1. Hans Christian Bruun Hansen a, Lars H. Rasmussen b, Frederik Clauson- Kaas a, Ole Stig Jacobsen c, Rene K. Juhler c, Søren Hansen a, and Bjarne W. Strobel a a Department of Plant and Environmental Sciences, KU- SCIENCE b Metropolitan University College c Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS) SOIL FATE AND LEACHING OF THE NATURAL CARCINOGEN PTAQUILOSIDE DWRIP 2014 KU-SCIENCE

2. Bracken form dense ”mats” Præstø Fed, Denmark Bracken is ”invasive” – and outcompetes other vegetation. Azores, Portugal

3. Why is this important? Bracken is one of very few plants known to cause cancer in animals Bracken is everywhere in Nature; 5 th most abundant plant on Earth The carcinogen in Bracken is produced in high amounts (up to 1 % dw) The carcinogen is very mobile in soil and water Several exposure routes for humans (air, milk, meat, drinking water) Little is known DWRIP 2014 KU-SCIENCE

4. A well known carcinogen in animals - Examples for cattle - Bovine enzootic haematuria (BEH): Tumours in the urinary bladder of cows and sheeps. Recognized worldwide. Test animals fed bracken produce similar symptoms. Upper digestive tract carcinomas: Ususally seen in conjunction with papillomavirus that infects the mucosa of the upper digestive tract in cattle. In presence of PTA papillomas transform to carcinomas DWRIP 2014 KU-SCIENCE

5. Exposure routes for humans DWRIP 2014 KU-SCIENCE Aranho, P (2013)

6. 6 Bracken norsesquiterpene glycosides and hydrolysis products R 1 = H; R 2 = CH 3 Ptaquiloside R 1 = H; R 2 = CH 3 Isoptaquiloside R 1 = H; R 2 = CH 2 OH Ptesculentoside R 1 = CH 3 ; R 2 = CH 2 OHCaudatoside R 1 = CH 3 ; R 2 = CH 3 Ptaquiloside Z R 1 = H; R 2 = CH 3 Pterosin B R 1 = H; R 2 = CH 2 OH Pterosin G R 1 = CH 3 ; R 2 = CH 2 OH Pterosin A Hydrolysis products DWRIP 2014 KU-SCIENCE

7. PropertyData CAS87625-62-5 Molecular formulaC 20 H 30 O 8 Mass398.45 Water solubility> 30 g L - -1 Melting point (acetone)85 – 89 o C Log K ow < 0 Soil sorption, K d < 0.25 L Kg -1 Half-life (25 o C), hydrol.hours - weeks Activation energy (pH 4.5), hydrolysis 74 kJ mol -1 Toxicitymutagenic, carcinogenic, clastogenic, genotoxic Threshold conc. drinking water (one hit model) 0.015 µg L -1 Hydrophobic Hydrophilic PTA amphiphilic DWRIP 2014 KU-SCIENCE

8. Methods used for determination of PTA and PTB MethodConditionsLOD µg L -1 Reference HPLC-UVReverse phase, 214 nm for PTA; 220 nm for PTB 5000 100 Agnew & Lauren (1991) LC-MS/MSReverse phase; 421.1  241.1 (PTA) 219.1  201.0 (PTB) 0.19 0.15 Jensen et al. (2008) GC-MSFormation of bromo- derivative of Pterosin B 0.3 Francesco et al. (2011) DWRIP 2014 KU-SCIENCE

9. PTA production, distribution and hydrolysis in soil and water DWRIP 2014 KU-SCIENCE

10. Bracken growth, PTA contents and PTA loads PTA contents in fronds during growing season at different sites in DK and UK PTA content in fronds per m 2 land surface during growing season at different sites in DK 300 mg m -2 = 3 kg ha -1 PTA in fronds (ug g -1 ) PTA load (mg m -2 ) Julian day number May Aug DWRIP 2014 KU-SCIENCE Rasmussen (2003)

11. PTA (min-max): (  g g -1 ) PTA (min-max): (mg m -2 ) Bracken Fronds 108 – 3,80015 – 500 Bracken Rhizomes 10 – 7,050N.D. Oi/Oe- horizons 0.09 – 6.430.3 – 160 Oa/A- horizons 0.01 – 0.710.9 – 57 High variation in PTA content between bracken populations DWRIP 2014 KU-SCIENCE Rasmussen (2003)

12. Hydrolysis of PTA k A = 25.7 h -1 M -1 ; k N = 9.49 10 -4 ; h -1 M -1 ; k B = 4.83 10 4 h -1 M -1 - Half-lives at pH 4, 6 and 8 (25 o C): 8 d, 20 d, and 0.6 d - Low temperatures increase half-lives considerably DWRIP 2014 KU-SCIENCE Ayala et al. (2006)

