sci tox linked in seminar

TOXICITY ANALYSIS

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

INTRODUCTION

The SciTOX Vision and Process

- Rapid

- WWTP specific

- Cost to Own

- Payback

ValueValue

Proposition

The Rapid Toxicity Measurement System10/05/2010

SciTOX, the company

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.

http://www.linkedin.com/companies/856897

- Rapid

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Proposition


SciTOX, the company

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.



- Rapid

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SciTOX: Board of Directors

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.

http://www.linkedin.com/profile?viewProfile=&key=20687152&authToken=Wyet&authType=name

http://www.ancare.co.nz/

http://www.linkedin.com/profile?viewProfile=&key=701383&authToken=Qmm2&authType=NAME_SEARCH&locale=en_US&srchindex=1&pvs=ps&goback=%2Efps_brent+ogilvie_*1_*1_*1_*1_*1_*1_*1_Y_*1_*1_*1_false_1_R_true_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2_*2


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http://www.linkedin.com/companies/integy-limited?trk=co_search_results&goback=%2Ecps_1273294160957_1




http://www.linkedin.com/profile?viewProfile=&key=26923040&authToken=bbQc&authType=name

http://www.linkedin.com/companies/413901/Canterbury+Development+Corporation?trk=pp_no_icon

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Proposition


Technology Patents

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

– Method and apparatus for measuring use of a substrate in a microbially catalysed reaction; Australia; 717224; Granted

– Method and apparatus for measuring use of a substrate in a microbially catalysed reaction; USA; 6,379,914; Granted

– Method and apparatus for measuring use of a substrate in a microbially catalysed reaction; Japan; 3479085; Granted

– Method and apparatus for measuring use of a substrate in a microbially catalysed reaction; Europe; 97946176.1; Allowed

– Method and apparatus for measuring use of a substrate in a microbially catalysed reaction; Canada; 2307603; Granted

SciTOX plans to file further patents as warranted.

The Market

Wastewater Treatment

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Proposition


Wastewater Treatment: The Bottom Line

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.

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Proposition


VALUE Measurements: Where

Raw Influent

Primary Treatment Secondary Treatment Secondary Clarification

Final Effluent

Anaerobic Digestion

Dewatered

Solids

Methane

Supernatant

Primary Solids

Secondary Solids

Contract Waste Hauler

1. Toxicity

2. BOD correlation

3. Food/Micro-Organism

1.

1.

2.

2.

2.

3.

Influent

2.

3.

The Technology

Electrochemistry and Biosensors: Theory and application

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Electrochemistry, the basics

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.

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Potentiostat basics

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.

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Electrode, types

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.

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Biosensors

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.

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Biosensors

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).

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Whole Cell Biosensors: The benefits

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.

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Proposition


Whole Cell Biosensors: The benefits

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.

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Proposition


Reagents: Ferricyanide Benefits

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.

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Proposition


Ferricyanide: A Chemical Equation

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

CH2O + O2microorganisms 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 SciTOXincubation, 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)

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Electrode Stability

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

0

10

20

30

40

50

60

70

80

0 5 10 15

KFCII conc. (mM)

i(n

A)

100mV

200mV

300mV

400mV

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

The Technology

Toxicity Measurement

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Proposition


Toxicant Measurement

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)

- Rapid

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Proposition

The Rapid Toxicity Measurement System


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

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Proposition


BPU and MIQ Examples

10/05/2010

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).

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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)

The Solution

The SciTOX ALPHA Toxicity analyzer

- Rapid

- WWTP specific

- Cost to Own

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Proposition


SciTOX: The first idea

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

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Proposition


The ALPHA Toxicity Analyzer

Sample Pod Crowns, Aluminium

Indicator LED’s

Transducer Array body

Cell Battery under cap

Firewire

Touchscreen

Wireless Antenna

Aluminium Chassis, Powder-Coated

Sample Pods, for heating and mixing

This is the production unit.

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Proposition


ALPHA strengths

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.

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Proposition


Initial Menu Screen

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Transducer Check

NOT WORKING

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Transducer Check

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Functional Checks Screen

Probe Check:

Test probe

electronically

Biological

Check: Check

performance of

inoculum

Recondition

probe: Electro-

conditioning

procedure to

clean probe

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Proposition


Biological Check Screen

Expressed as:

Metabolic

Inhibition

Quotient

BPU; Biological

Potential

Nano Amps

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Proposition


Prepare inoculum

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.

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Prepare inoculum

Filter the sludge, or concentrate it by some other means.

– Centrifugation possible; more difficult

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Proposition


Prepare inoculum

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

Prepare day before; store in refrigerator.

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Prepare Reagent, simple

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.

- Rapid

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Proposition


Sample Analysis, begin control test

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Sample Analysis, incubation

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Sample Analysis, take reading

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Sample Analysis, results

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Toxicity Analysis: Lead

1A. Pb2+ Standard Curve

Pb2+ (mg L-1)

0 100 200 300

% A

ctivity

30

40

50

60

70

80

90

100

110

1C. Current variation with [Pb2+]

Pb2+ (mg L-1)

0 20 40 60 80 100

Curr

ent (n

A)

0

2

4

6

8

1B. Rest potential variation with [Pb2+]

Pb2+ (mg L-1)

0 20 40 60 80 100

Rest P

ote

ntial (m

V

300

320

340

360

380

400

420

440

Conforms to published EC50 data for Lead

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Toxicity Analysis: Copper

2A. Cu2+ Standard Curve

Cu2+ (mg L-1)

0 20 40 60 80 100 120

% A

ctivity

0

20

40

60

80

100

120

2C. Current variation with [Cu2+]

Cu2+ (mg L-1)

0 20 40 60 80 100

Rest P

ote

ntial

0

1

2

3

4

5

6

7

8

2B. Rest potential variation with [Cu2+]

Cu2+ (mg L-1)

0 20 40 60 80 100

Curr

ent (n

A)

300

320

340

360

380

400

420

440

460Conforms to published EC50 data for Copper

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Toxicity Analysis: Zinc

4A. Zn2+ Standard Curve

Zn2+ (mg L-1)

0 50 100 150 200 250 300

% A

ctivity

-20

0

20

40

60

80

100

120

4C. Current variation with [Zn2+]

Zn2+ (mg L-1)

0 20 40 60 80 100

Curr

ent (n

A)

0

1

2

3

4

5

6

7

8

4B. Rest potential variation with [Zn2+]

Zn2+ (mg L-1)

0 20 40 60 80 100

Rest P

ote

ntial (m

V)

300

320

340

360

380

400

420

440

460

Conforms to published EC50 data for Zinc

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6A. Activity vs 3,5-DCP (17 Jun 08)Measured supernatant in Eppendorf Tube

3,5-DCP (mg L-1)

0 20 40 60 80 100 120A

ctiv

ity0.0

0.2

0.4

0.6

0.8

1.0

1.2

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

Acetone (g L-1)

0 20 40 60 80 100 120

Act

ivity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Toxicity Results, Acetone

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SciTOX solutions, the benefits

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)

SciTOX

Our second product, the UniTOX

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Proposition


SciTOX products: UniTOX

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.

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UniTOX, the market

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

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References: A suggested few

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.

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References: A suggested few

Pasco N & Hay J (2005) Biochemical Oxygen Demand. In: J Lehr (ed.), The Encyclopediaof 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.

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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: [email protected]: www.SciTOX.com

mailto:[email protected]

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