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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