degradation of 2-methyl-4chlorphenoxyacetic acid … · degradation of 2-methyl-4chlorphenoxyacetic...

MCPA and tfdA genes; Warwick, March 2006

Degradation of 2-methyl-4chlorphenoxyacetic acid (MCPA) in top- and subsoil is quantitatively linked to the class III

tfdA gene

Carsten Suhr Jacobsen1, 2 & 3 * Jacob Bælum1 & 2; Trine Henriksen1, & Hans Christian Bruun Hansen2

1) Geological Survey of Denmark and Greenland (GEUS), Department of Geochemistry 2) Royal Veterinary and Agricultural University (KVL), Department of Chemistry3) Royal Veterinary and Agricultural University (KVL), Department of Ecology

MCPA d fdA W i k M h 2006

Groundwater as source of

drinking water in Europe

(red)


GRUNDWATER QUALITY CONTROLEBased on: • 700 State owned (GEUS)

Grumo filters• 1700 Commercial groundwater

abstraction wells• Pesticide Leaching Assessment

Programme (PLAP) (run by GEUS and DJF). See poster B4 by Jeanne Kjaer – and poster B3 by Heid Barlebo for examples.

EU drinking water limit >0,1 µg/l• Exceeded in 10% of the Grumo-

filtres• Exceeded (since 2000) in

decreasing numbers of commercial groundwater abstraction wells


Groundwater contaminated with pesticides

• Red: over the 0.1 ug/l EU-limit

• Blue: detectable but below the 0.1 ug/l EU-limit

GRUMO boringer

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1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

Fund

(%)

Fund >=0,1 µg/l Fund <0,1 µg/l

Vandværksboringer

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1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

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(%)

Survey wells

Production wells


Geologic heterogeneity in a small country

• A large contract with the Danish EPA

• >800 soil samples is described geological and analyzed using geochemical, soil physical, and microbial key-parameters and these are correlated to pesticide sorption and degradation


An example on field variability in microbial activity and pesticide mineralisation

• 50 samples from A horizon and 50 samples from Bs horizon

• Vinther et al in prep.

0 40 80 120 160 200Meter in eastern direction


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a Clay, Ap

c

b

dClay, Bs

Org. C, Ap

Org. C, Bs

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A-horizon samples Vinther et al in prepFredslund et al in prep

• Metribuzine and triazineamine low mineralisation in all samples (higest 3.1% in 90 days)

• Glyphosate mineralisation was found to correlate with microbial activity (SIR and ASA ArylSulfatase Activity) (and with total C).

• MCPA mineralized brilliantly in all A horizon soils with absolutely no correlation to soil parametres of any kind



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c ASA d FDA

e Total CFU f Pseudomonas sp.0 40 80 120 160 200

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b metribuzin e triazinamin


MCPA (a pet herbicide for microbial ecologist)

• Phenoxyacetic acid herbicide (MCPA, 2,4-D og 2,4,5-T)

• Banned (restricted) in Denmark in 1995 but now used in several mixtures against weeds in grasses

Annual sale of MCPA in Denmark

0

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400000

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1000000

1960 1970 1980 1990 2000 2010

Act

ive

com

poun

d (k

g/ye

a

OCOOH

CH3

Cl

• 4-chloro-2-methylphenoxyaceticacid

• C9H9ClO3

• Mol. weight: 200,6 g/mol• Log KOW: 2,75 (pH 1)

0,46 (pH 5)• pKa: 3,07


Lessons learned from >800 soils in the Danish EPA supported pesticide area zonation project

• Higher maximum mineralization of MCPAin subsoils compared to surface soil.

• Metabolite formation in surface soil but not in subsoils ?

• Formation of less bioavailable metabolites?• Variation in subsoils (does numbers matter ?).

(KUPA)

MCPA Mineralization day 62, 5-20 cm depth

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0 25 50 75 100 125 150 175 200 225 250

meter east

met

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MCPA Mineralization day 63, 50 cm depth

0255075

100125150175200

0 25 50 75 100 125 150 175 200 225 250meter east

met

er n

orth

(KUPA)

OCOOH

CH3

Cl


Degradation pathway for MCPA

• The phenol compound MCP is a likely degradation product from MCPA

• Simultaneous and quantitative extraction and detection of MCP and MCPA from soils needed new methods

O

CH2COOH

CH3

Cl

OH

CH3

Cl

CH2COOH

OH

OH

CH3

Cl

COOHCOOH

CH3Cl

Cl-

COOH

CH3

O

O

COOH

CH3

O

O

COOH

CH3

O

O

TCA-cycle

MCP

tfdA tfdB tfdC tfdD tfdE tfdF

MCPA

tfdAαcadA ?

