Arch Environ Contam Toxicol DOI 10.1007/s00244-015-0128-9

A Simultaneous Extraction Method for Organophosphate, Pyrethroid, and Neonicotinoid Insecticides in Aqueous Samples Chloe´ de Perre • Sara A. Whiting • Michael J. Lydy

Received: 3 July 2014 / Accepted: 2 January 2015 Ó Springer Science+Business Media New York 2015

Abstract A method was developed for the extraction and analysis of 2 organophosphate, 8 pyrethroid, and 5 neonicotinoid insecticides from the same water sample. A salted liquid–liquid extraction (LLE) was optimized with a solidphase extraction (SPE) step that separated the organophosphates (OPs) and pyrethroids from the neonicotinoids. Factors that were optimized included volume of solvent and amount of salt used in the LLE, homogenization time for the LLE, and type and volume of eluting solvent used for the SPE. The OPs and pyrethroids were quantified using gas chromatography–mass spectrometry, and the neonicotinoids were quantified using liquid chromatography– diode array detector. Results showed that the optimized method was accurate, precise, reproducible, and robust; recoveries in river water spiked with 100 ng L-1 of each of the insecticides were all between 86 and 114 % with RSDs between 2 and 8 %. The method was also sensitive with method detection limits ranging from 0.1 to 27.2 ng L-1 depending on compounds and matrices. The optimized method was thus appropriate for the simultaneous extraction of 15 widely applied insecticides from three different classes and was shown to provide valuable information on their environmental fate from field-collected aqueous samples.

Electronic supplementary material The online version of this article (doi:10.1007/s00244-015-0128-9) contains supplementary material, which is available to authorized users. C. de Perre
S. A. Whiting
M. J. Lydy (&) Center for Fisheries, Aquaculture, and Aquatic Sciences, and Department of Zoology, Southern Illinois University, 1125 Lincoln Drive, Life Sciences II, Room 173, Mailcode 6511, Carbondale, IL 62901, USA e-mail: [email protected]

Insecticides are commonly applied in agricultural fields and urban environments to kill insect pests [United States Environmental Protection Agency (USEPA) 2013]. Some of the major classes of insecticides currently used include organophosphates (OPs), pyrethroids, and neonicotinoids. OPs were first introduced during World War II. Pyrethroids were introduced during the 1970s to replace persistent organochlorine insecticides, which were causing environmental concerns (Walker 2009). However, OPs and pyrethroids have been shown to be toxic to several nontarget organisms in both aquatic and terrestrial ecosystems (Siegfried 1993; Wijngaarden et al. 2005). Neonicotinoids were introduced in the 1990s and are frequently used in agriculture pest management due to their more specific mode of actions, which makes them less toxic to mammals, birds, and fish (Tomizawa and Casida 2005). Combinations of all three insecticide classes can be applied on the same agricultural field to target insect species as part of an integrated pest management plan to provide several different modes of action that may decrease the risks of developing insecticide resistant pests (Chandrasena et al. 2011; Onstad et al. 2011). For example, corn fields in Central Illinois, USA, are often planted with seeds coated with neonicotinoid insecticides, such as thiamethoxam (THX) or clothianidin (CLO); the pyrethroid insecticide tefluthrin (TEF) is applied during planting as an infurrow application. As another example, corn fields can have a granular insecticide formulation made of the OP insecticide phostebupirim (PHOS), and the pyrethroid insecticide cyfluthrin (CYF) is applied in-furrow at planting with CLO-coated seeds. Because fields from the same watershed may be treated with different active ingredients, complex mixtures containing several compounds of different classes may be detected simultaneously if these compounds are able to travel off-site. Similarly, urban

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waterways may contain several insecticides, especially OPs and pyrethroids, at concentrations greater than toxicity benchmarks (Ensminger et al. 2013). The presence of several active ingredients on site or downstream may be an issue because synergistic effects may occur if nontarget species are exposed to all of the different compounds. Indeed, the toxicity of mixtures may be greater in some cases than the addition of the individual toxicities of each compound (Lydy et al. 2004; Belden and Lydy 2006; Zhang et al. 2010). However, the first step in the determination of such effects is the development of analytical methods that permit the quantification of these active ingredients in environmental matrices, which may be simultaneously present in surface waters. The objective of the current project was to develop a single method to extract insecticides from the three pesticide classes mentioned above from water samples to examine their environmental fate. The method was developed for several analytes applied to a particular field study, i.e., PHOS, TEF, CYF, THX, CLO, and imidacloprid (IMI), and a few other compounds with similar physicochemical properties belonging to the same insecticidal classes were added for use of the method on a larger scale. The main challenge in developing this method came from the contrasting chemical properties of these chemicals (Table 1). Pyrethroids are very hydrophobic (log KOW between 5 and 7) and poorly soluble in water (in the lg L-1 range) unlike neonicotinoids, which are very hydrophilic (log KOW \1.3) and have an aqueous solubility of several hundreds of mg L-1; OPs are intermediate in their hydrophobicity (log KOW B5) but moderately soluble in water. Volatility also varies among these compounds, thus making them difficult to simultaneously extract from complex matrices with a single method. Commonly used pesticide-extraction methods for aqueous samples are liquid–liquid extraction (LLE) or solid phase extraction (SPE). LLE is usually the recommended method for use by the USEPA, especially in the presence of solid particles (unfiltered samples) (USEPA 2007a, b). However, SPE provides advantages compared with LLE by using less solvent, plus it is more time and cost-efficient when extracting a large number of samples. Because the objective of the current study was to quantify the target insecticides in whole aqueous samples, including in the dissolved and particulate phases, the LLE method was optimized. The SPE step was added to improve analyte selectivity after LLE and to separate the analytes into two fractions to be quantified on two different instruments. OPs have been analyzed using gas chromatography– mass spectrometry (GC–MS), GC with a flame photometric detector, GC with a nitrogen–phosphorus detector (NPD), liquid chromatography–tandem mass spectrometry (LC– MS/MS), LC-diode array detector (DAD), and capillary

