Recent Advances in Rapid Ecotoxicity Screening

Transcription

1 WATER CONTAMINATION EMERGENCIES CONFERENCE Manchester, UK, June 2005 Recent Advances in Rapid Ecotoxicity Screening Prof.em.Dr.G.Persoone Ghent University, Laboratory of Environmental Toxicology Microbiotests Inc.

2 WATER CONTAMINATION EMERGENCIES = Rapid detection of toxicity

3 How RAPID must RAPID be? = Case dependent!

4 Two possible approaches : CHEMICAL ANALYSES TOXICITY TESTING

5 CHEMICAL ANALYSES What to search for? Time needed?

6 TOXICITY TESTS Signal for the impact of all the chemicals present

7 TOXICITY TESTS - CONVENTIONAL TESTS - MICROBIOTESTS

8 CONVENTIONAL TOXICITY TESTS Dependent of the (year-round) culturing of live stocks of test species - Specialised - Time consuming - Costly

9 MICROBIOTESTS - Culture/maintenance free - Small-scale - User-friendly - Cost-effective - Suited for routine analyses

10 TOXKIT MICROBIOTESTS Make use of dormant eggs of selected aquatic species -Dormant eggs can be stored Dormant eggs can be hatched when test organisms are needed

11 TOXKIT MICROBIOTESTS Development of a battery of acute and short-chronic assays Worldwide application for toxicity screening and biomonitoring

12 Most acute toxicity tests (conventional and microbiotests) are based on mortality as the effect criterion Exposure time = 1 day to several days! WATER CONTAMINATION EMERGENCIES Answermaybeneededin onehour!

13 Bacterial luminescence inhibition tests (Microtox Lumistox Checklight, etc ) very rapid (15-30 minutes) sensitive to many chemicals but not sensitive to all compounds! Need for a «complementary» (non-bacterial) rapid assay

14 Sublethal endpoints for rapid toxicity detection with aquatic organisms Enzymatic Physiological Behavioral

15 FLUOTOX ENZYMATIC INHIBITION MICROBIOTEST 4-methylumbellifeyl- galactoside enzyme Normal Daphnia galactose methylumbelliferone Stressed Daphnia UV light

16 The Fluotox (developed originally at the Ghent University) has been commercialised in the USA under the name Daphnia IQ test In 2003 a USEPA ETV programme evaluated 8 commercially available rapid toxicity tests (6 bacterial assays, one chemiluminescence test and the Daphnia IQ) for Anti-terrorism Water Monitoring. Each test has been performed on 5 organic chemicals and on 4 biotoxins (Botulinum toxin, Ricin, Soman and VX) No bacterial or chemiluminescence assay was able to detect all 4 biotins whereas the Daphnia enzymatic test was very sensitive to each of them

17 Contrary to all the other assays applied in the USEPA/EV study, the Daphnia IQ test is, unfortunately, dependent on the continuous culturing/maintenance of live stocks of Daphnias, with daily feeding and water renewals Need for a culture/maintenance free alternative assay

18 Inhibition of uptake of particles by test organisms under toxic stress Application on the Thamnotoxkit F with activated carbon Toxic stress Normal particle uptake No particle uptake

19 The RAPIDTOXKIT microbiotest with larvae of the crustacean Thamnocephalus platyurus A very simple and practical test for detection of toxic hazard in water in minutes Each kit contains all the materials to perform 3 independent series of tests on 7 up to 15 water samples each

20 The RAPIDTOXKIT microbiotest in a nutshell Suspected water Control water minutes exposure minutes exposure Addition of red microspheres (15-30 minutes) No particle uptake Uptake of red particles YES NO Toxic hazard

