Hans Christian Bruun Hansena, Lars H. Rasmussenb, Frederik Clauson-Kaasa, Ole Stig Jacobsenc, Rene K. Juhlerc, Søren Hansena, and Bjarne W. Strobela
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
Bracken form
dense ”mats”
Præstø Fed, Denmark
Bracken is ”invasive” – and outcompetes other vegetation.
Azores, Portugal
Why is this important?
• Bracken is one of very few plants known to cause cancer in animals
• Bracken is everywhere in Nature; 5th 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
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
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Exposure routes for humans
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Aranho, P (2013)
6
Bracken norsesquiterpene glycosides and hydrolysis products
R1 = H; R2 = CH3
Ptaquiloside
R1 = H; R2 = CH3
Isoptaquiloside
R1 = H; R2 = CH2OH
Ptesculentoside
R1 = CH3; R2 = CH2OH
Caudatoside
R1 = CH3; R2 = CH3
Ptaquiloside ZR1 = H; R2 = CH3 Pterosin B
R1 = H; R2 = CH2OH Pterosin G
R1 = CH3; R2 = CH2OH Pterosin A
Hydro
lysi
spro
duct
s
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Property Data
CAS 87625-62-5
Molecular formula C20H30O8
Mass 398.45
Water solubility > 30 g L--1
Melting point (acetone) 85 – 89 oC
Log Kow < 0
Soil sorption, Kd < 0.25 L Kg-1
Half-life (25 oC), hydrol. hours - weeks
Activation energy (pH 4.5), hydrolysis
74 kJ mol-1
Toxicity mutagenic, carcinogenic, clastogenic, genotoxic
Threshold conc. drinking water (one hit model)
0.015 µg L-1
Hydrophobic
Hydrophilic
PTA amphiphilic
DWRIP 2014 KU-SCIENCE
Methods used for determination of PTA and PTB
Method Conditions LODµg L-1
Reference
HPLC-UV Reverse phase, 214 nm for PTA; 220 nm for PTB
5000100
Agnew & Lauren (1991)
LC-MS/MS Reverse phase;421.1 241.1 (PTA)219.1 201.0 (PTB)
0.190.15
Jensen et al. (2008)
GC-MS Formation of bromo-derivative of Pterosin B
0.3Francesco et al. (2011)
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PTA production, distribution and hydrolysis
in soil and water
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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 m2 land surface during growing season at different sites in DK
300 mg m-2 = 3 kg ha-1
PTA
in f
ronds
(ug g
-1)
PTA
load (
mg m
-2)
Julian day number
May
Aug
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Rasmussen (2003)
100-499 ppm Pta
500-999 ppm Pta
1000-4000 ppm Pta
N PTA
(min-max):
(g g-1)
PTA
(min-max):
(mg m-2)
Bracken Fronds
108 – 3,800 15 – 500
Bracken Rhizomes
10 – 7,050 N.D.
Oi/Oe-horizons
0.09 – 6.43 0.3 – 160
Oa/A-horizons
0.01 – 0.71 0.9 – 57
High variation in PTA content between bracken populations
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Rasmussen (2003)
Hydrolysis of PTA
-6
-4
-2
0
2
0 2 4 6 8 10
-log[H+]
logk
obs
]
[
log
log
Hkk
k
WB
obs
]
[
log
log
Hk
k
A
obs
Nobs kk loglog
2σ
kA = 25.7 h-1 M-1; kN = 9.49 10-4; h-1 M-1; kB = 4.83 104 h-1 M-1
- Half-lives at pH 4, 6 and 8 (25 oC): 8 d, 20 d, and 0.6 d
- Low temperatures increase half-lives considerably
OH CH3 HO
CH3
O
D - glucose
CH3
(p H > 7 .5 )
D - g lu c o se
CH3
OH
CH3
OCH3
D - g lu c o se
H + /H 2 O
H + /H 2 OOH
CH3
CH3
O
CH3
Bracken dienone Pterosin B
Ptaquiloside
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Ayala et al. (2006)
A0
25
50
75
100
0 200 400 600Time (hours)
Deg
rada
tion
(%)
B 0
25
50
75
100
0 200 400 600Time (hours)
Deg
rada
tion
(%)
C0
25
50
75
100
0 200 400 600Time (hours)
Deg
rada
tion
(%)
D0
25
50
75
100
0 200 400 600Time (hours)
Deg
rada
tion
(%)
Sandy soil Clayey soil
S
ub
soil
To
pso
il
Ovesen et al. (2008)
Microbial contribution to PTA degradation
1
1
1
100 1
F
S
c(t) a ( exp( k t))
( a) ( exp(k t))
open symbols: sterilized; closed symbols: untreated soil
Degradation of PTA in soils at field moisture and 10 oC with initial PTA concentration of 25 g kg-1
Fast reaction: Abiotic Slow reaction: Biotic + Abiotic
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-30
-10
10
30
0 200 400 600Time (hours)
De
gra
da
tion
(%
)
Hydrolysis in soil solution
Kinetics of PTA degradation in soil solutions from sandy and clayey top- and subsoils (10 oC).
Open symbols represent solutions filtered (0.2 µm) before incubation; closed symbols unfiltered solutions.
!! No significant hydrolysisPTA is stabilized in soil solution!
Can degradation in soil be attributed to hydrolysis in solution phase?
pH 4.5 - 7
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Ovesen et al. (2008)
Leaching
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PTA and PTB in shallow groundwater at Bracken infested areas
LocationGroundwater Surface water
Soil typeWater level
(m)pH
TOC(mM)
pHTOC(mM)
GadevangLoamy sand 1.7-2.5 6.3 0 5.9 3.3
Præstø Sand 0.8-1.1 4.9 2.3 7.1 3.8
Ravnsholt Organic 0.14-0.38 4.5 12.8 5.6 7.7
Study sites
Sampling in small inspection wells.Determination of PTA and PTB by a SPE-LC-MS/MS
Clauson-Kaas et al. (2014)
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PTA and PTB distribution in soil
Clauson-Kaas et al. (2014)
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- 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?
PTAw, PTBw: Extracted with water
PTBa: Extracted with methanol
Soil Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan.
Gadevang PTA 0.091 0.032 T 0.080 bd bd T bd
PTB bd bd bd xx bd T bd bd
Præstø PTA T T 0.034 bd bd T T bd
PTB 0.15 0.035 0.024 0.026 0.017 T bd bd
Ravnsholt PTA nm nm T bd bd bd 0.016 bd
PTB nm nm 0.47 0.39 0.49 0.24 0.039 0.083
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)
Soil Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan.
Gadevang PTA nd 0.030 nd 0.024 0.031 T nd nd
PTB 0.014 nd T 0.014 nd nd nd nd
Præstø PTA nd 0.035 0.20 nd nd nd nd nd
PTB nm nm 0.037 T nm nm nm nm
Ravnsholt PTA nm 1.11 nm nm nm 0.053 0.023 nd
PTB nm 0.56 nm nm nm 0.090 nd nd
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)
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)PTAsoil 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).
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decomposed
0
500
1000
1500
2000
(g h
a-1 y
-1)
soil content
0
50
100
150
200
(g h
a-1)
input
0
500
1000
1500
2000
(g h
a-1 y
-1)
PTA fast
PTA slow
Leaching - 110 cm
0
0.05
0.1
0.15
0.2
19
62
19
64
19
66
19
68
19
70
19
72
19
74
19
76
19
78
19
80
19
82
19
84
19
86
19
88
19
90
19
92
19
94
19
96
19
98
20
00
20
02
time
(g h
a-1 y
-1)
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
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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.
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