SEMI-QUANTITATIVE TEST FOR PARAQUAT IN PLASMA AND URINE Syngenta Crop Protection Basel

1. SEMI-QUANTITATIVE TEST FOR PARAQUAT IN PLASMA AND URINE Syngenta Crop Protection Basel Switzerland Test developed at Syngenta CTL United Kingdom by Bruce Woollen

2. WHAT SAMPLES ARE AVAILABLE ? • Blood plasma is the preferred sample • If only blood serum is available this can also be tested with limitations for interpretation of results (see slide 19) • Urine samples are useful to confirm that a poisoning case involves paraquat

3. DITHIONITE REDUCTION OF PARAQUAT Paraquat is converted to a blue colour by sodium dithionite Sodium dithionite alkali PARAQUAT PARAQUAT RADICAL ION (BLUE)

4. SUMMARY OF THE TEST PART 1 • A pre-screen will detect high concentrations of paraquat in plasma and urine If Part 1 is negative PART 2 • A more sensitive blue band test will detect paraquat down to 0.1 µg/mL

5. 1M WHAT IS NEEDED FOR THE TEST ? 1M NaOH ( FOR URINE) WATER SYNGENTA TEST KIT 15 ML PLASTIC TUBE 5 ML OR 10 ML SYRINGE PLASTIC MICROPIPETTE CEREX SILICA CARTRIDGE GLASS TUBES 1.2 ML SYRINGE ADAPTOR

6. Sachet B 1g Na2S204 Sachet A 2g NaHC03 PREPARATION OF DITHIONITE REAGENT 2 MINS 10 mL WATER MIX ALLOW TO SETTLE 2 mins

7. Video clip preparation of dithionite

8. PART 1 : PRE-SCREEN IN PLASMA OR URINE X 20 drops approx 0.9 mL MIX PLASMA or URINE DITHIONITE REAGENT COMPARE COLOUR

9. COLOUR CHART FOR PRE-SCREEN 0 3 10 30 100 µg/mL The limit of detection for paraquat in plasma and urine is about 2-3 µg/mL

10. Video clip pre-screen

11. RESULTS OF THE PRE-SCREEN • If a blue colour is produced the concentration of paraquat is estimated from the chart and no further testing is done • If a very faint blue colour or no blue colour is produced the blue band test is carried out • Urine samples require an additional step before the blue band test

12. 1M URINE ONLY PREPARATION OF URINE FOR THE BLUE BAND TEST IF TESTING PLASMA PROCEED TO SLIDE NO 14 DISCARD PELLET + URINE 5 mL 1M NaOH 0.5 mL CENTRIFUGE MIX 10 MINS URINE FOR TESTING 3000 RPM

13. PART 2 : BLUE BAND TEST FOR PARAQUAT IN PLASMA OR URINE DITHIONITE REAGENT approx 1 mL 2 MINS BLUE BAND PLASMA or URINE ~ 3 mL ~ 1 mL WATER COMPARE COLOUR

14. COLOUR CHART FOR BLUE BAND INTENSITY 0 0.1 0.2 0.5 1 2 µg/mL Chart is applicable to both plasma and urine

15. Video clip of blue band test

16. Plasma paraquat concentration µg/mL 5 3 a 1.0 0.7 0.5 0.3 b 0.1 0.07 0.05 4 8 2 12 14 6 10 20 22 16 18 24 Time after ingestion (hours) INTERPRETATION OF PLASMA PARAQUAT RESULTS Proudfoot curve – predictive line Examples : a) 1 µg/mL at 8h after ingestion survival unlikely b) 0.1µg/mL at 12h after ingestion survival likely a survival unlikely b survival likely Proudfoot A T, Stewart M S, Levitt T and Widdop B. Paraquat Poisoning : significance of plasma paraquat concentrations. Lancet 1: 330 1979

17. INTERPRETATION OF PLASMA PARAQUAT RESULTS From Syngenta paraquat treatment booklet Example : 1 µg/mL at 8h after ingestion approximately 30% chance of survival

18. WARNING ABOUT SERUM Plasma versus serum Paraquat concentrations in plasma and serum are NOT equivalent (serum concentrations are about 3 times lower than plasma concentrations) Proudfoot and Syngenta survival curves are based on plasma concentrations of paraquat

19. INTERPRETATION OF URINE PARAQUAT RESULTS From Schermann data Dark blue Navy blue Light blue Pale blue No blue Colour Schermann,J.M, Houze P, Bismuth C and Bourdon R. Prognostic value of Plasma and urine paraquat concentration. Human Toxicology : 6 91-93 1987

20. LIST OF EQUIPMENT AND SUPPLIERS • Paraquat test kit containing sodium bicarbonate and sodium dithionite (Syngenta) • Glass tubes 40 x 6mm 1.2mL (NLG Scientific) • Cerex silica cartridges 3mL 20mg Cat no 620-0203 ( Crawford Scientific) • Micropipettes Mini-Pastette LW4231 (Alpha Laboratories) • Syringe adaptor 120-1101 (Argonaut Technologies)