GFP – 17 mai 2017

Development of a LC-MS/MS multiresidu method for the quantification of traces of

pesticides in earthworms

G. Daniele, F. Lafay, C. Pelosi , C. Fritsch, V. Bretagnolle, E. Vulliet

GFP – 17 mai 2017

Background

Agriculture intensification

progressive loss of biodiversity

decline of the ecosystemic services

in the cultivated zones and the agricultural landscapes

The landscape’s organization can affect the spatial distribution of pesticides as well as the exposition of non-target organisms and the impacts on them

GFP – 17 mai 2017

Objectives RESCAPE project (RESistance of agricultural landSCAPEs to pesticide transfers in soils and living organisms)

Evaluate the impact of landscape’s characteristics and cultural practices on the resistance of the landscapes to pesticides transfers into the soils and living organisms Earthworms involved in the functioning of agroecosystems present in most of the soils recognized as indicators of use of lands and practices of culture In natura approach Pesticide content Actual exposition

GFP – 17 mai 2017

Objectives RESCAPE project (RESistance of agricultural landSCAPEs to pesticide transfers in soils and living organisms)

Evaluate the impact of landscape’s characteristics and cultural practices on the resistance of the landscapes to pesticides transfers into the soils and living organisms Earthworms involved in the functioning of agroecosystems present in most of the soils recognized as indicators of use of lands and practices of culture In natura approach Pesticide content Actual exposition Develop analytical methodology to detect and quantify traces of pesticides in earthworms

GFP – 17 mai 2017

Targeted pesticides – Zone atelier Chizé

Classe Pesticide Masse molaire (g/mol) log Kow Famille chimique IN

SEC

TIC

IDES

Thiamethoxam 291,7 -1,16

Neonicotinoide Thiacloprid 252,7 1,26

Imidacloprid 255,7 0,57

Pirimicarb 238,3 1,7 Carbamate

Tau-fluvalinate 502,1 4,26

Pyréthrinoide

Lambda-Cyhalothrin 449,9 7,0

Deltamethrin 505,2 5,43

Cypermethrin 416,3 6,3

Bifenthrin 422,9 >6

HER

BIC

IDES

Pyroxsulam 434,4 1,08 Sulfonamide

Pendimethalin 281,3 5,18 Dinitroaniline

Napropamide 271,2 3,36 Alcanamide

Metazachlor 277,1 2,49 Chloroacétanilide

Dimethachlor 255,7 2,33

Diflufenican 394,1 2,5 Carboxamide

Cycloxydim 325,5 3,23 Cyclohexane dione

Cloquintocet-mexyl 335,8 5,12 Dérivés de la quinoline

Clomazone 239,7 2,54 Isoxazolidine

FON

GIC

IDES

Prochloraz 376,7 4,1 Imidazole

Metrafenone 408,1 4,3 Benzophénone

Pyraclostrobin 387,1 3,99 Strobilurine

Fluoxastrobin 458,1 2,86

Propiconazole 342,2 3,72

Triazole Metconazole 319,1 3,85

Epoxiconazole 329,8 3,33

Cyproconazole 291,8 2,91

Boscalid 342,0 2,96 Carboxamide

GFP – 17 mai 2017

Analytical developments

GFP – 17 mai 2017

Analytical methodology

Sample preparation Analysis

- Extract the analytes

- Eliminate the interferences

- Concentrate the traces

QuEChERS

- Detect

- Identify

- Quantify

LC-MS/MS

GFP – 17 mai 2017

Detection optimization

Triple quadrupole mass spectrometer XeVO TQ-S (Waters®)

Detection in MRM mode

Infusion of individual standards :

Detection mode (positif or negatif)

Specific transitions for each compound

Optimisation of different tensions, gas,…

GFP – 17 mai 2017

Detection optimization CV (V) MRM 1 CE 1 (CV) MRM 2 CE 2 (CV)