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electrophoresis (Guardino et al. 1998; Kamel et al. 2009; Pucarevic´ et al. 2013). Pyrethroids are commonly analyzed by GC coupled with MS or electron capture detection (ECD) (Bonwick et al. 1995; Gamon et al. 2001; You and Lydy 2006, 2007; Amelin et al. 2012), and can be detected using LC-DAD; however, LC does not provide sufficient separation, and coelution is an issue when quantifying pyrethroids (Amelin et al. 2012). Neonicotinoids have been analyzed by different methods including GC–MS (Amelin et al. 2012), GC-ECD (Amelin et al. 2012), immunoassay (Watanabe et al. 2006), and induced fluorescence detection (Lo´pez Flores et al. 2007); however, the most common methods are based on LC and include the following: LC– ultraviolet (UV) detection (Zhou et al. 2006; Mohan et al. 2010), LC-DAD (Ying and Kookana 2004; Seccia et al. 2005; Watanabe et al. 2007), and LC–MS/MS (Kamel 2010; Hladik and Calhoun 2012; Zhang et al. 2012). Because of the universal and selective detection obtained with mass spectrometry, GC–MS and LC–MS/MS have been identified as techniques of choice for trace analyses of pesticides (Alder et al. 2006). Although LC–MS techniques have been shown to be more sensitive than GC–MS for many classes of pesticides (Alder et al. 2006), access to LC–MS equipment is often limited due to the cost and space requirements of the system. Using this rationale and the instruments available in our laboratory, extracts were quantified by GC–MS for the OPs and pyrethroids and by LC-DAD for the neonicotinoids. The following text describes the development and validation of a method to extract and analyze OP, pyrethroid, and neonicotinoid insecticides from a single aqueous sample.

Experimental Selection of Analytes of Interest This study focused on agricultural insecticide fate in Central Illinois, USA. The fields investigated were applied at planting with TEF, or a formulation containing CYF and PHOS, on corn treated with THX or CLO seed-coating and IMI from soybean seed-coating. A method was needed to analyze all of these insecticides, simultaneously if possible, in aqueous samples including groundwater, soil infiltration water, and runoff water. However, to establish the robustness and validate the method, several different aqueous matrices were used including moderately hard reconstituted water (MHRW), groundwater, lake water, and runoff water. After optimization for the analytes of interest for this specific study, additional OP, pyrethroid, and neonicotinoid insecticides were included in the method to extend its use to compounds of national interest. Adjustments of the

1995

4.19

5.5

318.37

Date of introduction

Log Kow

Aqueous solubility (20 °C, mg L-1)

M (g mol-1) 434.29

0.0066

6

1983

CLO

IMI

291.71

4100

-0.13

1991

249.7

340

0.91

2002

Neonicotinoid insecticides

255.66

610

0.57

1991

222.67

2950

2.1 9 10-5 (Yalkowsky 2010)

1995 0.8

498.66

THC

252.72

184

1.26

1999

Neonicotinoid insecticides

8.18 (Hawker and Connell 1988)

Polychlorinated biphenyl –

ACE

OP organophosphate, PHOS phostebupirim, CYF cyfluthrin, TEF tefluthrin, THX thiamethoxam, CLO clothianidin, IMI imidacloprid, DCBP decachlorobiphenyl, ACE acetamiprid, THC thiacloprid

418.73

0.016

6.4

1986

Pyrethroid insecticides

Pesticide Property Database accessed on 2 February 2013

OP insecticide

Class

Chemical structure

THX

DCBP

TEF

PHOS

CYF

Surrogates

Analytes

Table 1 Chemical formulas and properties of selected analytes and surrogates

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extraction and analytical methods were then performed for analysis of a total of 15 compounds including the organophosphate chlorpyrifos (CPF), the pyrethroids bifenthrin (BIF), k-Cyhalothrin (k-CYH), cypermethrin (CYP), esfenvalerate (ESF), fenpropathrin (FEN), and permethrin (PER), and the neonicotinoid insecticides acetamiprid (ACE), and thiacloprid (THC). Method detection limits (MDLs) for these 15 compounds were then calculated in matrices including deionized water, river water, and lake water.