21 Correlation of the effect data of the 1h Rapidtoxkit with the 24h Thamnotoxkit Nalecz-Jawecki (Poland, 2005) 29 drugs 1000 Thamnotoxkit Rapidtoxkit 100 TU 10 c 1 0,1 Sulfacetamide Phenobarbital Sotalol HCl Chloramphenicol Acebutolol HCl Ibuprofen Captopril Caffeine Enalapril maleate Theophiline Practolol HCl Carbamazepine Isoniazid Acetylsalicylic acid Lidocaine HCl Metoprolol Paracetamol Sulfanilamide Diclofenac 5,5-Diphenylhydantoin Diltiazem HCl Verapamil HCl Orphenadrine HCl Chloroquine phosphate Propranolol HCl Amitryptyline HCl Thioridazine HCl Chlorpromazine HCl

22 Correlation of the effect data of the 1h Rapidtoxkit with the 24h Thamnotoxkit Nalecz-Jawecki (Poland, 2005) 29 drugs y = 0,2165x R 2 = 0, Rapidtoxkit Thamnotoxkit

23 EILATOX Pecs A Technical Workshop on Toxicity for Biodefense University of Pecs, Hungary, September different types of tests (toxicity tests, mutagenicity tests, genotoxicity tests, biosensors, microbiotests) Application of the 1h (sublethal) Rapidtoxkit and the 24h (mortality) Thamnotoxkit -30 blind samples (not spiked and spiked natural waters and sediment pore waters)

24 Deep groundwater A34459 (not spiked and spiked) Sample number Sample type Rapidtoxkit (1h) Thamnotoxkit (24h) 2 Groundwater (not spiked) ppm MNNG ppm KCN ppm MNNG ppm KCN ppm HgCl ppm MNNG ppm KCN ppm HgCl 2 Not toxic Toxic Very toxic Extremely toxic Toxic Units >100 Color code

25 Deep groundwater A34459 (not spiked and spiked) Sample number Sample type Rapidtoxkit (1h) ToxScreen II PO ToxScreen II PM 2 Groundwater (not spiked) ppm MNNG ppm KCN ppm MNNG ppm KCN ppm HgCl ppm MNNG ppm KCN ppm HgCl 2 Not toxic Toxic Very toxic Extremely toxic Toxic Units >100 Color code

26 The 1h Rapidtoxkit is a good early warning tool for rapid prediction of important biological effects A second EPA/ETV study is now in progress in the USA with the same chemicals and biotoxins to determine the usefulness of the 1h Rapidtoxkit for rapid detection of water contamination

27 RAPID TOXICITY TESTS THE ECOTOX A fully automated on-line biomonitoring system based on real time image analysis of movement and behavior of a flagellated protozoan Developed at the Friedrich-Alexander University of Erlangen in Germany by Prof.Dr.D.Häder and associates

28 The freshwater flagellate Euglena gracilis Stock cultures can be stored on the shelf for long periods of time without maintenance

29 Biotest system ECOTOX: an overview

30 The ECOTOX instrument

31 Measurement parameters Movement parameters: velocity orientation motility Cellular properties cell form

32 The measurement principle 1. Cells are mixed with (control) water 2. Measurement parameters are determined 3. Cuvette is rinsed 4. Cells are mixed with sample 5. Measurement parameters are determined 6. Parameters are compared and an alarm is elicited when a significant deviation is found

33 Results: control vs. sample

34 Overall, the sensitivity of the rapid Euglena assay was found to be similar to that of the bacterial luminescence inhibition test with Vibrio fischeri

35 The ECOTOX instrument has been developed specifically for on line detection of chemical hazards in water

36 A low cost manual version : the EUGLENOTOX is now near completion for one by one analysis of individual water samples The EUGLENOTOX is a portable instrument for lab and field analyses. The EUGLENOTOX intrument will be supported by an EUGLENOTOXKIT with all the materials for 30 rapid toxicity tests

37 CONCLUSIONS 1. Several culture/maintenance free toxicity tests with bacteria, unicellulars and crustaceans are now available for rapid toxicity detection in contaminated waters 2. Since there is not one single species which is most sensitive to all toxicants, it is advised to apply 2 assays with species from different phylogenetic groups in order to avoid false negatives