Toxkit microbiotests for detection and quantification of ... toxins Pisa 2007.pdf · Microsoft PowerPoint - Cyanobacterial toxins Pisa 2007.ppt Author: php Created Date: 3/7/2008

Toxkit microbiotests for detection and quantification of cyanobacterial toxins

NATO Advanced Study InstituteSensor Systems for Biological Threats : The Algal Toxins Case

(Pisa, September 30 – October 10, 2007)

Prof.em.Dr.G.Persoone- Ghent University, Laboratory of Environmental Toxicology- MicroBioTests Inc

Cyanobacterial toxins

CHEMICAL ANALYSES : the causes

BIOASSAYS : the effects

Why to look for causesIf there are no effects… ?


WHEN ARE BIOASSAYS NEEDED ?(from Ken-Ichi Harada, Fumio Kondo and Linda Lawton

in : TOXIC CYANOBACTERIA IN WATER)

Bioassays are recommended if any of these conditions are fulfilled :

1. A laboratory can easily establish them, but has little or no access to adequate equipment and expertise for establishingphysico-chemical analysis, or little means of subcontracting analysis





2. There is indication of cyanotoxins other than(or in addition to) the known toxicants





3. Cyanobacterial taxa dominate, the toxins of whichhave not yet been well studied and which maytherefore contain unknown toxic metabolites





4. Confirmation of results from physico-chemicalanalysis is required, especially to confirmbioactivity





5. Validation of physico-chemical methods by analternative method is desired


BIOASSAYS ARE NEEDED

Because the effects expressed by the test species are an integration of all the toxins present, each

in their respective concentrations

Toxin A Toxin C

Toxin B Toxin D

Cyanobacterial toxins are not only microcystins…


Mixing of different paintsin different volumes

+ +=

+ + Final color ?


TYPES OF BIOASSAYS

VertebratesInvertebrates

BacterialBiochemical

Mammalian cells


TYPES OF BIOASSAYS

Tests with aquatic invertebratesare performed in most cases on

“water extracts” of algal biomassobtained through sonication


MICROBIOTESTS OTHER

Artemia franciscana Mosquito larvae(Artoxkit M) House flies and fruit fliesDaphnia magna Locusts(Daphtoxkit F magna) CopepodsThamnocephalus platyurus Protozoans(Thamnotoxkit F) Coelenterates

NematodesOligochaetes


ADVANTAGES OF TOXKIT MICROBIOTESTS

Culture/maintenance freeYear-round availability


ADVANTAGES OF TOXKIT MICROBIOTESTS

MiniaturizationUser-friendlinessStandardizationValidationReproducibilityCost-effectiveness


TOXKIT MICROBIOTESTS

Have been developed specifically for :- routine toxicity monitoring in contaminatedenvironments

- toxicity ranking or pure chemicals and plantextracts

- detection and quantification of the toxicity ofpollutants in : surface and groundwaters, industrialeffluents, sediments, soils, solid wastes and wasteleachates


TOXKIT MICROBIOTESTS

Have only recently been earmarked for theirsensitivity to, and their possible use as lowcost tools for routine screening of aquaticenvironments for biotoxins

Cyanobacterial toxinsBattery of acute and short-chronic

Toxkit microbiotests available presently


Artemia cysts and hatching larvae


Artoxkit Mwith Artemia franciscana


Daphnia ephippium and freshlyhatched Daphnia neonates


Daphtoxkit F magnawith Daphnia magna


Adult Thamnocephalus platyurus with cysts, and freshly hatched larva


Thamnotoxkit Fwith Thamnocephalus platyurus


TOXKIT MICROBIOTESTS SENSITIVITY

ARTOXKIT M

- Lower sensitivity than Daphtoxkitand Thamnotoxkit

- Sensitivity can be increased by prolongingthe exposure time from 24h to 48h (but still far below sensitivity of Thamnotoxkit)


TOXKIT MICROBIOTESTS SENSITIVITY

THAMNOTOXKIT F

- 24h LC50 for microcystin LR around 1 µg/ml- More sensitive than Daphtoxkit F magna

and Daphtoxkit F pulex- More sensitive than mouse assay- Less sensitive than Spirotox

(culture dependent ciliate bioassay)


TOXKIT MICROBIOTESTS SPECIFICITY

Challenged statement :“No single method is currently available to replace the mouse for the detection of all cyanotoxins using a single assay, and further validation and comparison of methods is needed before general recommendations ontheir application be given”.

(Ken-ichi Harada, Fumio Kondo and Linda LawtonChapter 13. Laboratory analysis of cyanotoxins.In : Toxic Cyanobacteria in Water, 1999)


THAMNOTOXKIT F ASSETS FOR DETECTION OF CYANOBACTERIAL TOXINS (1)

-Sensitive to pure microcystins, neurotoxins and crudeextracts of cyanobacteria

-Good correlation of effects found on natural sampleswith mouse assay data

-Signals the presence of additional (unknown) toxinsin cyanobacteria whereas chemical analysis can onlydetect the known toxic compounds targeted by thedetection method(Törökne et al., 2000)

Cyanobacterial toxinsTHAMNOTOXKIT F ASSETS FOR DETECTION

OF CYANOBACTERIAL TOXINS (2)

- Sensitivity lower than that of some biochemical assays(ELISA) and mammalian cell tests (rat hepatocytes),but the latter are much more “toxin specific”

- Sensitivity can be increased by addition of DMSOand/or increase of the test temperature (Tarczynska, 2000)

- Rapid “direct feeding” bioassays on algal biomassare possible, and have been shown to lower the toxindetection threshold substantially (Aquasense, 1998)


CONCLUSIONS-Over the last few years, several Toxkit microbiotests, and in particular the Thamnotoxkit F, have been earmarked and validated as simple and low cost tools for monitoring the toxicityof cyanobacterial blooms.