Materials and Reagents Chemicals The OPs used in the current study were PHOS (96.9 % pure, ChemService, West Chester, PA, USA) and CPF (99.9 % pure, Fluka Analytical, Sigma-Aldrich, St. Louis, MO, USA). Pyrethroids included BIF (99.5 % pure, ChemService), CYF (98 % pure, mix of isomers, ChemService), k-CYH (99.1 % pure, ChemService), CYP (98 % pure, mix of isomers, ChemService), ESF ([98 % pure, ChemService), FEN (99.5 % pure, ChemService), PER (40.1 % cis, 58.7 % trans, ChemService), and TEF (96.4 % pure, Fluka Analytical, Sigma-Aldrich). Neonicotinoids included THX (99.7 % pure, Sigma-Aldrich), IMI (99.5 % pure, ChemService), and ACE, CLO, and THC, all of which were 99.9 % pure and purchased from ChemService. All of these standards were purchased as neat material. The surrogate for the GC–MS analysis was decachlorobiphenyl (DCBP; 200 lg mL-1 in acetone, [98 % pure, Supelco Analytical, Bellefonte, PA, USA). The internal standards were FLU (100 lg mL-1 in methanol, [99 % pure, AccuStandard, New Haven, CT, USA) and 2,2’,4,4’,5,5’hexachlorobiphenyl (PCB 153, 100 lg mL-1 in isooctane, [97 % pure, Supelco Analytical).

Reagents Solvents were all purchased from Fisher Scientific (Fair Lawn, NJ, USA) and included pesticide-grade hexane, acetone, and dichloromethane (DCM), acetonitrile Optima, and high-performance liquid chromatography (HPLC)– grade submicron filtered water. Trifluoroacetic acid (C98 %) was purchased from Sigma-Aldrich. Acidified hexane solution was prepared from hexane and glacial acetic acid (99.5 % pure; Mallinckrodt, Paris, KY, USA) at 0.6 % (v/v). Na2SO4 (anhydrous certified, American Chemical Society; Fisher Scientific) and NaCl (Fisher Scientific) were baked at 450 °C before use.

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Optimized LLE/SPE Procedure Although the current article relates the steps of the analytical development, this paragraph describes the final optimized procedure for easy use and reproduction of the method. A flow chart of the final protocol is provided in supporting information (Supplementry Fig. S1). Insecticides were extracted from 200-mL water samples using 2 9 100 mL of DCM after surrogate addition to the water in a 1000-mL separatory funnel. After a brief shake to homogenize the water and the first 100 mL of DCM, 10 g of previously baked NaCl were added to the separatory funnel, which was then homogenized until complete salt dissolution. Separatory funnels were then secured on an automated Glas-Col shaker (Terre Haute, IN, USA) at 138 rotations/min and shaken for 10 min. For most of the samples, the DCM was directly drained from the separatory funnel into a Turbovap vial. In the case of complete emulsion of water and DCM (due to a highly charged matrix), the emulsion was drained into a disposable 50-mL Fisherbrand polypropylene centrifuge tube and centrifuged with an Eppendorf 5810 centrifuge for 10 min at 500 g. The upper layer of water was then transferred back to the separatory funnel and the DCM poured into a Turbovap vial. Another 100-mL aliquot of DCM was added to the separatory funnel, which was shaken using the same conditions as the first extraction. After draining the second aliquot of DCM from the separatory funnel into the Turbovap vial, the DCM was concentrated in a Turbovap II evaporator (Zymark, Hopkinton, MA, USA) using nitrogen gas to 0.5 mL. 10 mL of a hexane/acetone solution (75/25 [v/v]) was then added and evaporated using the Turbovap II evaporator after gentle mixing of the vial. After the second concentration of the eluate to 0.5 mL in the TurboVap vial, the extracts were transferred into SPE cartridges (dual-layer Supelclean ENVI-Carb II/PSA 300/600 mg 6 mL, Supelco Analytical), which had been previously conditioned with 3 mL of hexane/acetone solution. A total of 7 mL of a hexane/DCM solution (90/10 [v/v]) was then added to the cartridges and drained into test tubes to elute the OP/pyrethroid fraction. New test tubes were then placed under each cartridge, and 5 mL of a DCM/acetonitrile solution (50/50 [v/v]) were added and drained to elute the neonicotinoid fraction. Each fraction was placed on a Rapidvap evaporator (Reacti-Therm III; Pierce, Rockford, IL, USA) at a temperature of 30 °C until the solvent evaporated to 0.5 mL due to the application of nitrogen gas. Each extract was then transferred into injection vials and evaporated to dryness. The OP/pyrethroid fractions were reconstituted to 0.5 mL by addition of acidified hexane and internal standards (final concentration of 50 ng mL-1). The neonicotinoid fractions were reconstituted to 0.5 mL by addition of a 0.1 % trifluoroacetic

