Transcript SciTOXTM - a low cost analyser for environmental

TM
SciTOX
- a low cost toxicity analyser for environmental applications
N Glithero1, J Hay1, N Pasco1, D Patterson2, R Wattinger2 and R Weld1
1 Lincoln Ventures Ltd, PO Box 133, Christchurch, NEW ZEALAND
2 Int2egy NZ Ltd www.integy-llc.com
To address an unmet need for technology capable of rapidly assessing toxic effects on microorganisms. A miniaturised method with rapid analysis based on limiting
current microelectrode amperometry is presented
Aim
Background
SciTOXTMunit quantifies
reduced mediator
Features
• Control via touch screen
Applications
• Measuring impact of contaminants and toxicants
in aqueous environments
KFC(II)
H+
• Rapid quantification of toxicity
MED++
Toxicants inhibit catabolism
e-
KFC(III)
e-
Assay
• 15 minute incubation with optional simultaneous
replicates followed by 20 second analysis
• Direct toxicity assessment using microelectrode
amperometry
• Microorganisms generate electrons from
catabolic reactions, these can be disrupted by
toxicants
Non-toxic substrate
promotes catabolism
• Electrons donated by microorganisms reduce
ferricyanide mediator to ferrocyanide
• Toxicant dose dependent response quantifies toxicity
• Tolerant of variable sources of microorganisms - a
wastewater treatment plant can use bacteria from its
own effluent
H+
Bacteria
• Toxic inhibition of microorganisms slows electron
production and reduces catabolic activity
depending on dosage
Experimental Method
Applied trials
Analysis
• Ferrocyanide quantified
immediately upon completion of
secondary incubation using
microelectrode amperometry
1. Microorganisms sourced from both laboratory
cultures and wastewater treatment plants and
adjusted to desired concentrations
Toxicants
•
•
•
•
• SciTOXTM unit bioreactors facilitate incubation
(heating and agitation) of toxic sample and
microorganisms
MED-
Phenol
2,4-dichlorophenol (2,4-DCP)
3,5-dichlorophenol (3,5-DCP)
Acetone
2. Toxicants diluted to produce dose dependent
response curve, zero toxicant control included
3. Primary incubation: equal volumes of toxicant and
microorganisms incubated together 5 mins at 25°C
with mild agitation for mixing
Microorganisms
• Incubated sample with toxicant
added compared to incubated
sample having no toxicant
4. Secondary incubation: mediator (potassium
ferricyanide) added and incubation continued for 10
mins
• Activated sludge flora from
wastewater treatment plant
• Escherichia coli
• Bacillus subtilis
• Microorganism activity calculated
from difference between the two
Potassium ferricyanide reduced to potassium
ferrocyanide by microorganisms
Results
Conclusions
• Mean generated from three replicates for each toxicant concentration to quantify ferrocyanide
production and displayed as ratio of activity compared to zero-toxicant control
• The EC50 is defined as the concentration of toxicant that provokes a response from the
microorganisms halfway between the baseline and maximum response. Graphs and tabled EC50
values generated by non-linear Hillslope analysis (Sigma Plot 10)
• The SciTOXTM unit produces usable toxicity measurements within
16 minutes
Activated sludge activity vs. 3,5-DCP
1.0
0.8
0.8
0.6
0.4
0.4
0.2
0.2
0.2
200
400
0
600
20
40
60
80
100
0
120
40
60
80
3,5-DCP (mg/L)
Acetone (g/L)
E. coli activity vs. 2,4-DCP
E. coli activity vs. 3,5-DCP
E. coli activity vs. phenol
0.8
0.6
Activity
1.0
0.8
Activity
1.0
0.8
0.6
0.4
0.4
0.2
0.2
0.2
0.0
0.0
40
60
80
100
0
120
20
40
60
80
100
B. subtilis activity vs. 2,4-DCP
120
• Activated sludge microorganisms more resistant to toxicants
than laboratory microorganism cultures with 3,5-DCP maximum
inhibition at 0.55 activity for activated sludge and 0.25 for E. coli.
For 2,4-DCP, maximum inhibition at 0.65 activity for activated
sludge and activity still declining at 100mg/L for both E. coli and
B. subtilis
• Despite differences in the degree of signal attenuation between
microorganism sources, the EC50 values to 2,4-DCP and 3,5-DCP
remained similar
0.0
1000
1500
3,5-DCP (mg/L)
2,4-DCP (mg/L)
100
0.6
0.4
20
20
2,4-DCP (mg/L)
1.0
0
• This technology can accommodate a range of microorganisms,
based on our trials: E. coli, B. subtilis and activated sludge
(comprising dozens of bacteria species depending on sludge age
and waste source)
0.0
0.0
0
• A potential step of 200mV is adequate to drive the reactions as
both electrodes exhibit the same equilibrium potential vs.
Ag/AgCl electrode
0.6
0.4
0.0
Activity
Activity
1.0
0.8
0.6
• A 1mm ø Au reference/auxillary electrode and 50µm ø Pt working
electrode are capable of facilitating chronoamperometric
measurements while microorganisms are present in analyte
Activated sludge activity vs. Acetone
1.0
Activity
Activity
Activated sludge activity vs. 2,4-DCP
• Quantification of ferrocyanide is reproducible with <5% variance
between toxicity test replicates
2000
2500
3000
3500
Phenol (mg/L)
-1
Microoorganisms
Toxicant
EC50 (mg L )
activated sludge
2,4-DCP
37.9 ± 4.5
activated sludge
3,5-DCP
22.7 ± 2.5
activated sludge
1
acetone
34.8 ± 12.8
E. coli
2,4-DCP
39.1 ± 7.2
E. coli
3,5-DCP
9.5 ± 0.9
E. coli
phenol
2419.4 ± 96.4
B. subtilis
2,4-DCP
48.5 ± 0.9
EC50 (2,4-DCP, E.coli) vs exposure
90
1.0
Acknowledgements
Activity
0.6
0.4
EC50 (mg L-1)
75
0.8
60
Research funded by New Zealand
Foundation for Research, Science and
Technology
45
30
0.2
15
0.0
Linear Regr
0
0
20
40
60
80
100
120
2,4-DCP (mg/L)
5
15
30
45
60
Exposure (min)
1
concentration g L
-1
EC50 values for toxicants with target
microorganisms
EC50 values for 2,4-DCP and E. coli with
varying exposure times
Int2egy NZ Ltd.