-Interferences due to other compounds present in the algae, butnot necessarily toxic to man, have to be investigated more in depth(e.g by biomass fractionation)

-A Standardized Operational Procedure (SOP) for pre-screeningand early warning for cyanobacterial toxins in waters for humanuse (drinking and recreational) should be worked out for the Thamnotoxkit F.

Since the time of the former presentation in 2001, extensive research has been performed on the application of Toxkit microbiotests, and in particularof the Thamnotoxkit to cyanotoxins.

The following scientific groups have been particularly active in this regard :

- Department of Water Hygiene, National Institute for EnvironmentalHealth, Budapest, Hungary (Dr.A.Törökne)

- Institute of Botany, Czech Academy of Sciences, Department of Experimental Phycology and Ecotoxicology, Brno, Czech Republic(Dr.B.Marsalek)

- Department of Applied Ecology, University of Lodz, Poland(Dr.M.Tarczynska)

- Department of Environmental Health Sciences, Medical University of Warsaw, Warsaw, Poland (Dr.G.Nalecz-Jawecki)

Dr.A.Törökne, a leading authority on the applicationof invertebrate bioassays to cyanotoxins, has

a) published several recent papers on the application of the Thamnotoxkit and the 1h Rapidtoxkit to cyanotoxins

b) written the chapter on “Toxicity Testing” in the new version of the WHO book on Toxic Cyanobacteria in Water (to bepublished in the spring of 2008).

c) emphasizes in this chapter that “the Thamnotoxkit seemspresently to be the most standardised and routinely usedalternative method for all known cyanotoxins”

d) gives a detailed description of the test methodology of the 24h Thamnotoxkit microbiotest

e) recently made a comparison of the 24h mortality Thamnotoxkitmicrobiotest and the 1h sublethal Rapidtoxkit microbiotest

Andrea Törökné PhDDept.Hygienic Water Biology

NCPH-NIEHISTA 12 Skiathos 12-17.06.2005

Rapid test for detecting

cyanobacterial toxins

Problems caused by cyanobacterial toxins

Surface water burdened by cyanobacterial blooms and scums

•Bathing in natural water •Drinking water supply


• IDENTIFIED– Hepatotoxins microcystins (more than 70 variants)

(tumor promoters) nodularin

– Neurotoxins saxitoxin, anatoxins, homoanatoxin

– Cytotoxin cylindrospermopsin

• UNIDENTIFIED

Detection of cyanotoxins

• IDENTIFIED– Biological assays

• Mouse assay• Alternative assays

– Chemical analyses• HPLC• LC/MS-MS• MALDI TOF etc.

• UNIDENTIFIED– Biological assays

• Mouse assay• Alternative assays

(e.g. Thamnotoxkitmicrobiotest)

MethodsThamnocephalus platyurus

sensitive to cyanotoxins

Thamnotoxkit24 hour test

16-24 h larvae hatched from cysts

dilution seriesmortality of larvae

comparison with control

Rapidtoxkit1 hour test

30-45 h larvaehatched from cysts

dilution seriesuptake of red beads in digestive tract

Comparison with control

Results

20.00mcyYR0.160.89Aph., M.754.50mcyYR0.340.11M.610.16mcyLR0.690.63Aph., L.5

132.9mcyRR,YR,LR

0.360.21M., P.446.86mcyYR,RR0.430.81An., L., O.310.66mcyYR1.492.7An., M.2

260.14mcyLR,RR0.430.32Aph., M.1

Toxin content(µg/g)

Toxin detected

Rapidtoxkit 1h EC50(mg/ml)

Thamnotoxkit24h LC50(mg/ml)

Dominant species

Results (2)

-Nd. mcy or cyn0.140.23An.15-Nd.cyn0.080.30C.14

950.0cyn0.420.14C.13-Nd. cyn0.290.70Aph., P.12

152.4mcyLR,YR, RR

0.010.17Aph., M.1146.5mcyLR,YR,RR1.050.97Aph., M.10

-Nd mcy0.020.16Aph. M.9-Nd.mcy or cyn0.140.49P.8

Toxin content(µg/g)

Toxin detected

Rapidtoxkit 1h EC50(mg/ml)

Thamnotoxkit 24h LC50(mg/ml)

Dominant species

Results (3)

R= 0.82

Correlation betweenthe 24h Thamnotoxkit testand the 1h Rapidtoxkit test

Conclusions

• We need rapid, routine methods for detecting unkown cyanotoxins in source water

• Thamnocephalus platyurus is sensitive to numerous types of cyanotoxins

• The 1h Rapidtoxkit test can replace the 24h Thamnotoxkit test for rapid detectionof cyanotoxins

Documents

Toxkit microbiotests for detection and quantification of ... toxins Pisa 2007.pdf · Microsoft PowerPoint - Cyanobacterial toxins Pisa 2007.ppt Author: php Created Date: 3/7/2008