Pesticide and Trace Metal Occurrence and Aquatic Benchmark Exceedances in Surface Waters

Pesticide and trace metal occurrence and aquatic benchmark exceedances in surface waters and sediments of urban wetlands and retention ponds in Melbourne, Australia, 2010

Supplementary Information

Graeme Allinson 1,2, Pei Zhang 3,1, AnhDuyen Bui 3,1, Mayumi Allinson 1,2, Gavin Rose 1,3,

Stephe Marshall 1, Vincent Pettigrove 1

1 Centre for Aquatic Pollution Identification and Management (CAPIM), The University of Melbourne, Parkville, Victoria 3010 Australia

2 Future Farming Systems Research, Department of Environment and Primary Industries, DPI Queenscliff Centre, Queenscliff, Victoria 3225, Australia

3 Future Farming Systems Research Division, Department of Environment and Primary Industries, Ernest Jones Drive, Macleod, Victoria 3085, Australia

SI1: Determination of pesticides in water and sediment samples

No one analytical method is appropriate for the measurement of all of the herbicides investigated in this study. Several different methods based on solid-phase extraction (SPE) were used to prepare the different sample matrices and chemicals. These SPE methodologies were validated and accredited by the Australian National Association of Testing Authorities to ISO 17025 standard. The method limit of reporting (LOR) was determined as the lowest concentration of a chemical that can be reliably quantified (95 % confidence interval) in the matrix in question.

Sediment samples were dried and ground, and then shaken (5g) for 30 min with 30 mL of 35% water/65%acetone (v/v, adjusted to pH <3) on a mechanical shaker. After shaking the mixture was sonicated for 15 min, and then centrifuged at 2800 rpm for 5 min. The supernatant liquid was passed through a glass fibre filter and collected in a 250mL flask. The extraction was repeated with 30 mL of solvent mixture and the combined filtered extract was concentrated to around 20mL on a rotary evaporator at 30°C under 95 kPa vacuum. The concentrated extract was transferred into a 250 mL measuring cylinder. The rotary evaporator flask was rinsed with 1mL MeOH, which was added to the concentrated extract along with sufficient deionised water to make the final volume 240mL. This aqueous sediment extract was split into two parts, which were subsequently subjected to the different SPE clean up procedures described below.

For analytes most appropriately measured by gas chromatography (GC), such as a range of volatile organophosphates (OP), fungicides, organochlorine (OC) and synthetic pyrethroid (SP) chemicals, a sub-sample (water, 500 mL pH adjusted to <2; sediment aqueous extract, 160 mL) was extracted with a [C18, 1500mg/83 mL] Enviro Clean® Universal 525 Extraction Cartridge; (UCT, Bristol, PA, USA) to remove interferences before GC determination. The cartridge was pre-conditioned with 10 mL of 50:50 ethyl acetate/dichloromethane (v/v), and then 10 mL methanol before loading the aqueous extract. Compounds of interest were eluted from the cartridge with first 10 mL ethyl acetate, then 2 x 10 mL 50:50 ethyl acetate/dichloromethane (v/v). The combined eluates were concentrated using a rotary evaporator at 30 ̊C under 95 kPa vacuum to about 5mL, and dried using anhydrous sodium sulphate. The extract was then transferred into a test tube and evaporated to near dryness under nitrogen. Residues were reconstituted in 0.2 mL acetone and 1.8 mL hexane. Sulphur was removed using copper granules, and the extract was split into two.

The first aliquot (1mL) of the extract was used to determine a range of volatile fungicides. The concentrations of the fungicides bupirimate, chlorothalonil, fenarimol, iprodione, procymidone, buprofezin (insecticide) and the benzamide herbicide propyzamide, were determined using a Varian 3400 CX or 3800 capillary gas chromatograph fitted with nitrogen-phosphorus detector (Varian, Mulgrave, Australia). An aliquot of hexane extract was simultaneously injected onto two parallel columns (15 m, 0.32 ID 0.25µm film, 95 % dimethyl - 5 % diphenylpolysiloxane stationary phase (J&W™ DB-5) and a 15 m, 0.32 ID 0.25µm film, 50 % dimethyl – 50 % diphenylpolysiloxane stationary phase (J&W™ DB-17)) via a split/splitless injector with a split ratio of 1:20. The GC oven was temperature-programmed (120oC 0–2 min, 120–300oC at 20oC/min, held 300oC for 1 min) for optimum separation efficiency. The injector and detector temperatures were set at 280oC and 320oC, respectively. Helium was used as carrier gas. Varian Star software (V6.0) was used to manage the chromatographic data. The organic residues were quantified by comparison with external standards. The LORs in water samples were: bupirimate and buprofezin, 0.1 mg/L; chlorothalonil, fenarimol, and iprodione, 0.2 mg/L; procymidone, 0.5 mg/L; propyzamide 1 mg/L, respectively. The LORs for these pesticides in sediment samples were: bupirimate and buprofezin, 50 mg/kg; fenarimol, iprodione, procymidone, 150 mg/kg; propyzamide 250 mg/kg; and chlorothalonil, 500 mg/kg (dry weight), respectively.