Arch Environ Contam Toxicol

acid water/acetonitrile solution (80/20 [v/v]). Each fraction was then quantified using GC–MS or LC-DAD. GC–MS Conditions The OP/pyrethroid fractions were analyzed by GC–MS and quantified using internal standard calibration. Two GC–MS systems were used interchangeably with the same method because they had similar sensitivities. One system was composed of a 6850 Agilent gas chromatograph coupled with a 5975C inert XL electron impact (EI)/chemical ionization (CI) mass selective detector (MSD); the other was a 7890A Agilent GC system coupled with a 5975C inert XL EI/CI MSD with triple-axis detector. Both systems were purchased from Agilent Technologies (Santa Clara, CA, USA). The EI mode was used for initial samples containing three OP/pyrethroids during the method development, and negative chemical ionization (NCI) mode was performed in samples containing the 15 insecticides due to its superior sensitivity for most compounds, especially pyrethroids. Standards were injected at known concentrations at the beginning of the sequence to trace the calibration curve used for quantification. To correct for matrix effects, four samples (randomly selected from the samples to analyze) were injected before the standards at the beginning of the sequence, and one was injected in between each standard. The injection of 2 lL of sample was performed at 260 °C in the pulsed splitless mode with a pressure of 30 pounds per square inch until 0.75 min. The purge flow to the split vent was then set at 45 mL min-1 until 2 min, when the gas saver turned on at 20 mL min-1. Ultra high–purity helium was used as carrier gas (Airgas Mid America, Bowling Green, KY, USA). An Agilent HP-5MS column (30 m 9 0.25 mm 9 0.25 lm) was installed in both instruments. The initial samples analyzed by EI had a total run time of \25 min starting with an oven temperature of 90 °C, increased to 275 °C at a rate of 15 °C min-1, then to 285 °C at a rate of 2 °C min-1, and finally to 300 °C at a rate of 10 °C min-1 and held at 300 °C for 6 min. In the NCI mode, with additional OP and pyrethroids, the temperature ramp was modified as follow: initial temperature of 50 °C, increased to 200 °C at 20 °C min-1, then to 295 °C at 10 °C min-1, and held at 295 °C for 5 min. The MSD was operated in the single ion monitoring mode with appropriate time windows for each ion to provide a dwell time of 100 ls and 2.36 cycles s-1 for each ion. The MS source and MS quadrupole temperatures were both set at 150 °C. Each analyte was searched using two m/z: one for quantification and the second for confirmation. The ion pairs were as follows: m/z = 183 and 167 for PHOS, m/z = 313 and 315 for CPF, m/z = 386 and 241 for BIF, m/z = 207 and 209 for CYF and CYP, m/z = 241 and 205 for k-CYH, m/z = 211 and 213 for ESF, m/z = 141 and

142 for FEN, m/z = 207 and 336 for PER, and m/z = 241 and 243 for TEF. For the surrogate and internal standards, the ions were as follows: m/z = 498 and 464 for DCBP, m/z = 360 for PCB 153, and m/z = 243 for FLU. The internal standards were PCB 153 for DCBP and PHOS and FLU for all of the other compounds. The peaks for CYF and CYP isomers were not well resolved, especially the third and fourth peaks of each compound that coeluted; therefore, the four isomers were integrated together for each compound (Supplementry Fig. S2). LC-DAD Conditions Neonicotinoids were analyzed using a 1260 Agilent LCDAD equipped with a guard column and an Agilent PrepC18 (4.6 9 250 mm, 5-lm column). Twenty lL of each sample was injected, and a constant flow of 0.75 mL min-1 was used during the run (28 min). Solvent A consisted of a mixture of water and acetonitrile (95/5 [v/v]), and solvent B was 100 % acetonitrile. The mobile phase gradient during the run was as follows: 13.7 % of solvent B for 1 min, increased to 36.8 % of solvent B in 7 min, increased to 63.2 % of solvent B for 6 min, increased to 100 % of solvent B in 1 min and held for 5 min; at 20 min, decreased back to 13.7 % of solvent B within 2 min and held until the end of the run. The visible lamp was off, and only the UV lamp was used as the detector. Three wavelengths were acquired during the run, including 242, 252, and 269 nm, with a bandwidth and slit of 4 nm. The spectrum from 190 to 400 nm was recorded at a step of 1 nm for each peak (threshold of 0.1 mAU). The peak width was [0.1 min, which is equivalent to a 2-second response time. Acetamiprid and THC were quantified using k = 242 nm (confirmed with k= 269 nm); CLO and IMI were quantified using k= 269 nm (confirmed with k= 252 nm); and THX was quantified using k= 252 nm (confirmed with k= 269 nm). To increase confidence in analyte identification, a spectral library was built from the neonicotinoid standards, and analytes were compared with any peak requiring additional confirmation. Samples Field runoff water was collected near Taylorville, Illinois, on a corn field that had previous applications of neonicotinoids among other chemicals. The objective of the current study was to analyze agricultural field runoff water; however, additional aqueous matrices, i.e., deionized, MHRW, lake, and river water, were investigated to assess the robustness of the method and its applicability to a wider range of matrices. The MHRW was made in the laboratory from deionized water and mineral salts (USEPA 2000). Lake water was collected from Campus Lake in

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Carbondale, Illinois, and river water was collected from the Cache River near Vienna, Illinois. All water samples were collected using an aluminum scoop or directly into a mason jar previously washed and rinsed with acetone. Only glass jars were used, to minimize glassware-binding, and the jars were thoroughly rinsed to remove any bound pesticides. Samples were stored at 4 °C in the dark until extraction. No insecticides were expected to be detected in this lake and river, and these samples were only used as complex matrices for field-matrix spiked samples. Field blanks were processed with every batch of 20 samples, and when traces of insecticides were detected in the blanks, they were accounted for in the results, i.e., subtracted from the total amount for recovery calculation. None of the samples were filtered before extraction and therefore included suspended material. All matrices were measured for pH by an Orion 4 Star pH meter (Thermo Scientific, Chelmsford, MA, USA), for conductivity using a YSI 30 salinity, conductivity, and temperature meter (YSI, Yellow Springs, OH, USA), and for total organic carbon (TOC) using a Shimatzu TOC V-SCN instrument (Columbia, MD, USA) (Table 2). In addition, field samples, including runoff water, soil infiltration water, and groundwater, were collected from an agricultural field near Taylorville, Illinois, in which corn was planted with CLO-coated seeds and TEF applied infurrow to apply the utility of the dual method.

control standards were reinjected using a new calibration curve. To determine MDLs, 7 or 8 spiked samples and a blank were extracted and analyzed according to the optimized method. The SDs obtained on the concentrations of the 7 or 8 samples were multiplied by the corresponding 99 % Student t test values of 3.143 or 2.998, respectively, for each compound (Wisconsin Department of Natural Resources 1996). Instrument detection limits (IDLs) were calculated using the SD of 10 samples at concentrations close to detection limits and multiplied by the 99 % Student t test value of 2.821 (Wisconsin Department of Natural Resources 1996).

