Sci Tox Linked In Seminar

TOXICITY ANALYSIS
A Real-World, Real-Time Technique
for the Wastewater Market.

INTRODUCTION
The SciTOX Vision and Process

- Payback - WWTP specific

SciTOX, the company
- Rapid - Cost to Own
Value
Value
Proposition

 Created in December of 2008
 Started as a result of a public investment offering.
– The offering closed oversubscribed by 30%, at 1.3M NZD.
 Company has exclusive license to a technology developed
and patented by Lincoln Ventures Ltd. (www.lvl.co.nz)
– A subsidiary of Lincoln University, New Zealand.
– Technology is a biosensor developed under government FRST
(Foundation for Research in Science and Technology) funding.

10/05/2010
The Rapid Toxicity Measurement System


SciTOX, the company
Value
Value
Proposition

 Application development of the technology and product done at
several sites
– Lincoln Ventures, Dr. Neil Pasco, Manager of Biosensor Group
– Griffith University, Australia. PhD thesis of Dr. Kylie Catterall
– Gold Coast Water, Australia (www.goldcoastwater.com.au)
• Dr. Kylie Catterall, Manager. Young Environmental Scientist of the Year, Australia,
2008
– Maarten van Eerten, Tomari Technology, Contract scientist for SciTOX.
Developed significant calibrations for other analytical technologies in NZ and
Australia. www.tomari.co.nz
– Dr. Aaron Marshall, University of Canterbury. Chemical Engineering and
Electrochemistry. (www.canterbury.ac.nz)
 Company located in Christchurch, New Zealand
– About 15 minutes drive from Lincoln Ventures.

10/05/2010


SciTOX: Board of Directors
Value
Value
Proposition

 Dr. Merv Jones, Chairman. Chemical Engineer. Former (retired) Asia-
Pacific Vice President for URS, a global Environmental Engineering
company. (http://www.urscorp.com/)
 Colin Harvey. Founder and Managing Director of Ancare, New
Zealand. (www.ancare.co.nz) Veterinary products. Sold company in
2007 and now is Venture Capital provider.
 Brent Ogilvie: Pacific Channel Ltd., investment advisor. New Zealand
Venture Investment Fund. (www.pacificchannel.com)
 Ralph Wattinger: CEO and Managing Director, SciTOX. Managing
Director of Int2egy Limited and Int2egy (NZ) Ltd. (www.Integy-Ltd.com)
Formerly with Emerson Company and Teledyne Technologies. Co-
founder of SciTOX.
 Peter Barrowclough: CEO of Lincoln Ventures Ltd. Director at
Canterbury Development Corporation. Former R&D Manager for
PGG Wrightson.

10/05/2010


Technology Patents
Value
Value
Proposition

 Generated by Lincoln Ventures and licensed exclusively to SciTOX.
 SciTOX Patent Portfolio
– Method and apparatus for measuring use of a substrate in a microbially catalysed
reaction; New Zealand; 336072; Granted
reaction; Australia; 717224; Granted
reaction; USA; 6,379,914; Granted
reaction; Japan; 3479085; Granted
reaction; Europe; 97946176.1; Allowed
reaction; Canada; 2307603; Granted
 SciTOX plans to file further patents as warranted.
10/05/2010

The Market
Wastewater Treatment

Wastewater Treatment: The Bottom Line Value
Value
Proposition

 Virtually any secondary treatment will be biological in nature, and
susceptible to toxicity.
 Any secondary treatment requires considerable electric power, and is
expensive.
 If the WWTP is advanced and uses nitrification and/or denitrification,
or biological phosphorous removal, it is more expensive to operate
and more susceptible to toxins.
 Any control measurement must be as close to real-time as possible, so
possible problems can be dealt with before they affect the treatment
process.

10/05/2010


VALUE Measurements: Where
Value
Value
Proposition
Influent
3.

Primary Treatment Secondary Treatment Secondary Clarification

2.
1. 2.

3.
Primary Solids

Methane Secondary Solids
2.

Supernatant
Dewatered
Solids 1. Toxicity
2. BOD correlation
Anaerobic Digestion 3. Food/Micro-Organism

1. 2.
Contract Waste Hauler

Raw Influent Final Effluent

10/05/2010

The Technology
Electrochemistry and Biosensors:
Theory and application


Electrochemistry, the basics
Value
Value
Proposition

 It is a well-known analytical technique
– Yet, most of us have probably not used or thought about it since university.
 Sensitive and robust
 Easy to maintain and operate
– Minimal sample, or reagent needed.

 Measures differences in the electric potential in samples before and
after either oxidation or reduction. It is a Redox measurement.
 Uses a Potentiostat to measure the current.

