Environmental Bio-Detection Products Inc (Biotoxicity.com...
Transcript of Environmental Bio-Detection Products Inc (Biotoxicity.com...
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
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