Statistics, Quality Assurance/Quality Control, and Detection Limit Calculations

LLE Development

Results and Discussion The development of LLE and SPE steps were performed using a limited number of compounds, i.e., PHOS, CYF, TEF, DCBP (surrogate), THX, CLO, IMI, ACE (surrogate), and THC (surrogate). The use of deuterated neonicotinoids as surrogates was not possible because they would appear as coeluted with the corresponding native neonicotinoid when using LC-DAD to quantify; therefore, THC and ACE, which are not applied in Illinois, were used as surrogates instead.

Solvent Volume During LLE and SPE optimization, triplicate samples were evaluated to compare recoveries and statistically tested for differences using two-way analysis of variance (ANOVA) tests and two-sample Student t tests [IBM Statistics 20 (SPSS, Chicago, IL, USA)]. For both GC–MS and LC-DAD analyses, one of the standards used for the calibration curve at the beginning of the sequence was injected every eight samples as a quality control. The quantification recoveries of the reinjected standards had to be within 100 ± 20 % to be considered acceptable; otherwise the samples between two of these Table 2 Water-quality parameters of matrices tested during method development Matrix

pH

Conductivity (lS cm-2)

TOC (mg L-1)

MHRW

7.80

290

\1

Lake water

7.32

178

8.5

Field runoff Cache river water

7.25 7.50

48 249

46 5.9

MHRW moderately hard reconstituted water, TOC total organic carbon

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To optimize the extraction method, several parameters were investigated including the volume of solvent (DCM) used, number of extractions, amount of time to homogenize the LLE, and amount of NaCl added to the LLE. The first set of experiments examined solvent volume. Analytes and surrogates were added to 200 mL of MHRW to obtain a concentration of 250 ng L-1 of each compound in triplicate for each experimental condition. Conditions for the solvent volume experiment included 50, 100, or 200 mL of DCM/200 mL of water. Each extraction was repeated three times for three different homogenization times maintaining NaCl amount constant at 10 g. Each LLE fraction was concentrated to 1 mL using the Turbovap II evaporator and divided equally into an OP/pyrethroid portion for GC–MS analysis and the neonicotinoid portion for LC-DAD analysis. Results showed that for all conditions, the OP, pyrethroids, and surrogate (DCBP) were extracted after the first homogenization with recoveries ranging from 52 to 124 %. For neonicotinoids, a significant amount of THX and CLO was present in the second fraction, especially for samples extracted with 50 mL of DCM. CLO showed the lowest

Arch Environ Contam Toxicol

recoveries in the first fraction (ranging from 51 to 89 %), and the third fraction contained no CLO when 200 mL of DCM were used, 0 to 6 % with a volume of 100 mL of DCM, and 7 to 12 % when using 50 mL of DCM. The second extraction recovered 12 to 28 % of the CLO and was thus essential. Overall, recoveries for all compounds after two successive extractions are listed in Table 3. When the two first extractions were considered, the volume of DCM had no significant effect (two-way ANOVA, p [ 0.05) on the total recoveries except for THX and CLO. For both THX and CLO, the use of 200 mL of DCM provided significantly (p \ 0.05) greater recoveries than the use of 50 mL, but these were not significantly (p [ 0.05) greater than those obtained with 100 mL. The use of two 100-mL DCM extractions, instead of two 200-mL DCM extractions, decreased the amount of solvent and the time needed to blow down the extracts, thus decreasing extraction costs. Therefore, two extractions with 100 mL of DCM were chosen as the optimum conditions for number of extractions and volume of solvent. Homogenization Time Analytes and surrogates were added to triplicates of 200 mL of MHRW to reach a concentration of 250 ng L-1 in water for each experimental condition as detailed in the section ‘‘Solvent Volume’’ of the methods. Conditions for the

homogenization time experiment were 10, 20, or 30 min for each fraction with three fractions collected using 50, 100, or 200 mL of DCM. The amount of NaCl used was kept constant at 10 g. Results showed that a 30-min extraction time may significantly increase the recoveries of some compounds (two-way ANOVA, p \ 0.05, for PHOS, TEF, and THX). However, as listed in Table 3 with two extractions for 10 min using 100 mL of DCM, all of the recoveries were C88 % except for TEF (64 %). An increase from 10 to 30 min for each extraction represents an increase in extraction time B2 h for a batch of 12 samples (considering 4 samples simultaneously extracted) and was not considered an efficient strategy because most of the compounds already had satisfactory recoveries. Therefore, 10 min was chosen as the optimal homogenization time. NaCl Amount A second set of experiments was then performed to assess extraction efficiency based on the amount of NaCl added to the LLE. Three experimental conditions were tested including: (1) 2 9 50 mL of DCM, 10-min extraction, 10 g of NaCl; (2) 2 9 50 mL of DCM, 10 min-extraction, 20 g of NaCl; and (3) 2 9 100 mL of DCM, 10-min extraction, 20 g of NaCl. Recoveries were similar (p [ 0.05) for the three conditions for all compounds, ranging from 78 to 109 %, except for CYF, whose

