the Water Framework Directive (WFD), but not yet mandated

France, Germany, Sweden and Holland have all adopted genotoxicity testing as part of their biological testing strategies to ensure safe water Supplies.

Germany

Implemented Regulation based on the UMU-Test

Genotoxicity testing only applies to effluent from the chemical production industrial sector

• Induction Ration must be greater than 1.5

• Growth Factor must be greater than 0.5

0

2

4

6

8

10

12

0 1 2 3 4 5 6

DILUTION FACTOR

2AA

Growth Factor Rel gal activity Induction ratio IR Threshold

Application to Environmental Research

Deepwater HorizonOil Spill

Crude oil to Gulf released over 86 Days

20 April 2010

Deepwater Horizon 4.9 Million Barrels of oil released

or

185 Million Gallons of crude oil (700 million litres)

90-95% of the worlds dispersants were usedIn an attempt to disperse the oil

Use of Dispersants to prevent

Research

University of South Florida

High Levels of Mutagenic activity was detected

US EPA (United States Environmental Protection Agency)

Minimal Levels of Acute Toxicity

24 – 48 acute toxicity test

Regulation

Application to Health Research

Detection of mutagenic activity from leaching rubber pellets from artificial turf.

How do rates of mutation differ in Space from those on earth?

Forward Thinking CompaniesBeyond Regulation

NASA - National Aeronautics and Space Administration

Genotoxic effects of Rocket Fuel in wetlandsadjacent to the launch site

asymmetric dimethylhydrazine (ADMH)

EBPI Educational Kits

Development of Educational Kits

Focus on synthetic and bacterial based

• High Schools• Colleges• Universities

www.highschoolbiotechnology.com

Education of Students

• Tell students something and they immediately forget it

• Teach students something well and they will remember it

• Involve the students in the process and they will learn it

Air Particulate Mutagenicity Monitoring

• Chronic dangers from air pollution and particulate matter (PM)• Small particulate from air pollutants penetrate airways and can

be absorbed by lung tissue in the alveoli• Chemicals adsorbed to these particles also carried in and

represent long-term exposure scenario• Many known and suspected carcinogens are contained in

polluted air- PAHs- Nitroaromatics- Nitrosamines- VOC

• A system to filter and quickly test air samples for long term effects would be valuable for human health risk assessments

EBPI PM Mutagenicity Assays

• Designed assay system to• Filter air pollution for particulate matter (PM between 2.5

and 10 µM)• Extract filtrate using optimized solvent system

• Dichloromethane and acetone• Sonication and mixing = high extraction of mutagenicity

• Test filtrates on EBPI kits

Can we improve on sensitivity over conventional methods?• Reduce number of tests done• Eliminate the need for S9 addition (expensive and time-

consuming)• Take advantage of known metabolic pathways for priority

pollutants

AMES-EXPRESS

EBPI PM Mutagenicity Assays

Early testing demonstrates significant increase of mutagenicity detection using Ames-Express Strains

Comparable mutagenic detection to systems using S9 bioactivation

Sensitive! User friendly!Cheap!Fast!

0

10

20

30

40

BG PC A B C DRev

erta

nt

Wel

ls/

48

To

tal w

ells

Cigarette Smoke Extract TA98 strains MOD ISO Procedure read on Day 3

TA 98 TA98 + S9 TA98 1A2

0

10

20

30

40

BG PC A B C D

Rev

erta

nt

wel

ls/

48

wel

ls

Cigarette Smoke Extract TA100 strains MOD ISO Procedure read on Day 3

TA100 TA100 + S9 TA100 1A2

Nanotechnologies and Aptamers

• Development of nanoparticulates for the concentration and accumulation of lipophilic environmental contaminants.

• Construction of biosensors using modified oligonucleotides (DNA, RNA) to detect priority contaminants at low concentrations and at low cost.

Questions?

Biotoxicity.com