Hans Christian Bruun Hansen a, Lars H. Rasmussen b, Frederik Clauson- Kaas a, Ole Stig Jacobsen c,...

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


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


Exposure routes for humans


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


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


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)


PTA production, distribution and hydrolysis

in soil and water


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


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


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


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


-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


Ovesen et al. (2008)

Leaching


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)


PTA and PTB distribution in soil



- 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



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



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).


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


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.


Hans Christian Bruun Hansen a, Lars H. Rasmussen b, Frederik Clauson- Kaas a, Ole Stig Jacobsen c,...

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