Table 3 Mean recoveries of each compound [percent – SD (n = 3)] obtained after two successive LLEs to compare homogenization time and DCM volume DCM volume (mL)

DCBP (surrogate)

PHOS

10 min

20 min

30 min

10 min

50 100

106 ± 1 119 ± 1

110 ± 4 113 ± 1

84 ± 1 76 ± 1

102 ± 1 106 ± 1

200

121 ± 1

103 ± 1

72 ± 1

98 ± 1

DCM volume (mL)

TEF

CYF 20 min

30 min

10 min

20 min

30 min

97 ± 3 94 ± 1

118 ± 1 124 ± 1

78 ± 1 88 ± 1

71 ± 4 74 ± 1

76 ± 1 81 ± 1

101 ± 1

107 ± 1

85 ± 1

77 ± 1

79 ± 1

THX

CLO

10 min

20 min

30 min

10 min

20 min

50 100

65 ± 1 64 ± 1

67 ± 3 61 ± 1

86 ± 2 99 ± 1

85 ± 6 88 ± 3

90 ± 1 85 ± 4

200

63 ± 1

52 ± 1

93 ± 1

84 ± 3

86 ± 5

DCM volume (mL)

IMI 10 min

30 min

10 min

20 min

86 ± 2 99 ± 1

87 ± 4 94 ± 2

78 ± 1 85 ± 2

75 ± 3 90 ± 2

112 ± 3

96 ± 2

92 ± 7

105 ± 18

ACE (surrogate) 20 min

30 min

10 min

20 min

30 min

THC (surrogate) 30 min

10 min

20 min

30 min

50

102 ± 10

97 ± 2

83 ± 8

88 ± 3

93 ± 4

85 ± 10

105 ± 1

103 ± 4

95 ± 9

100

89 ± 1

92 ± 1

93 ± 1

89 ± 2

92 ± 2

95 ± 1

105 ± 5

99 ± 1

102 ± 1

200

102 ± 15

97 ± 12

114 ± 3

89 ± 4

92 ± 7

101 ± 19

115 ± 5

98 ± 7

115 ± 16

PHOS phostebupirim, CYF cyfluthrin, TEF tefluthrin, THX thiamethoxam, CLO clothianidin, IMI imidacloprid, DCBP decachlorobiphenyl, ACE acetamiprid, THC thiacloprid

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recoveries slightly decreased with an increase of solvent volume or salt. Therefore, 10 g of NaCl was determined to be the optimized condition, and the 200 mL of salted water were extracted successively twice with 100 mL of DCM for 10 min each time. SPE Development The SPE step in this method was optimized to improve analyte selectivity in the presence of matrix interferents and to separate the analytes into two fractions. One fraction contained the OPs and pyrethroids for GC–MS analysis, and the other fraction contained the neonicotinoids for LCDAD analysis. ENVI-Carb II/PSA cartridges are reversephase (ENVI-Carb) and anion-exchange (primary secondary amine, PSA) cartridges suitable for extraction of both polar and nonpolar analytes from organic or aqueous matrices. After LLE, analytes were extracted from the ENVI-Carb II/PSA cartridges after a normal-phase extraction, i.e., using nonpolar solvent first to extract lesspolar analytes followed by an elution with more polar solvent to extract more-polar compounds. In our case, the OPs and pyrethroids were thus eluted in the first fraction (less polar), and the neonicotinoids eluted in the second fraction (more polar). The first test to optimize SPE conditions was performed to confirm the elution of the OPs and pyrethroids in the first fraction. The cartridges were conditioned with a hexane/ acetone solution (75/25 v/v), which was also used as the loading solvent. After the OPs and pyrethroids were loaded into the cartridge, 3.5, 7, or 10 mL (n = 3) of a hexane/ DCM (70/30 v/v) solution was used to elute the OPs and pyrethroids. PHOS and CYF recoveries increased with the amount of solution used for elution, whereas the opposite was observed for TEF. Therefore, the intermediate elution volume (7 mL) was considered optimum providing recoveries ranging from 85 to 108 % for the OPs and pyrethroids. Because this step was successful, neonicotinoids were tested to determine if they would be eluted using this solvent solution. Therefore, 3.5 mL of a hexane/ acetone solution (75/25 v/v) was spiked with neonicotinoids and added to the cartridges. After adding and draining 7 mL of a hexane/DCM solution (70/30 v/v) to mimic the OP/pyrethroid elution, the cartridges were eluted for neonicotinoids with 3.5, 7, or 10 mL (n = 3) of a DCM/ acetonitrile solution (50/50 v/v). Recoveries of the neonicotinoids ranged from 97 to 107 % regardless of the elution volume, with the exception of ACE, which had recoveries ranging from 54 to 69 % and were not enhanced by using more solvent. Further tests showed that ACE was eluted with the OP/pyrethroid fraction due to ACE exhibiting a high affinity for DCM. To remedy this situation, simultaneous tests were performed using a hexane/DCM solution