Thiaméthoxame 7 292.2 > 211.1 11 292.2 > 181.1 19

Imidaclopride 11 256.2 > 209.2 15 256.2 > 175.2 17

Thiaclopride 20 253.0 > 126.0 20 253.0 > 186.0 12

Pyroxsulame 16 434.9 > 194.9 26 434.9 > 123.9 46

Pirimicarbe 30 239.1 > 72.0 18 239.1 > 182.1 18

Métazachlore 15 278.0 > 134.1 22 278.0 > 210.0 10

Diméthachlore 20 256.2 > 224.2 15 256.2 > 148.2 25

Clomazone 23 240.0 > 125.0 18 240.0 > 89.0 46

Cyproconazole 27 292.2 > 70.2 18 292.2 > 125.1 24

Boscalid 30 343.2 > 307.2 20 343.2 > 140.0 20

Napropamide 21 272.1 > 129.1 16 272.1 > 171.1 18

Epoxiconazole 30 330.0 > 121.0 23 330.0 > 123.0 20

Metconazole 29 320.1 > 70.0 22 320.1 > 125.0 36

Fluoxastrobine 27 459.0 > 427.0 18 459.0 > 188.0 36

Propiconazole 37 342.0 > 159.0 34 342.0 > 69.0 22

Prochloraz 27 376.2 > 308.1 11 376.2 > 70.0 23

Cycloxydime 23 326.0 > 280.0 16 326.0 > 180.0 22

Metrafenone 19 409.0 > 209.1 14 409.0 > 226.9 16

Pyraclostrobin 20 388.1 > 193.9 12 388.1 > 163.0 25

Diflufenican 30 394.9 > 266.0 28 394.9 > 246.0 32

Cloquintocet-mexyl 30 336.1 > 238.0 18 336.1 > 192.0 35

Pendiméthaline 12 282.2 > 212.2 10 282.2 > 194.1 17

Lambda-Cyhalothrine 6 467.3 > 225.1 12 467.3 > 141.1 46

Cyperméthrine 20 433.3 > 191.1 12 433.3 > 416.2 8

Deltaméthrine 10 523.2 > 281.0 14 523.2 > 506.1 8

Bifenthrine 32 440.3 > 181.2 14 440.3 > 166.0 44

Tau-fluvalinate 15 503.0 > 181.1 30 503.0 > 208.1 12

GFP – 17 mai 2017

Chromatographic separation

Column Kinetex PhenylHexyl (100 × 2.1 mm ; 2.7 µm) (A) water + 0.01 % AA + 0.4 mM AcAm (B) Methanol Flow : 0.7 mL/min Vinj = 2 µL T=60°C

Optimisation parameters (H-Class UPLC System Waters®) - Chromatographic column

- Mobile phases (aqueous and organic, salts)

- Elution gradient

- Column temperature

- Injection volume

GFP – 17 mai 2017

Chromatographic separation

GFP – 17 mai 2017

Analytical methodology Extraction

(QuEChERS)

X mL of ultra pure water

250 mg of earthworms

X mL of heptane

10 mL of acetonitrile

Buffer salts

Shake

Centrifuge

GFP – 17 mai 2017

Analytical methodology Extraction

(QuEChERS) Purification

X mL of ultra pure water

250 mg of earthworms

X mL of heptane

10 mL of acetonitrile

Buffer salts

Shake

Centrifuge

Purification (dSPE)

GFP – 17 mai 2017

Analytical methodology Extraction

(QuEChERS) Purification Analysis

X mL of ultra pure water

250 mg of earthworms

X mL of heptane

10 mL of acetonitrile

Buffer salts

Shake

Centrifuge

Purification (dSPE)

Concentrate X times

Injection of 2 μL in mobile phase

Separation + Detection (LC-MS/MS)

H-Class Xevo TQS - Waters®

GFP – 17 mai 2017

Optimization of QuEChERS extraction - Earthworms-

GFP – 17 mai 2017

Choice of the extraction salt

Citrate salt

0

20

40

60

80

100

120

Norm

alized a

rea (

%)

Citrate salt

Acetate salt

GFP – 17 mai 2017

Choice of the ACN/water ratio

Ratio ACN/water (2x5)/6

0

10

20

30

40

50

60

70

80

90

100

Norm

alized a

rea (

%)

ACN/water 10/3

ACN/water 10/6

ACN/water 10/9

ACN/water 2x5/6

ACN/water 2x5/9

GFP – 17 mai 2017

Choice of the purification phase (dSPE)

Purification dSPE with PSA/C18 phase

0

20

40

60

80

100

Norm

alized a

rea (

%)

PSA

PSA/C18

EMR lipid

No dSPE

GFP – 17 mai 2017

Analytical methodology Extraction

(QuEChERS) Purification Analysis

6 mL of ultrapure water

250 mg of earthworms

3 mL heptane

5 mL acetonitrile

Citrate buffer

Agitation + Vortex + GenoGrinder +

Centrifugation

2d extraction with 5 mL ACN

Purification with dSPE (PSA/C18)

Concentrate x 6

Injection of 2 μL in mobile phase

Separation + Detection (LC-MS/MS)