13. Ovesen et al. (2008) Microbial contribution to PTA degradation open symbols: sterilized; closed symbols: untreated soil Degradation of PTA in soils at field moisture and 10 o C with initial PTA concentration of 25 g kg -1 Fast reaction: Abiotic Slow reaction: Biotic + Abiotic DWRIP 2014 KU-SCIENCE

14. Hydrolysis in soil solution Kinetics of PTA degradation in soil solutions from sandy and clayey top- and subsoils (10 o C). Open symbols represent solutions filtered (0.2 µm) before incubation; closed symbols unfiltered solutions. !! No significant hydrolysis PTA is stabilized in soil solution! Can degradation in soil be attributed to hydrolysis in solution phase? pH 4.5 - 7 DWRIP 2014 KU-SCIENCE Ovesen et al. (2008)

15. Leaching DWRIP 2014 KU-SCIENCE

16. PTA and PTB in shallow groundwater at Bracken infested areas Location GroundwaterSurface water Soil type Water level (m) pH TOC (mM) pH TOC (mM) Gadevang Loamy sand 1.7-2.56.305.93.3 PræstøSand 0.8-1.14.92.37.13.8 RavnsholtOrganic 0.14-0.384.512.85.67.7 Study sites Sampling in small inspection wells. Determination of PTA and PTB by a SPE-LC-MS/MS Clauson-Kaas et al. (2014) DWRIP 2014 KU-SCIENCE

17. PTA and PTB distribution in soil Clauson-Kaas et al. (2014) DWRIP 2014 KU-SCIENCE - PTA concentrations highest in the litter layer, but much higher total quantities in the mineral soil -Higher PTB than PTB concentrations in mineral soil -PTB as ”memory” effect of PTA? PTA w, PTB w : Extracted with water PTB a : Extracted with methanol

18. SoilJun.Jul.Aug.Sep.Oct.Nov.Dec.Jan. GadevangPTA0.0910.032T0.080bd T PTBbd xxbdT PræstøPTATT0.034bd TT PTB0.150.0350.0240.0260.017Tbd RavnsholtPTAnm Tbd 0.016bd PTBnm 0.470.390.490.240.0390.083 Observed groundwater concentrations of PTA and PTB (µg L -1 ) - PTA could be detected at all sites -Max. PTA concentration observed 0.09 ug L -1 ; max PTB observed 0.49 ug L -1. -Big variations over time! T = trace DWRIP 2014 KU-SCIENCE Clauson-Kaas et al. (2014)

19. SoilJun.Jul.Aug.Sep.Oct.Nov.Dec.Jan. GadevangPTAnd0.030nd0.0240.031Tnd PTB0.014ndT0.014nd PræstøPTAnd0.0350.20nd PTBnm 0.037Tnm RavnsholtPTAnm1.11nm 0.0530.023nd PTBnm0.56nm 0.090nd Observed concentrations of PTA in pond water near Bracken stands (µg L -1 ) -PTA detected in all surface waters -max. PTA concentration 1.1 g L -1 ; max. PTB concentration 0.56 g L -1. -Large temporal and spatial variation T = trace DWRIP 2014 KU-SCIENCE Clauson-Kaas et al. (2014)

20. Modelling of PTA leaching from a sandy soil using the DAISY Plant-Soil-Water model - First attempt - PTA production: Biomass production data of Rasmussen and Hansen (2002) PTA in biomass: 200 g g -1 DM (low) PTAsoil transfer:Leaching from fronds (Rasmussen et al., 2003), and decaying plants (frost for 3 consecutive days) Soil: Sandy soil (Præstø), 2 - 6 % of clay Hydraulic properties estimated according to Mualen and van Genuchten PTA degradation: Model from Ovesen et al. (2008) Climate data:Data for Zealand (Højbakkegaard) 1962 - 2001 used. Modelling:Leaching modelled for the period 1962 - 2001, and for a selected 1-year period (1967 - 1968). DWRIP 2014 KU-SCIENCE

21. Separate degradation rate constants have been used for O, A and B soil horizons for fast and slow degrading PTA pools Annual total PTA addition to soil 1.6 kg ha -1. Note the extremely variable soil contents and amounts of PTA leached Modelling results for the period 1962 - 2002 DWRIP 2014 KU-SCIENCE

22. Conclusions PTA proven animal and suspected human carcinogen. PTA production of kg ha -1 y -1. High spatial and temporal variation. Initial PTA degradation due to hydrolysis; highly sensitive to pH and temperature. Apparent stabilization in soil water Fast abiotic and slower biotic degradation of PTA in soil; stabilization of PTA in soil by clays. Sorption of PTA in soil is insignificant  fast leaching PTA and PTB present in groundwater and surface water; µg L -1 to ng L -1 range Groundwater and surface water monitoring is strongly needed; high time and spatial resolution is critical. DWRIP 2014 KU-SCIENCE