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(90/10 v/v) as the eluting solvent for the OP/pyrethroid fraction and a DCM/acetonitrile solution (50/50 v/v) as the eluting solvent for the neonicotinoids. Recoveries from 85 to 110 % were obtained for the OPs and pyrethroids, and neonicotinoid recoveries ranged from 89 to 107 % with no loss of ACE in the OP/pyrethroid fraction. Therefore, the final SPE method included cartridge conditioning and transfer of samples with a hexane/acetone solution (75/25 v/v); the OP and pyrethroids were eluted with 7 mL of a hexane/DCM solution (90/10 v/v); and the neonicotinoids were eluted into a separate collection tube with 3.5 mL of a DCM/acetonitrile solution (50/50 v/v). When these final SPE conditions were tested, recoveries ranged from 86 to 103 % for all compounds. Combination of LLE and SPE Before applying the LLE and SPE extraction combination to field-collected samples, both steps were tested with four replicates of 200 mL of MHRW with all analytes and surrogates added at a concentration of 250 ng L-1 of water. The LLE was performed using two 10-min extractions with 100 mL of DCM and 10 g of NaCl. The SPE step included elution of the OPs and pyrethroids with 7 mL of the hexane/DCM solution (90/10 v/v) and elution of the neonicotinoids with 3.5 mL of the DCM/acetonitrile solution (50/50 v/v). Recoveries ranged between 77 and 97 % depending on the analyte, and RSDs were between 2 and 11 %. To document the robustness of the method, additional field matrices were spiked at the same concentration, i.e., 250 ng L-1, and extracted according to the same protocol. Recoveries ranged from 81 to 100 % and from 82 to 108 % in lake and field runoff water, respectively. The RSDs ranged from 2 to 11 %, which was similar to the MHRW results. In the runoff samples, a thick emulsion was observed after LLE. Different procedures can be used to eventually separate the aqueous from organic phases; however, chemical-based and cooling methods to break moderate and severe emulsions were shown to not be successful for pyrethroids (Wu et al. 2010). Centrifugation at 500 g for 10 min was sufficient with our samples to separate both phases. However, it was necessary to reextract the aqueous phase after centrifugation to limit loss of the neonicotinoids. Therefore, centrifugation could not be performed after the second LLE extraction. The amount of emulsion was smaller in the second extraction, and draining the DCM very slowly was sufficient to obtain good recoveries with almost no aqueous phase in the extract. If any traces of water were visible in the extract, the addition of small amounts of Na2SO4 directly in the extract or in the SPE cartridge was sufficient to dry the sample. It is noteworthy that if too much water is present in

Arch Environ Contam Toxicol

the DCM extract and the sample is transferred to the SPE cartridge, the polarity of the loading solvent is changed and the neonicotinoids could be lost into the OP/pyrethroid fraction. As shown by the acceptable recoveries, complex matrices were not an issue for accuracy using the current method and centrifugation of the LLE extracts. Expansion to the 15 Insecticides Once the method was optimized and its robustness validated using several different matrices, additional analytes were included to the method to expand its use across different agricultural and urban areas. Common insecticides detected in the United States were thus added, including CPF (an OP) and six pyrethroids. Because ACE and THC (neonicotinoid insecticides) may be used in other agricultural areas of the country, they were then considered as analytes instead of surrogates (i.e., they were not added to the blanks). In addition, we noted slight losses of THC and/ or THX in some samples that were corrected when eluting the last SPE fraction with 5 mL of a DCM/acetonitrile solution (50/50 v/v). Because of the addition of several pyrethroids and their better sensitivities in NCI mode, samples were analyzed for OPs and pyrethroids by GC– NCI-MS. These conditions were also used for the calculation of MDLs. Validation of the method for the 15 insecticides included thorough investigation of the capabilities of the GC–NCI-MS and LC-DAD as well as accuracy and sensitivity in different matrices including deionized, lake, and river water. Tables 4 and 5 list the linear calibration ranges with their corresponding regression coefficients and IDLs for each compound using GC– MS and LC-DAD, respectively. The OPs and pyrethroids had overall wide linear calibration ranges from \1 to 250 or 500 ng mL-1 injected, except for PHOS and PER, which were less sensitive with IDLs [1 ng mL-1; other compounds had IDLs between 0.02 and 0.45 ng mL-1. The use of a less sensitive detector for neonicotinoid analysis lead to lower sensitivities for neonicotinoids than for most OPs and pyrethroids, yet IDLs were still low, ranging from 1.1 to 3.3 ng mL-1 injected, and calibration curves were linear at least from 5 to 500 ng mL-1. The MDLs of all compounds for the three matrices are listed in Table 6. They ranged from 0.1 to 27.2 ng L-1 with the lowest for FEN and the highest for PER. It is noteworthy that PHOS, which had a relatively high MDL using GC–NCI-MS, had a 10-fold greater sensitivity using GC–EI-MS. However, the priority was given to pyrethroids, which are more widely used and more likely to be present in environmental water samples; therefore, EI mode, which is less sensitive for pyrethroids, was not performed. Sensitivities of pyrethroids with several isomers, i.e., PER, CYF, and CYP, were overall less sensitive