Extraction and quantification of MCPA and MCP in soil:Extraction with ”Accelerated solvent extraction” (ASE) (MeOH:H2O 50:50, temp. 50 oC, at 1200 psi) quantification using LC-MS/MS (100×2.1 mm RP18)


Time (days)0 20 40 60 80 100 120

MC

PA (%

) of i

nitia

l con

c.

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MC

P (%

) of i

nitia

l M

CPA

con

c.

0

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20Top2.3

Top20

Sub2.3

MCP in Top2.3

MCP in Top20

Dissipation of MCPA and formation and dissipation of MPC

- Semi-quantitative- Not corrected for

extraction efficiency- Low extraction efficiency


Mineralization of MCPA Mineralization

Time (days)0 20 40 60 80 100 120 140 160

% o

f ini

tial 1

4 C m

iner

aliz

ed

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% o

f ini

tial 14

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iner

aliz

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100Single plots of 2,3 mg/kg in subsoilfrom panel A

MCPA Mineralisering dag 63, 50 cm dybde

0255075

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150175200

0 25 50 75 100 125 150 175 200 225 250meter i østlig retning

met

er i

nord

lig r

etni

ng

2.3 mg/kg MCPA in the topsoil20 mg/kg MCPA in the topsoil200 mg/kg MCPA in the topsoil2.3 mg/kg MCPA in the subsoil200 mg/kg MCPA in the subsoil

x


Linking microbial degradation activity with analysis of degrader populations

• Microbial degradation activity/kinetics are analyzed using nonlinear regression analysis.

• Brunners & Foghts (1984; Appl. Environ. Microbiol.47:167-172) 3/2 models indicates logarithmic growth of degrader populations as validated by F-test analysis of Chi2 values (Robinson 1985; Adv. Microb. Ecol. 8:61-114).

• The role of different gene families of tfdA and alike genes was investigated.


Primer selection and validation

No PCR product58f: AAGCTGCARTTTGARAAY*

r: MGGATTGAAGAAATCCTGRTA*HW13 (cadA-gene)cadA

One band (app. 350 bp)-qPCR maybe possible

65f: ACSGAGTTCKSCGACATGCG*

r: GCGGTTGTCCCACATCACAlignment of HW13, HWK12 and BTH (tfdAα-genes)

tfdAα**

No clear band on the agarose gel-fluffy PCR products.

N.D. ****f: CCGGCGTCGATCTGCGCAAGr: GTTGACGACGCGCGCCGA

Alignment of RD5-C2, HWK12 and HW13 (tfdAα-genes)

tfdAα*

One band on agarose gel (app. 220 bp.) - qPCR possible

64f: GAGCACTACGCRCTGAAYTCCCG*, **

r: GTCGCGTGCTCGAGAAGSequence alignment of 23 known tfdA-genes

tfdA**

Used for probe preperation to southern blot hybridization

65f: GCATACGACGCGCTGCCTCGr: CTTCGGCCACCGGAAGGCCT

R. eutropha JMP134tfdA-intern

Unspecific binding-qPCR not possible 58f: AACGCAGCGRTTRTCCCA*

r: ACGGAGTTCTGYGAYATG*R. eutropha JMP134/ Burkholderia sp.RASC

tfdA*

NoteOptimal annealing temperature Used (°C) ***

Primer (5’-3’) forward (f) / reverse (r) Model genesPrimer set

* R=(G/A), Y=(T/C), S=(G/C), K=(G/T) and M=(A/C).** GC clamp added to the 5’ end of the primer for the PCR/DGGE, 5’-CGCCCGCCGCGCGCGGCGGGCGGGGCG *** The optimal annealing temperature is based on gradient ramping (58-69 °C).**** N.D no optimal annealing temperature could be determined.


Primer selection and validation

• Soil DNA from days 0, 6, 12, 33, 50, 68 and 115 of topsoil amended with 20 ppm MCPA were used as template (panel A left)

• R. eutropha JMP 134 inoculated into the soil in concentrations of 8×106, 8×105 and 8×104 cells/g soil (panel A right)

• Panel B shows Southern hybridization analysis of the agarose gel shown in panel A) using a tfdA probe. Similarity can be detected for band no. 2 and 3.

• Results from the use of Vallaeys, T. et al. 1996. FEMS Microbiol. Ecol. 20:163-172. primers against tfdA genes


New primers where developed

• A BLAST search in 2004 revealed a large group of new tfdA genes not covered by the old consensus primers

• Forward primer: GAGCACTACGCRCTGAAYTCCCG• Reverse primer: GTCGCGTGCTCGAGAAG• DGGE using GC clamp


DGGE on tfdA genes

• ”Denaturing gradient gel electrophoresis” (DGGE) on the tfdA gene

• Clear shift in populations in soils with MCPA

• Different populations (if DGGE are trusted) are involved in degradation.