The first aliquot (1mL) of the extract was also used to determine a range of OPs. The concentrations of the OPs azinphos ethyl, chlorpyrifos, chlorpyrifos methyl, diazinon, ethion, fenchlorphos, fenitrothion, fenthion, malathion, mevinphos, methidathion, parathion ethyl, parathion methyl and prothiofos were determined by external calibration using a Varian 3400 CX or 3800 capillary gas /-programmed (120oC 0–2 min, 120–300oC at 20oC/min, held 300oC for 1 min) for optimum separation efficiency. The injector and detector temperatures were set at 280oC and 320oC, respectively. Helium was used as carrier gas. Varian Star software (V6.0) was used to manage the chromatographic data. The organic residues were quantified by comparison with external standards. The LORs in water samples were: chlorpyrifos, 0.04 mg/L; azinphos ethyl, chlorpyrifos methyl, diazinon, ethion fenchlorphos, fenitrothion, fenthion, malathion, parathion ethyl and parathion methyl, 0.05 mg/L; methidathion, mevinphos, and prothiofos, 0.1 mg/L; respectively. The LOR for these pesticides in sediment samples were: ethion, fenitrothion, fenthion, parathion methyl, 3 mg/kg; chlorpyrifos, chlorpyrifos methyl, diazinon, malathion, methidathion, mevinphos, parathion ethyl, prothiofos 4 mg/kg; azinphos ethyl 10 mg/kg; and fenchlorphos 12 mg/kg (dry weight), respectively. Note that the OP pesticides azinphos methyl, fenamiphos, dimethoate, omethoate, dichlorvos and trichlorfon were determined by LC-MS/MS and conditions and LORs are reported below

The second aliquot of the extract (1 mL) was subjected to further clean up by first conditioning a Bond Elute® 500 mg florisil cartridge with 1 mL of hexane. Using a Rapid Trace® automated SPE instrument. Then loading 1 mL of the sample to separate organochlorine (OC) and synthetic pyrethroid (SP) compounds. The OC and SP analytes were eluted from the florisil cartridge with 3 mL of 50: 48.5: 1.5 dichloromethane, hexane and acetonitrile (v/v/v). The eluate was evaporated to dryness under nitrogen and reconstituted in 1 mL hexane. This hexane solution was directly injected into a capillary gas chromatograph with dual electron capture detectors(GC-ECDs) to determine the SPs, whereas a further one to ten dilution of the aliquot was performed before injection into GC-ECD for the determination of the OCs.

The concentrations of the SPs bifenthrin, cyhalothrin, cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenvalerate and permethrin were determined using a Varian 3400 CX or 3800 capillary GC-ECD (Varian, Mulgrave, Australia). An aliquot of hexane extract was simultaneously injected onto two parallel columns (15 m, 0.32 ID 0.25µm film, 95 % dimethyl - 5 % diphenylpolysiloxane stationary phase (J&W™ DB-5) and a 15 m, 0.32 ID 0.25µm film, 50 % dimethyl – 50 % diphenylpolysiloxane stationary phase (J&W™ DB-17)) via a split/splitless injector with a split ratio of 1:20. The GC oven was temperature-programmed (260oC 0–5 min, to 300oC at 10oC/min, held 300oC for 6 min) for optimum separation efficiency. The injector and detector temperatures were set at 280oC and 350oC, respectively. Helium was used as carrier gas. Varian Star software (V6.0) was used to manage the chromatographic data. The organic residues were quantified by comparison with external standards. The LORs in water samples were: cyhalothrin 0.01 mg/L; bifenthrin, cyfluthrin, deltamethrin and fenvalerate 0.02 mg/L; cypermethrin and esfenvalerate 0.05 mg/L; and permethrin 0.1 mg/L; respectively. The LORs for these pesticides in sediment samples were: cyhalothrin, 2 mg/kg; cyfluthrin, deltamethrin, esfenvalerate and fenvalerate, 4 mg/kg; bifenthrin and cypermethrin 5 mg/kg; and permethrin 20 mg/kg (dry weight), respectively.