10/05/2010


Potentiostat basics
Value
Value
Proposition

 Definition
– A potentiostat is an electronic instrument that controls the voltage
difference between a working electrode and a reference
electrode. Both electrodes are contained in an electrochemical
cell. The potentiostat implements this control by injecting current
into the cell through an auxiliary, or counter, electrode.
– In almost all applications, the potentiostat measures the current
flow between the working and auxiliary electrodes. The controlled
variable in a potentiostat is the cell potential and the measured
variable is the cell current.

10/05/2010


Electrode, types
Value
Value
Proposition

 Material can be fabricated from a variety of materials
 Cathode usually larger. Reaction is not measured at this
pole.
 Anode is smaller.
– Micro-electrodes (anodes) typically are limited to a maximum of 50
microns
– Larger anodes increase the degree of interference from other
reactions.

10/05/2010


Biosensors
Value
Value
Proposition

 Biosensors are analytical devices that detect, transmit and
record information about a physiological or biochemical
change.
 They are composed of two essential elements:
– a bio-recognition component (bio-component, cells) and,
– a transducer.

ref: D'Souza SF (2001) Biosens. Bioelect. 16(6), 337-353. –
Excellent review of whole cell biosensors, including functionality,
immobilization, transduction and applications.

10/05/2010


Biosensors
Value
Value
Proposition

 Whole cell, or microbial, biosensors may
incorporate either prokaryotic or eukaryotic
cells as the bio-component.
 Bio-sensing strategies based on cellular
respiration have, historically, used a number of
monitoring techniques including measuring:
– Oxygen depletion (due to the breakdown of carbon
structures and terminal electron accepting activity);
– Generation of CO2 (through the Kreb’s cycle);
– Accumulation/production of reduced co-factors (using
redox mediators/dyes); and
– Production of ATP (via luminescent proteins).

10/05/2010


Whole Cell Biosensors: The benefits
Value
Value
Proposition

 Cost: Microorganisms are cheaper, quicker and easier to produce and
do not require extensive purification. Nor do they need the addition
of expensive co-factors.
 Stability: The cellular environment protects sub-cellular components
from inactivation and preserves intracellular enzyme systems in their
natural environments.
 Broad spectrum range: Microorganisms are present ubiquitously and are
able to metabolize a wide range of substrates. Whole cells also
provide a multi-purpose catalyst, particularly useful when the biosensor
requires the participation of a number of enzymes in sequence.
 Shelf-life: Whole cells can be immobilized onto the sensor and stored
for many months, requiring only a re-hydration step before use. By
comparison, enzyme biosensors can only last for a few days.

10/05/2010


Whole Cell Biosensors: The benefits
Value
Value
Proposition

 Adaptability: Microorganisms have a great capacity to adapt to adverse
conditions and can develop the ability to degrade new compounds over
time.
 Modification: Microorganisms are amenable to genetic modifications
through mutation or recombinant DNA technology.
 Growth rate: Microorganisms have a large population size, are self
replicating, have a rapid growth rate and are easy to maintain.
 Generality: A major strength of whole cell bio-sensing is not the
specificity of their response, but the generality. Unlike enzyme-based
biosensors, whole cell biosensors often assay the effect of the target
chemical(s) rather than identify the chemical itself.
– However, catabolic biosensors based on the ability of some microorganisms to
metabolize potentially toxic compounds (often called ‘bio-reporters’) are capable
of specificity.

10/05/2010


Reagents: Ferricyanide Benefits
Value
Value
Proposition

 The most commonly used inorganic mediator in bio-sensing
is hexacyanoferrate (III) and it has many of the characteristics
of an ‘ideal’ mediator including:
– A well-defined stoichiometry,
– A known formal potential,
– Fast heterogeneous and homogeneous electron transfer,
– Is ready soluble in aqueous media at ph 7,
– Is stable in both oxidised and reduced forms, and
– Has no interaction with the biocomponents that alter its redox
potential.

10/05/2010


Ferricyanide: A Chemical Equation
Value
Value
Proposition

A representative stoichiometric equation for the aerobic oxidation of an organic
substrate is:

CH2O + O2 microorganisms H2O + CO2
organic substrate electron acceptor
Like aerobic oxidation, the hexacyanoferrate (III)-mediated degradation of organic
compounds by microorganisms involves the oxidation of organic substrates to CO2
(Eq. 1a). When the microorganisms oxidise organic compounds in a SciTOX
incubation, the hexacyanoferrate (III) acts an electron acceptor and is reduced to
hexacyanoferrate (II) (Eq. 1b), which in turn is re-oxidised to hexacyanoferrate (III) at
a working electrode (anode).

