SciTOX TM - a low cost toxicity analyser for environmental applications N Glithero 1, J Hay 1, N Pasco 1, D Patterson 2, R Wattinger 2 and R Weld 1 1 Lincoln Ventures Ltd, PO Box 133, Christchurch, NEW ZEALAND 2 Int 2 egy NZ Ltd 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 EC 50 is defined as the concentration of toxicant that provokes a response from the microorganisms halfway between the baseline and maximum response. Graphs and tabled EC 50 values generated by non-linear Hillslope analysis (Sigma Plot 10) Assay Microorganisms generate electrons from catabolic reactions, these can be disrupted by toxicants Electrons donated by microorganisms reduce ferricyanide mediator to ferrocyanide Toxic inhibition of microorganisms slows electron production and reduces catabolic activity depending on dosage Background Features Control via touch screen SciTOX TM unit bioreactors facilitate incubation (heating and agitation) of toxic sample and microorganisms 15 minute incubation with optional simultaneous replicates followed by 20 second analysis Direct toxicity assessment using microelectrode amperometry Toxicant dose dependent response quantifies toxicity Tolerant of variable sources of microorganisms - a wastewater treatment plant can use bacteria from its own effluent Toxicants Phenol 2,4-dichlorophenol (2,4-DCP) 3,5-dichlorophenol (3,5-DCP) Acetone Microorganisms Activated sludge flora from wastewater treatment plant Escherichia coli Bacillus subtilis Applied trials Experimental Method 1. Microorganisms sourced from both laboratory cultures and wastewater treatment plants and adjusted to desired concentrations 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 4. Secondary incubation: mediator (potassium ferricyanide) added and incubation continued for 10 mins Potassium ferricyanide reduced to potassium ferrocyanide by microorganisms Analysis Ferrocyanide quantified immediately upon completion of secondary incubation using microelectrode amperometry Incubated sample with toxicant added compared to incubated sample having no toxicant Microorganism activity calculated from difference between the two Results Applications Measuring impact of contaminants and toxicants in aqueous environments Rapid quantification of toxicity EC 50 values for toxicants with target microorganisms EC 50 values for 2,4-DCP and E. coli with varying exposure times Aim 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 Conclusions The SciTOX TM unit produces usable toxicity measurements within 16 minutes Quantification of ferrocyanide is reproducible with <5% variance between toxicity test replicates 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 A potential step of 200mV is adequate to drive the reactions as both electrodes exhibit the same equilibrium potential vs. Ag/AgCl electrode 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) 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 EC 50 values to 2,4-DCP and 3,5-DCP remained similar Research funded by New Zealand Foundation for Research, Science and Technology Acknowledgements Int 2 egy NZ Ltd. Non-toxic substrate promotes catabolism e - H + e - H + Toxicants inhibit catabolism Bacteria MED - KFC(II) MED + + KFC(III) SciTOX TM unit quantifies reduced mediator