Xevo TQS - Waters®

Stirring + Vortex + GenoGrinder +

Centrifugation

GFP – 17 mai 2017

Analytical performances of whole protocol

17 LOQ < 1 ng/g 88% LOQ < 5 ng/g 3 LOQ : >5 ng/g

LOD (ng/g) LOQ (ng/g) Pirimicarb 0,05 0,1 Clomazone 0,1 0,2 Thiacloprid 0,05 0,1 Dimethachlor 0,2 0,5 Imidacloprid 0,2 0,4 Napropamide 0,2 0,5 Metazachlor 0,2 0,5 Pendimethalin 1,5 4,5 Thiamethoxam 0,2 0,4 Cyproconazole 0,5 1,4 Metconazole 0,2 0,5 Cycloxydime 1,5 5,1 Epoxiconazole 0,2 0,4 Cloquintocet-mexyl 0,2 0,5 Propiconazole 0,3 0,7 Boscalid 0,5 1,6 Prochloraz 0,2 0,4 Pyraclostrobin 0,1 0,3 Diflufenican 0,2 0,4 Metrafenone 0,5 1,4 Cypermethrin 9,8 32,7 Pyroxsulame 0,2 0,5 Bifenthrin 0,2 0,4 Fluoxastrobine 0,4 1,5 Lambda-Cyhalothrin 15,2 50,8 Tau-fluvalinate 1,3 4,2 Deltamethrin 1,2 3,9

Extraction recoveries :

All comprised between 80-120 %

(except pyroxsulam)

Limits of quantification (LOQ) :

All comprised between 0.1-51 ng/g

GFP – 17 mai 2017

Analysis of earthworm samples - collected from Zone atelier Chizé -

GFP – 17 mai 2017

Results of earthworm collected in Chizé ZA

24 hedge

6 meadow

8 wheat

Earthworms collected in 60 different plots (1 km x 1 km) Analysis of 38 earthworm samples :

GFP – 17 mai 2017

Results of earthworm collected in Chizé ZA

24 hedge

6 meadow

8 wheat

Earthworms collected in 60 different plots (1 km x 1 km) Analysis of 38 earthworm samples :

34 samples with at least 1 targeted pesticide (onto 38 analysed)

13 different pesticides quantified in the samples (onto 27 targeted)

1 to 7 targeted pesticides/sample

GFP – 17 mai 2017

Results of earthworm collected in Chizé ZA

Classe Pesticide Famille chimique LOQ (ng/g) Nb

détections/38 Cmin

(ng/g) Cmax

(ng/g)

INSECTICIDES Thiacloprid

Neonicotinoide 0,1 8 <LQ 2,8

Imidacloprid 0,4 32 2 631

HERBICIDES

Pyroxsulam Sulfonamide 0,5 3 <LQ 78,8

Pendimethalin Dinitroaniline 4,5 1 51,5

Napropamide Alcanamide 0,5 1 3,2

Diflufenican Carboxamide 0,4 20 <LQ 493

FONGICIDES

Prochloraz Imidazole 0,4 7 1,2 487

Metrafenone Benzophénone 1,4 1 21,8

Pyraclostrobin Strobilurine 0,3 1 48

Propiconazole

Triazole

0,7 1 3,5

Epoxiconazole 0,4 7 3,5 204

Cyproconazole 1,4 7 2,7 22

Boscalid Carboxamide 1,6 3 2,3 12,4

GFP – 17 mai 2017

Results of earthworm collected in Chizé ZA

Around 30% imidacloprid > 100 ng/g & 50% diflufenican > 10 ng/g

More frequent combinations : Imidacloprid/Diflufenican/Epoxyconazole (7)

Imidacloprid/Diflufenican/Cyproconazole (7)

Imidacloprid/Diflufenican/Prochloraz (6)

0

5

10

15

20

25

30

35

40 N

um

ber

of

sam

ple

s

[ ] > 100 ng/g

10 < [ ] < 100 ng/g

LQ-10 ng/g

<LQ

GFP – 17 mai 2017

Conclusions

GFP – 17 mai 2017

Conclusions

Development of a sensitive multiresidue method

27 pesticides, LOQ ≤ 1,5 ng/g for 74% of the targeted pesticides

Low amount of sample (250 mg)

13 different pesticides quantified in the collected samples

The most often detected : imidacloprid and diflufenican

Up to 7 targeted pesticides by sample

The most frequent combination : 1 insecticide/1 herbicide/1 fongicide (epoxyconazole, cyproconazole or prochloraz)

Analysis of the remaining earthworm samples (142 samples)

GFP – 17 mai 2017

Thank you for your attention