The concentrations of the OCs aldrin, BHC-alpha, BHC-beta, BHC-delta, cis-chlordane, dieldrin endosulfan sulphate, endosulfan-alpha, endosulfan-beta, endrin, HCB, heptachlor, heptachlor epoxide, lindane, oxychlordane, p,p'-DDD, p,p'-DDE, p,p'-DDT, and trans-chlordane were determined using a Varian 3400 CX or 3800 capillary gas chromatograph fitted with electron capture detectors (Varian, Mulgrave, Australia). An aliquot of hexane extract was simultaneously injected onto two parallel columns (15 m, 0.32 ID 0.25µm film, 95 % dimethyl - 5 % diphenyl polysiloxane stationary phase (J&W™ DB-5) and a 15 m, 0.32 ID 0.25µm film, 50 % dimethyl – 50 % diphenyl polysiloxane stationary phase (J&W™ DB-17) or alternatively a 15 m, 0.32 ID 0.25µm film, 50 % phenyl polysiloxane/ 50% cyanopropylmethyl polysiloxane stationary phase (Rtx™ 225)) via a split/splitless injector with a split ratio of 1:20. The GC oven was set at 200oC, isothermal. The injector and detector temperatures were set at 280oC and 350oC, respectively. Helium was used as carrier gas. Varian Star software (V6.0) was used to manage the chromatographic data. The organic residues were quantified by comparison with external standards. The LORs in water samples were: aldrin, BHC-delta, cis-chlordane, endrin, HCB, heptachlor, heptachlor epoxide, lindane, oxychlordane, p,p'-DDD, p,p'-DDE, and trans-chlordane 0.002 mg/L; BHC-alpha and dieldrin 0.003mg/L; BHC-beta, endosulfan sulphate, endosulfan-alpha, endosulfan-beta and p,p'-DDT 0.005 mg/L; respectively. The LORs for these pesticides in sediment samples were: BHC-delta and heptachlor epoxide 2 mg/kg; BHC-alpha, HCB, Lindane and p,p'-DDE 3 mg/kg; aldrin, cis-chlordane, dieldrin, endrin, heptachlor, oxychlordane and trans-chlordane 4 mg/kg; BHC-beta, endosulfan-alpha, endosulfan-beta, p,p'-DDD and p,p'-DDT 5 mg/kg; and endosulfan sulfate 7 mg/kg (dry weight), respectively.

For analytes most appropriately measured by liquid chromatography (LC), such as a range of triazine/triazine herbicides, and many fungicides, a sample (water, 100 mL; sediment, 80 mL of sediment aqueous extract solution) was loaded onto to a Bond Elute® PPL 500 mg/ 3mL SPE cartridge for LC-tandem mass spectrometry (LC-MS/MS). The cartridge was pre-conditioned with 5mL MeOH followed by 5 mL deionised water, followed by loading of the sample and by elution with 5mL of acetonitrile. The eluate was evaporated to dryness under a stream of nitrogen. The residues were dissolved in 1mL 50% MeOH: H2O (v/v). The final extract was filtered through a 0.45 mm PTFE syringe filter before analysis by LC-MS/MS

The concentrations of the herbicides atrazine, cyanazine, hexazinone, metribuzin, prometryn, simazine, terbutryn and the atrazine metabolites desethyl atrazine (DEA), deisopropyl atrazine (DIA) and hydroxyl atrazine (HA) were determined using a ‘triazines screen’ using a Varian 1200L Quadrupole LC-MS/MS (Varian, Mulgrave, Australia) operating in the positive ion electrospray mode. The triazine/triazole herbicides were separated from other extract components with a Varian C18, 5µm, Luna column (150 mm x 2.0 mm). The HPLC column was maintained at 25oC. The mobile phase consisted of (A) 20 % methanol in 5 mM ammonium acetate and (B) 90 % methanol in 5 mM ammonium acetate with the following gradient: 80 % A (7 min), 70% A (12 min), 100 % B (13 min) and 100% A (7 min) at a flow rate of 0.2 ml/min. Varian Workstation (V6.0) was used for data processing. Residues were quantified using external standards and each standard set was assayed a minimum of three times during each sample batch run. Sample and recovery concentrations were calculated from a linear regression of the standards. The tandem mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. Method LORs in water were 0.001 mg/L for atrazine, cyanazine, hexazinone, prometryn simazine and terbutryn; and were 0.002 mg/L for metribuzin, DEA, DIA and HA, respectively. The LORs for these pesticides in sediment were 5 mg/kg (dry weight) for all analytes except prometryn (2 mg/kg).