• Fylogenetisk træ over tfdA-og tfdA lignende gener

• De fremvoksede gener tilhører klasse III tfdA gener

• Baggrunds indholdet af tfdA,Tilhører klasse I tfdA gener

tfdA containingorganisms

Organisms containingtfdA-like genes


Melting profiles used in analysis of changes in functional diversity of tfdA genes

• Days 0, 12 and 22 in topsoil amended with 20 ppm MCPA using the tfdA-II primer set.

• Compare to dotted line R. eutrophusJMP 134 (pJP4).

Temperature, oC

65 70 75 80 85 90 95

- d(R

FU)/d

T

0

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800

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1200R. eutropha JMP134Day 0Day 12Day 22


Quantitative PCR on tfdA genes in soil


Linking function and tfdA gene quantification in top soils

2.3 mg/kg in topsoil

Time (days)0 20 40 60 80 100 120 140

MC

PA re

mai

ning

(%) /

(%

) of i

nitia

l 14 C

min

eral

ized

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Log

(tfdA

gen

es/g

soil)

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20 mg/kg in topsoil

Time (days)0 20 40 60 80 100 120 140

MC

PA re

mai

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(%) /

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min

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ized

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Log

(tfdA

gen

es/g

soil)

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Mineralization curveDegradation curveNo. of tfdA genes


Linking function and tfdA gene quantification in B horizon soils

2.3 mg/kg in subsoil

Time (days)0 20 40 60 80 100 120 140 160

MC

PA re

mai

ning

(%) /

% in

itial

14C

min

eral

ized

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Log

(tfdA

gen

es/g

soil)

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7

2.3 mg/kg in subsoil

Time (days)0 20 40 60 80 100 120 140

MC

PA re

mai

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(%) /

% in

itial

14C

min

eral

ized

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Log

(tfdA

gen

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soil)

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Mineralization curveDegradation curveNo. of tfdA genes


tfdA gene analysis of 50 field variation samples plotted against MCPA

mineralisation

• High tfdA gene copy number (low Ct) is coupled to fast onset of MCPAmineralisation.

• But after 2 weeks the picture disappear.

35 40 450

5

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208 days

ct

min

eral

isat

ion

35 40 4530

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7015 days

ct

min

eral

isat

ion

35 40 4530

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7022 days

ct

Min

eral

isat

ion

35 40 4530

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7029 days

ct

min

eral

isat

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7039 days

ct

min

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isat

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35 40 4530

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7067 days

ct

min

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isat

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Fredslund et al in prep.


Bs horizon samples

• Fever soils produced Ct values

• High gene number is associated with faster onset for mineralization.

• But some microbes never wake up ☺

25 30 35 400

0.5

1

1.5

28 days

ct

min

eral

isat

ion

25 30 35 400

0.5

1

1.5

215 days

ct

min

eral

isat

ion

25 30 35 400

2

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622 days

ct

min

eral

isat

ion

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3029 days

ct

min

eral

isat

ion

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6039 days

ct

min

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8067 days

ct

min

eral

isat

ion


Conclusions

• Fieldvariation of some pesticides can not be explained by soil microbial activity.

• Analysis of MCPA mineralization kinetics revealed growth dependent microbial mineralization in top-soil as well as B-horizon soil.

• New PCR-primers shows sensitive and highly reproducible quantitative results linked to degradation.

• Functional diversity analysis of tfdA genes shows population shifts during degradation. The detected tfdA-I populations present in the top-soil from the beginning of the experiment where not associated with growing microbial populations. The growing populations where associated with tfdA-III class genes.

• LC- MS/MS analysis revealed formation of MCP in top-soil but not in B-horizon soil. MCP formations is likely associated with tfdA-I class genes.

• tfdA gene quantification can predict the speed of degradation but the growth kinetics of MCPA is really fast


Acknowledgments

• Mette Andersen for skilled technical support on Real Time PCR.• Line Fredslund Nielsen for work with the tfdA gene in relation to

fieldvalidation.• Finn Vinther, Lars Elsgaard and Ulla Brinch for analysis of soil

microbial activity and pesticide mineralization.• Kaare Johnsen and Mette Nicolaisen for help with phylogeny of

tfdA sequences data. • René Juhler for discussions on MCP and MCPA quantification.• The Danish research Council (STVF) for financial support

through the “SOUND” research center.• The Danish EPA for founding the Danish pesticide zonation

project.


Thank you for your attention !

2,4-D

OCH2COOH

Cl

Cl

Cl

Cl

OH

Cl

Cl

OH

OH

COOHCOOH

ClCl

3,5-dichlorocatechol

2,4-dichloromuconic acid

2,4-dichlorophenol

tfdA

tfdB

tfdC

Tricarboxylic acid cycle

degradation of 2-methyl-4chlorphenoxyacetic acid … · degradation of 2-methyl-4chlorphenoxyacetic...

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