**2.4.2 Organophosphate pesticides analyses**

5 g of muscle sample was shaken with 60 ml dichloromethane and 10 g anhydrous sulfate. Mix was then filtered trough a Whatman 1 PS membrane, and evaporated under vacuum at 40°C. Dry samples were diluted in 3 ml ethanol, and underwent an ultrasonic step. Extract was then purified with a Sep pack R 300 (Silica Waters, 020810; 500 mg) column conditioned with 2 ml methanol and 2 ml ethanol. 2 ml dichloromethane were used for column elution. Purified samples were dried and diluted with 3 ml dichloromethane. Organophosphate (OP) and 2 carbamates (CA) pesticides (Dichlorvos, Carbofuran, Mevinphos, Phorate, Phorate oxon, Phorate sulfone, Methiocarbe, Terbufos, Diazinon, Disulfoton, Chlorpyriphos methyl, Chlorpyriphos ethyl, Fenitrothion, Pyrimiphos methyl, Malathion, Fenthion, Parathion, Methidathion, Disulfoton sulfone, Triazophos) concentrations were determined by GC/MS in SIM mode (OP + carbofuran and methiocarbe). A 5973N MS coupled with a 6890 GC (Agilent®) was used, with a 30m HP5-MS column (0.25 mm ID, 0.25µm thickness). For each samples standard and spiked sample, 2 µL were injected. The temperature program was 100°C (2 min), 55°C/min up to 200 °C (held for 5 min), 50 °C up to 220 °C (held for 3 minutes), followed by 60 °C/min up to 300°C. A final, post-run time of 2 min at 300°C was maintained. Total run time was 13.55 min. Injector was set at 250°C and the He flow was set at 2.5 ml/min. Each OP or CA was identified based on the following criteria: retention time and 3-4 fragmentation ions with pre-defined relative amounts and 20% variability acceptance for each ion. Linearity was confirmed between 25 and 500 ng.g-1 with 5 point calibration curves and r2 >0.99. Recovery was determined between 76% and 104% for all spiked samples and repeatability was considered acceptable with coefficients of variation <15%.

#### **2.4.3 Pyrethroids pesticides analyses**

5 g of tissue (liver or muscle) sample was shaken in 60 ml ethanol and 10 g anhydrous sulfate, and then filtered trough a Whatman 1 PS membrane. Extract was dissolved in 5 ml methanol and underwent a second filtration procedure. Concentrations were determined by GC / ECD and confirmed by GC/MS according to a modified method of the French Food Safety Authority (Anses Met AFSSA). An Agilent GC-ECD 6850 with a 30m HP1 column (0.32 mm ID, 0.25µm film) was used. For each samples standard and spiked sample, 2 µL were injected. The temperature program was common to OCs', PCBs and pyrethroids (initial temp: 100°C, first ramp 6°C/min up to 220 °C held for 10 min, 2nd ramp 7 °C/min up to 285°C, held for 1 min, total run time 42.29 min) Injector was at 230°C, detector at 300°C. Total He flow was 9 ml/min. Pyrethroids were identified according to their retention times. Linearity was confirmed between 10 and 100 ng.g-1 with 5 point calibration curves and r2 >0.99. Recovery was determined between 82% and 94% for all spiked samples and repeatability was considered acceptable with coefficients of variation <15%. For all positive samples, a confirmatory analysis was performed with GC/MS in SIM mode. Identification was based on retention times and 3 or 4 ions.

#### **2.4.4 Herbicides analyses**

296 Pesticides in the Modern World - Risks and Benefits

2.0-8.0 g of tissue were sampled and 30 ml of hexane/acetone 75/25 mix was added. Each sample was blended twice with an Ultraturrax® (Ika, Werke, Germany) and filtered trough a phase separator membrane. The extract was evaporated at 60 °C in a rotary evaporator. The

Two ml of fuming sulphuric acid (SO3 7%) were added, and after centrifugation at 4x *g*, 1 ml of the supernatant was used for OC pesticides quantification by gas chromatography with electron capture detection material. Temperature program and injection conditions are described in Lemarchand et al. (2007; 2010). Each sample was run in duplicate. Organochlorine pesticides concentrations were calculated by using different mix standards. Recovery level on standard mixtures was always greater than 92%. All standards were purchased from CIL (St Foy la Grande, France), and purity was > 99%. Linearity was determined between 5 and 100 ng.g-1 (*r*² > 0.99 on standards and spiked samples, 5-point calibration curves). Limits of detection were between 0.5 and 1.0 ng.g-1 lipids for individual PCB congeners. Cod liver oil (BCR349) certified material was used as a regular quality