CH2O + H2O → CO2 + 4H+ + 4e‾ (1a)
[Fe(CN6)]3‾ + e‾ → [Fe(CN)6]4‾ (1b)
CH2O + H2O + 4[Fe(CN)6]3‾ → CO2 + 4H+ + 4[Fe(CN)6]4‾ (1c)

10/05/2010


Electrode Stability
Value
Value
Proposition

Potential Equation R2
100mV y = 6.5986x + 0.3208 R2 = 0.9999
200mV y = 6.9707x + 0.3773 R2 = 1
300mV y = 7.0327x + 0.4037 R2 = 0.9999
400mV y = 7.0294x + 0.6497 R2 = 0.9999

22-04-08. Calibration of Scitox electrode (Pt 50µm/Au) in Scitox transducer v1.0.

80

70

60

50
i(nA)

40 100mV
30 200mV
300mV
20
400mV
10

0
0 5 10 15

KFCII conc. (m M)

10/05/2010

The Technology
Toxicity Measurement


Toxicant Measurement
Value
Value
Proposition

 How is toxicity measured?
– It is not a mg/l type of measurement
– Can be compared to a pharmaceutical tablet; content given in IU, not mg.
– It has no absolute standard, like testing lead, chloroform, etc.
– Customers ask: Is this a LD50 test?
• No. Simply stated, the LD50 test means what amount of a toxin will kill fifty percent of a given
population.
• The EC50 or IQ50 measurement determines how much of a toxin reduces the metabolism of a
given population by fifty percent.
• The toxicity assay is a sort of precursor to the LD50 test.
• Often, if an organism has its metabolism reduced by fifty percent, it is going to be dead, but is not
yet.
 SciTOX utilizes two relative measurements
– Biological Potential Units (BPU)
– Metabolic Inhibition Quotient (MIQ)

10/05/2010


Value
Value
Proposition

 Biological Potential Units: This represents the relative bacterial activity
of a sample compared to a control sample.
– Note: If the activity (and hence nA reading) of a sample is greater than the
Control, then the calculated BPU will be greater than 100. This can often happen
when the sample activity is similar to the control, and the difference is just due to
experimental variation of the bacteria. Other times, it can be due to an increased
biological activity.
 Metabolic Inhibition Quotient
– MIQ is a measure of Metabolic Inhibition in the test sample compared to the
Control. It represents the percent drop in metabolic activity.
– If a negative value is displayed, disregard the MIQ value, and pay attention to the
BPU – it represents the relative activity compared to the control (water).

10/05/2010


BPU and MIQ Examples
Value
Value
Proposition

MIQ is a measure of Metabolic Inhibition in the test sample compared to the
Control. It represents the percent drop in metabolic activity.

MIQ is calculated from the relative activity of the test sample (BPU) in the
following formulae:

BPU = 100 * (nA of Sample) / (nA of Control)
MIQ = 100 - BPU

Example:

Control: 53.6 nA
Sample: 39.4 nA
BPU = 100 * 39.4 / 53.6 = 73.5
MIQ = 100 - BPU = 26.5

In the above example, the sample had 73.5% metabolic activity (BPU) due to a
26.5% metabolic inhibition (MIQ).

10/05/2010


Value
Value
Proposition

 This is a measurement of the effect of a toxicant on the
metabolism of bacteria
 The bacteria act on the mediator (Ferricyanide) reducing it
to Ferrocyanide.
 We measure the change in Redox potential from this action.

 BIG THING: Because this test measures the effect on
biological metabolism, it is also INDICATIVE of the results
from the standard BOD assay.
– Much, much faster, though. (Five days vs. fifteen minutes)

10/05/2010

The Solution
The SciTOX ALPHA Toxicity analyzer


SciTOX: The first idea
Value
Value
Proposition

 This was our ‘Proof of Concept’
unit.
 Six units were produced.
 Basic hardware design remained
the same on commercial
production.
 Biggest change is in the chassis,
sample handling, and the software

10/05/2010


The ALPHA Toxicity Analyzer
Value
Value
Proposition

Wireless Antenna

Touchscreen
Cell Battery under cap

Transducer Array body
Firewire

Sample Pod Crowns, Aluminium

Indicator LED’s

Sample Pods, for heating and mixing
Aluminium Chassis, Powder-Coated

This is the production unit.

