Juha Lappalainen, PhD
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 Daphnia
Ceriodaphnia
Consumers
Thamnocephalus
Decomposers
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
Inkuboi
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
Luminescence after contact
time vs. beginning/ %
Standard Method ISO 11348-3
100
10
1
0.01
0.10
1.00
Concentration/ g-1
10.00
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
Control
Soil
Toxic
Toxic soil
110
Luminescence
90
70
50
30
10
-10 0
2
4
Time/ s
6
8
10
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
EC50
11348
method
Sample 1
1.3 g/l
EC50
Kinetic
method
2.1 g/l
Results of the interlaboratory
comparison
Sample 2
EC50
11348
method
8.4 g/l
EC50
Kinetic
method
198 g/L
Results of the interlaboratory
comparison
Sample 1
EC50
Standard
method
1.3 g/l
EC50
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
Commercial aspects
Bio-Orbit
Labsystems
2002
Commercial aspects
Labsystems
Thermo Fisher Scientific
Commercial aspects
Berthod Detection
Systems
Titertek-Berthold
Oriola
Mediq
Commercial aspects
Commercial aspects
Commercial aspects Competitor
Microtox, the standard
11348-3 method
Commercial aspects Competitor
Microtox by Beckman 1990
Commercial aspects Competitor
Microtox by Microbics 1994
Commercial aspects Competitor
Microtox by Azur 1996
Commercial aspects Competitor
Microtox by SDI 2000
Commercial aspects Competitor
Microtox by Modern Water 2013
Thank you for your attention!