Juha Lappalainen Ph D Aboatox Oy Finland www

Juha Lappalainen, Ph. D Aboatox Oy, Finland www. aboatox. com

CASE: Kinetic luminescent bacteria test (the Flash test) Water quality – Kinetic determination of the inhibitory effect of sediment and other solids and colour containing samples on the light emisssion of Vibrio fischeri International Standard 21338: 2010

Agenda • • Ecotoxicology Photobacterium Standardization Commercial aspects

Ecotoxicology Tests are used to monitor environmental impact of • effluents • chemicals • waste ……to ecosystems

Ecotoxicity tests – test groups Producers Micro-algae Selenastrum Crustaceans Ceriodaphnia Consumers Decomposers Daphnia Thamnocephalus Rotifers Brachionus Bacteria Vibrio Protozoa Tertrahymena

Ecotoxicity tests - microscopic

Ecotoxicity tests - macroscopic

Ecotoxicity tests – automated alarm systems for drinking water Algae toximeter Tox Protect

Standard Method ISO 11348 -3 Luminescent bacteria test 30 min Unstressed bacteria + sample end point reading

Standard Method ISO 11348 -3 • Photobacterium Vibrio fischeri is widely used • Superior speed: 30 minutes contact time – Algal growth inhibition test 72 h – Daphnia magna immobilisation test 24 – 48 h – Fish mortality test 92 h

Standard Method ISO 11348 -3

Standard Method ISO 11348 -3 • interferences – volatile or insoluble substances – oxygen demand – hyperosmotic effects – light absorption

Standard Method ISO 11348 -3 • Light intensity is reduced due to - toxicity - colour - particles: turbidity and sedimentation - combination of all these factors

Proposed solutions • Extraction - some toxic compounds are not soluble, they are attached to sample particles • Double walled cuvette – difficult to operate – not applicable to solid samples • Centrifugation method – a non-toxic reference sample is needed for each sample • Filtration – Limited amount of solids; colour correction needed afterwards! • Spectrophotometric correction – Limited amount of solids

Kinetic luminescent bacteria test (the Flash test) history • scientific work from 1997 • standardization started in 2000 – annex to 11348 -3; 1998: failed, too complicated to add • WHY to standardize, the standard for luminescent bacteria already exists? • new standard proposal – difficulties with the local authorities in keeping the time tables etc. • intercalibration with reference chemicals 2006 • intercalibration with samples 2007

Standardization 5 years (optimistic)

The Flash test 30 min 1. Start measuring 2. Dispence BACTERIA and record luminescence maximum 3. Record luminescence after the contact time (mix first)

The Flash test 21338

The Flash test Features and benefits • No correction steps. Same protocol can be used for all samples • Direct contact test • High solid contents possible (200 g/l) • Comparable resuls to the standard method with reference chemicals

Results of the interlaboratory comparison • 2 sediment samples, 2 water samples • 8 laboratories, 10 systems • 5 different instruments: – Tube luminometer • Single tube (4) • Carousel luminometer (4) – Microtiter Plate Luminometer (2)

Results of the interlaboratory comparison EC 50 11348 method Sample 1 1. 3 g/l EC 50 Kinetic method 2. 1 g/l

Results of the interlaboratory comparison Sample 2 EC 50 11348 method 8. 4 g/l EC 50 Kinetic method 198 g/L

Results of the interlaboratory comparison Sample 1 EC 50 Standard method 1. 3 g/l EC 50 Kinetic method 2. 1 g/l Sample 2 8. 4 g/l 198 g/l FALSE

Results of the interlaboratory comparison • All laboratories were able to separate toxic samples from the non toxic samples with the kinetic method • The standard method failed (with very good cv)

Instrument requirements • dispence 200 - 500 µl reagent and read simultaneously (peak value) • 5 determinations/second • incubate at 15 °C (outside the luminometer)

Present situation • The kinetic method has been evaluated with different instruments, protocols and samples during the last 10 years • Instruments available from different manufacturers • The kinetic method has been accepted by critical users - after they have used the method themselves Standard as of 15. 7. 2010

Users of the method • • Compost maturity Biodegradable plastic compost toxicity PCB contaminated river remediation Fly ash measurements Biocide detection from pulp Packing material toxicity Oil shale waste water Oil contaminated sediment testing