10/05/2010


ALPHA strengths
Value
Value
Proposition

 Customer/market focus: SciTOX specifically targets the
wastewater treatment market.
 Is there a payback? It comes from three possible sources
– Potential to charge industrial contributors to the waste stream
based on the toxicity (and biodegradability) of their waste.
– Potential to monitor incoming waste and take corrective action if a
toxic surge comes to the plant.
– Potential to increase operational performance with real-time
biodegradability data.

10/05/2010


Initial Menu Screen
Value
Value
Proposition



Transducer Check
Value
Value
Proposition

NOT WORKING



Transducer Check
Value
Value
Proposition



Functional Checks Screen
Value
Value
Proposition

Probe Check:
Test probe
electronically

Biological
Check: Check
performance of
inoculum

Recondition
probe: Electro-
conditioning
procedure to
clean probe


Biological Check Screen
Value
Value
Proposition

Expressed as:

Metabolic
Inhibition
Quotient

BPU; Biological
Potential

Nano Amps



Prepare inoculum
Value
Value
Proposition

 Inoculum, what is it? It is the bacterial sample from the
WWTP that is used to measure the toxic effect.
– It makes this test specific to each individual WWTP.
– Remember all the kinds of bacteria in wastewater treatment.

10/05/2010


Prepare inoculum
Value
Value
Proposition

 Filter the sludge, or concentrate it by some other means.
– Centrifugation possible; more difficult



Prepare inoculum
Value
Value
Proposition

 Pipette and suspend the sludge in perhaps 10-20ml of buffer.

 Prepare day before; store in refrigerator.



Prepare Reagent, simple
Value
Value
Proposition

 Use Potassium Ferricyanide, reagent grade
 Measure out the appropriate amount of Potassium
Ferricyanide and dissolve in water.
 Store in a dark glass or plastic bottle.

 The buffer solution is a weak Potassium Chloride solution
with trace Magnesium Sulphate added.

10/05/2010


Sample Analysis, begin control test
Value
Value
Proposition



Sample Analysis, incubation
Value
Value
Proposition



Sample Analysis, take reading
Value
Value
Proposition



Sample Analysis, results
Value
Value
Proposition



Toxicity Analysis: Lead
Value
Value
Proposition

1A. Pb2+ Standard Curve Conforms to published EC50 data for with [Pb ]
1C. Current variation Lead
1B. Rest potential variation with [Pb2+] 2+

110 8
440
100
420
90 6

Rest Potential (mV
400

Current (nA)
80
% Activity

380
70 4

60 360

50 340 2

40 320

30 300 0
0 100 200 300 0 20 40 60 80 100 0 20 40 60 80 100

Pb2+ (mg L-1) Pb2+ (mg L-1) Pb2+ (mg L-1)

10/05/2010


Toxicity Analysis: Copper
Value
Value
Proposition

2A. Cu2+ Standard Curve 2B. Rest potential variation with [Cu2+] 2C. Current variation with [Cu2+]
120 460
Conforms to published EC50 data for Copper
8

440 7
100
420 6
80

Rest Potential
Current (nA)
400 5
% Activity

60 380 4

360 3
40
340 2
20
320 1

0 300 0
0 20 40 60 80 100 120 0 20 40 60 80 100 0 20 40 60 80 100

Cu2+ (mg L-1) Cu2+ (mg L-1) Cu2+ (mg L-1)

10/05/2010


Toxicity Analysis: Zinc
Value
Value
Proposition

4A. Zn2+ Standard Curve 4B. Rest potential variation with [Zn2+] 4C. Current variation with [Zn2+]
Conforms to published EC50 data for Zinc
120 460 8

100 440 7

420 6

Rest Potential (mV)
80

Current (nA)
400 5
% Activity

60
380 4
40
360 3
20
340 2
0 320 1

-20 300 0
0 50 100 150 200 250 300 0 20 40 60 80 100 0 20 40 60 80 100

Zn2+ (mg L-1) Zn2+ (mg L-1) Zn2+ (mg L-1)

10/05/2010

0.4

Toxicity Results, Acetone
Value
Value
0.2
Proposition
0.0
0 20 40 60 80 100 120

3,5-DCP (mg L-1)

6B. Activity vs Acetone (17 Jun 08)
Measured supernatant in Eppendorf Tube
1.2

1.0

0.8
Activity

0.6

0.4

0.2

0.0
0 20 40 60 80 100 120

Acetone (g L-1)

10/05/2010


SciTOX solutions, the benefits
Value
Value
Proposition

 Simplicity and time needed: Non technical people can run the
test, and total time is 15 minutes, including incubation.
– Less than time required for standard and non-standard BOD tests
or COD analysis
 Application focus: This is an analyzer dedicated to the
wastewater industry.
– Designed specifically for the wastewater industry
– Results correlation possible to BOD (non-regulatory)

10/05/2010

SciTOX
Our second product, the UniTOX


SciTOX products: UniTOX
Value
Value
Proposition

 Announced in December 2009
– A ‘University’ product, the UniTOX.
• Focused on teaching labs to give an easy-to-use analyzer for training and
method development research
• Software focused on method development and techniques
• Supplied with three or four types of electrodes
• Possible uses
– Hazardous waste bioremediation. Test using bacteria developed for treatment.
– Physical treatment of hazardous waste (irradiation). Is the treatment reducing
toxicity?
– Other cell cultures, like animal liver cells. Biotech/Physiology Research.