The concentrations of 40 polar pesticides (azinphos methyl, azoxystrobin, boscalid, carbaryl, cyproconazole, cyprodinil, dichlorvos, difenoconazole, dimethoate, dimethomorph, fenamiphos fenoxycarb, fipronil, imidacloprid, indoxacarb, linuron, metalaxyl, methiocarb, methomyl, myclobutanil, omethoate, oxadixyl, penconazole, pendimethalin, pirimicarb, prochloraz, propargite, propiconazole, pymetrozine, pyraclostrobin, pyrimethanil, spinosad, tebuconazole, tebufenozide, tetraconazole, thiodicarb, triadimefon, triadimenol, trichlorfon, and trifloxystrobin) were determined using a multi-residue screen using a Varian 1200L Quadrupole LC-MS/MS (Varian, Mulgrave, Australia) operating in the positive ion electrospray mode. The pesticides were separated from other extract components with a Varian Pursuit C18 column (150 mm x 2.0 mm) fitted with a Pursuit C18 guard column. The HPLC column was maintained at 25oC. The mobile phase consisted of (A) 20 % methanol in 5 mM ammonium acetate and (B) 90 % methanol in 5 mM ammonium acetate with the following gradient: 100 % A–100% B (0–15 min), 100 % B (15–28 min), 100 % B–100 % A (28–30 min) with a flow rate of 0.2 ml/min. Varian Workstation (V6.0) was used for data processing. Residues were quantified using external standards and each standard set was assayed a minimum of three times during each sample batch run. Sample and recovery concentrations were calculated from a linear regression of the standards. The tandem mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The method LORs in water were: metalaxyl, myclobutanil, pyraclostrobin, fenamiphos and trifloxystrobin, 0.001 mg/L; azoxystrobin, boscalid, carbaryl, cyprodinil, dimethoate, dimethomorph, fenoxycarb, imidacloprid, methiocarb, methomyl, omethoate, penconazole, pirimicarb, propiconazole, tebufenozide and triadimefon, 0.002 mg/L; difenoconazole, indoxacarb, linuron, oxadixyl, prochloraz, pymetrozine, pyrimethanil tetraconazole and trichlorfon, 0.004 mg/L; fipronil and dichlorvos and triadimenol 0.005 mg/L; azinphos methyl, cyproconazole and tebuconazole 0.01 mg/L; pendimethalin and propargite 0.05 mg/L; spinosad and thiodicarb 0.1 mg/L, respectively; LORs for these pesticides in sediment were: carbaryl, metalaxyl, and oxadixyl tebufenozide, 1 µg/kg; boscalid, linuron, methiocarb, myclobutanil and triadimenol, 2 mg/kg; azoxystrobin, fenoxycarb pirimicarb and trifloxystrobin, 3 mg/kg; pyraclostrobin tebuconazole, tetraconazole and triadimefon, 4 mg/kg; dimethoate, fenamiphos, fipronil, imidacloprid, indoxacarb, penconazole, spinosad and trichlorfon, 5 mg/kg; cyproconazole, omethoate and pyrimethanil, 10 mg/kg; dimethomorph and propiconazole, 12 mg/kg; azinphos methyl, cyprodinil and propargite, 15 mg/kg; methomyl, difenoconazole and prochloraz, 20 mg/kg; dichlorvos and pendimethalin, 25 mg/kg; pymetrozine, 250 mg/kg; thiodicarb, 500 mg/kg (dry weight), respectively.

For every analytical batch of water and sediment samples extracted, a sample was randomly selected for spike recovery determinations. Water samples were spiked with the reported OCs at 0.02mg/L, reported OPs at 0.1mg/L NPD screen at1mg/L, SPs at 0.2mg/L triazines at 0.1mg/L and LC-MS/MS screen compounds at 0.1mg/L. Water OC recoveries ranged from 112% for α-endosulfan to 26% for HCB with DDT and isomers in the range of 107-64% and dieldrin = 99% . Recoveries in the OP screen ranged from 143% for mevinphos to 81% for fenchlorphos. Recoveries for SPs ranged from cyhalothrin at 96% to bifenthrin at 54%. Recoveries for NPD screen analytes ranged from 145% for chlorothalonil to 25% for buprimate. Triazine recoveries ranged from hexazinone 98%, atrazine 81%, simazine 82%, to 2-hydoxy-atrazine, 37%. Recoveries of LC-MS/MS analytes were above 70% except for methiocarb, 54%, prochloraz, 63%, propargite, 2%, dimethoate, 68%, dichlorvos 41%, cyprodinil, 67%, indoxacarb,665, omethoate, 52%, pendimethalin, 11%, spinosad, 6%, thiodicarb, 1% and trichlorfon 7%. The measured recoveries are reflected in the water LORs reported above.