5 g of muscle sample was shaken with 60 ml dichloromethane and 10 g anhydrous sulfate. Mix was then filtered trough a Whatman 1 PS membrane, and evaporated under vacuum at 40°C. Dry samples were diluted in 3 ml ethanol, and underwent an ultrasonic step. Extract was then purified with a Sep pack R 300 (Silica Waters, 020810; 500 mg) column conditioned with 2 ml methanol and 2 ml ethanol. 2 ml dichloromethane were used for column elution. Purified samples were dried and diluted with 3 ml dichloromethane. Organophosphate (OP) and 2 carbamates (CA) pesticides (Dichlorvos, Carbofuran, Mevinphos, Phorate, Phorate oxon, Phorate sulfone, Methiocarbe, Terbufos, Diazinon, Disulfoton, Chlorpyriphos methyl, Chlorpyriphos ethyl, Fenitrothion, Pyrimiphos methyl, Malathion, Fenthion, Parathion, Methidathion, Disulfoton sulfone, Triazophos) concentrations were determined by GC/MS in SIM mode (OP + carbofuran and methiocarbe). A 5973N MS coupled with a 6890 GC (Agilent®) was used, with a 30m HP5-MS column (0.25 mm ID, 0.25µm thickness). For each samples standard and spiked sample, 2 µL were injected. The temperature program was 100°C (2 min), 55°C/min up to 200 °C (held for 5 min), 50 °C up to 220 °C (held for 3 minutes), followed by 60 °C/min up to 300°C. A final, post-run time of 2 min at 300°C was maintained. Total run time was 13.55 min. Injector was set at 250°C and the He flow was set at 2.5 ml/min. Each OP or CA was identified based on the following criteria: retention time and 3-4 fragmentation ions with pre-defined relative amounts and 20% variability acceptance for each ion. Linearity was confirmed between 25 and 500 ng.g-1 with 5 point calibration curves and r2 >0.99. Recovery was determined between 76% and 104% for all spiked samples and

repeatability was considered acceptable with coefficients of variation <15%.

5 g of tissue (liver or muscle) sample was shaken in 60 ml ethanol and 10 g anhydrous sulfate, and then filtered trough a Whatman 1 PS membrane. Extract was dissolved in 5 ml methanol and underwent a second filtration procedure. Concentrations were determined by GC / ECD and confirmed by GC/MS according to a modified method of the French Food Safety Authority (Anses Met AFSSA). An Agilent GC-ECD 6850 with a 30m HP1 column

**2.4 Pesticides quantification methods 2.4.1 Organochlorine pesticides** 

dry extract was dissolved in 10 ml hexane.

**2.4.2 Organophosphate pesticides analyses** 

**2.4.3 Pyrethroids pesticides analyses** 

control.

2 g of muscle sample was shaken during 5 minutes in 8 ml acetone, and then centrifuged at 4x g; supernatant was placed in separate tubes, and this extraction was performed twice. Samples were evaporated under nitrogen, and dry extract was dissolved in 1 ml aceton/methanol (50:50) solution. Extract was then purified with a SPE C18 500 mg column conditioned with 2 ml acetone and 2 ml methanol. Column was vacuum dried and purified samples were diluted in 3 ml acetone. After drying under nitrogen, samples were diluted in 1 ml methanol. Herbicides (Trifluraline, Atrazine, Simazine, Terbuthylazine, Diuron, Alachlor, Metolachlor, Cyanazine, Epoxyconazloe) concentrations were determined by GC/MS spectrometry. A 5973N MS coupled with a 6890 GC (Agilent®) was used, with a 30m HP5-MS column (0.25 mm ID, 0.25µm thickness). For each samples standard and spiked sample, 2 µL were injected. The temperature program was 85°C held 1 min, followed by 6°C/min up to 170°C (held for 12 min), then followed by 20°C/min up to 280°C, held for 4.33 min (total run time 37 min). Injector was at 250°C and in the splitless mode. Each herbicide was identified based on the following criteria: retention time and 3-4 fragmentation ions with pre-defined relative amounts and 20% variability acceptance for each ion. Linearity was confirmed between 100 and 500 ng.g-1 with 5 point calibration curves and r2 >0.99. Recovery was determined between 67% and 98% for all spiked samples and repeatability was considered acceptable with coefficients of variation <15%.