10/05/2010


UniTOX, the market
Value
Value
Proposition

 Additional areas of interest
– Antibiotic residue screening
– Bacterial contamination in prepared foods.
– Bacterial metabolism research, can be applied to fuel cell research.
– Incorporation of antibodies in reagent mix, targeting specific
chemical analysis (catabolic biosensor)
– Biotechnology departments: Development of Analytical methods
and biosensors.
– Physiology Departments: Development of new test procedures and
screening procedures using different cell types or mixes.
• This is a unique aspect to the SciTOX products. They are not limited to a
single type of bacteria.
10/05/2010


References: A suggested few
Value
Value
Proposition

 D'Souza SF (2001) Biosens. Bioelect. 16(6), 337-353. – Excellent review of whole cell
biosensors, including functionality, immobilisation, transduction and applications.
 Pasco NF, Hay JM, Webber J (2001) Biomarkers 6(1), 83-89. – Original publication
describing application of a mediated bioassay for monitoring DTA.
 Keane A, Phoenix P, Ghoshal S, Lau PCK (2002) J. of Microbiol. Methods 49, 103-119.
– Review of whole cell biosensors application for monitoring the toxicity of organic pollutants.
 Kissinger PT (2005) Biosensors & Bioelectronics 20, 2512-2516.
 Hansen LH & Sorensen SJ (2001) Microbial Ecology 42, 483-94. – Good review of bio-
reporter whole cell biosensors, including construction, applications and environmental
monitoring.
 Leveau JHJ & Lindow SE (2002) Curr. Opin. Microbiol. 5, 259-265.
 Lei Y, Chen W, Mulchandani A (2005) Anal. Chim. Acta (In press). – Excellent review of
whole cell biosensors, including applications, immobilization and transducers.
 Rogers KR (2006) Anal. Chim. Acta 568, 222-231. – Overview of biosensors for
environmental monitoring, including whole cell biosensors.
 Tizzard, A (2006) Unpublished degree in Doctor of Philosophy, Lincoln University,
New Zealand.
 van der Meer JR, Tropel D, Jaspers M (2004) Environ. Microbiol. 6(10), 1005-1020.
– Excellent review of bacterial bio-reporter biosensors, including functionality and detection.
10/05/2010


References: A suggested few
Value
Value
Proposition

 Pasco N & Hay J (2005) Biochemical Oxygen Demand. In: J Lehr (ed.), The Encyclopedia
of Water, Vol in press. John Wiley & Sons, New Jersey. – Review of BOD monitoring
including use of biosensors.
 Karube I, Matsunaga T, Mitsuda S, Suzuki S (1977) Biotechnol. Bioeng. 19, 1535-1547 –
Original rapid BOD biosensor publication.
 Pasco NF, Baronian KH, Jeffries C, Hay J (2000) Appl. Microbiol.Biotechnol. 53(5), 613-
618. – Original publication describing application of a mediated bioassay for BOD
monitoring.
 Pasco N, Baronian K, Jeffries C, Webber J, Hay J (2004) Biosens. Bioelect. 20, 524-532.
 Yoshida N, Yano K, Morita T, McNiven SJ, Nakamura H, Karube I (2000) Analyst 125,
2280-2284.
 Catterall K, Morris K, Gladman C, Zhao HJ, Pasco N, John R (2001) Talanta 55(6),
1187-1194.
 Catterall K, Zhao H, Pasco N, John R (2003) Anal. Chem. 75, 2584-90.
 Morris K, Catterall K, Zhao H, Pasco N, John R (2001) Anal. Chim. Acta 442(1),129-
139.

10/05/2010

Value
Value
Proposition

THANK YOU!
Questions?
SciTOX Limited
1 Tussock Lane, Unit 2,
Ferrymead
Christchurch 8023
New Zealand
P: 64 (3) 376-4996
F: 64 (3) 359-1018
E: Enquiries@SciTOX.com
W: www.SciTOX.com
10/05/2010

Sci Tox Linked In Seminar

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