when the total compound was considered because the total amount of compound was distributed over several peaks; however, the sensitivity of each isomer was considerably better, especially for CYF and CYP, which presented four peaks each on the chromatogram (Supplementry Fig. S2). Therefore, if fewer isomers were present in the sample, as would be the case after application of b-CYF, better sensitivities would be achieved. For k-CYH and ESF, their isomerization was considered negligible because one of the two peaks they each presented was always \5 % of the other peak and did not lower the sensitivity. The method developed in the current study provided an improvement in sensitivity from a method developed by Bonwick et al. (1995), who, after extraction of 1 L of water and GC–NCIMS analysis, obtained MDLs of 0.05 lg L-1 for CYF and PER. The current study’s MDLs were in the same range or slightly greater than that of Wang et al. (2009), who found MDLs from 0.4 to 1.7 ng L-1 for OPs and pyrethroids in four different water matrices using GC-lECD, GC-ECD, and GC-NPD. The MDLs for the neonicotinoids ranged from 2.6 to 12.3 ng L-1 depending on the compound and the matrix. These values were much lower than the ones obtained by other groups using GC-ECD, LC-DAD, or even LC-MS (Ying and Kookana 2004; Seccia et al. 2005; Amelin et al. 2012), and in the same range as that of Zhou et al. (2006), who extracted 500 mL of water and used LC–UV, or the ones obtained for the same compounds after a 1-L water extraction analyzed by LC–MS/MS (Hladik and Calhoun 2012). However, the selectivity of a LC–MS/MS would likely be necessary to achieve such low MDLs in very complex matrices. To calculate MDLs, water samples were spiked close to detection and quantification limits of each compound (\10 times the MDL), and recoveries ranged from 52 % (THC) to 143 % (FEN), with most compound recoveries being between 70 and 110 % (Table 6). For samples spiked at 100 ng L-1, recoveries ranged from 86 to 114 % for all compounds with good precision as shown by SDs \10 % (Table 6). Accuracy was thus excellent at concentrations of 100 ng L-1, which may be found in environmental waters in urban waterways or agricultural wetlands (Ensminger et al. 2013; Main et al. 2014), and acceptable accuracy was obtained when samples were spiked close to detection limit concentrations, thus allowing the use of the method on samples coming from less affected areas. Examples of chromatograms obtained for the 15 insecticides in this lake water sample spiked at 100 ng L-1 are given in the supporting information (Supplementry Figs. S2 and S3). Low baseline and high resolution were obtained for most compounds, especially OP/pyrethroids analyzed by GC–NCIMS. Due to the wide range of compounds detectable by DAD, the baseline was greater for neonicotinoids, and thus

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Arch Environ Contam Toxicol Table 4 Quantification ions, linear calibration ranges, and calibration curves for OP and pyrethroid analysis using GC–NCI-MS Analytes

Quantification m/z

Linear calibration range (ng mL-1 injected)

Calibration curve area = a 9 concentration ? b

R2

IDL (ng mL-1 injected)

Phostebupirim

183

4.00–500

a = 0.0411, b = 0.0023

0.99466

1.22

Chlorpyrifos

313

0.25–500

a = 1.9530, b = 0.3009

0.99840

0.10

Tefluthrin

241

0.25–25

a = 0.1152, b = 0.1569

0.99657

0.02

Bifenthrin

386

0.45–500

a = 5060, b = -3.1120

0.99893

0.45

Fenpropathrin

141

0.25–250

a = 2.1420, b = -0.0532

0.99799

0.05

k-Cyhalothrin

241

0.25–500

a = 2.6460, b = 0.0013

0.99947

0.06

Permethrin cis Permethrin trans

207 207

2.50–90 2.50–30

a = 0.0637, b = -0.0009 a = 0.0693, b = -0.0002

0.99528 0.99716

2.27 2.38

Cyfluthrin 1

207

0.06–55

a = 2.9920, b = -0.0120

0.99893

0.03

Cyfluthrin 3

207

0.08–75

a = 2.9870, b = -0.0028

0.99915

0.02

Cyfluthrin 2 & 4

207

0.20–120

a = 3.0340, b = -0.0029

0.99830

0.19

Cypermethrin 1

207

0.15–70

a = 1.406, b = -0.0144

0.99830

0.13

Cypermethrin 3

207

0.10–60

a = 1.388, b = -0.0126

0.99813

0.09

Cypermethrin 2 & 4

207

0.20–120

a = 1.472, b = -0.0294

0.99824

0.19

Esfenvalerate

211

0.25–250

a = 3.291, b = 0.1227

0.99870

0.04

Calibration ranges were performed using standards at 10 different concentrations. Because differences in sensitivities and linear ranges were evident between isomers, detailed results are given here for each CYF and CYP resolved isomer, although only total CYF and CYP were reported for quantification of these compounds Table 5 Quantification wavelengths, linear calibration ranges, and calibration curves for neonicotinoid analysis Analytes

DAD quantification wavelength (nm)

Linear calibration range (ng mL-1 injected)

Calibration curve Area = a 9 concentration ? b

R2

IDL (ng mL-1 injected)

Thiamethoxam

252

5–500

a = 0.0967, b = -0.1749

0.99996

1.6

Clothianidin

269

5–500

a = 0.1191, b = -0.2855

0.99988

1.6

Imidacloprid Acetamiprid

269 242

5–500 5–500

a = 0.1475, b = -0.2035 a = 0.0643, b = -0.1202

0.99996 0.99995

1.1 1.5

Thiacloprid

242

5–500

a = 0.0634, b = -0.0277

0.99996

3.3

Calibration ranges were performed using standards at eight different concentrations

interferences were more likely to occur; however, great improvement of selectivity and sensitivity are expected if a LC–MS/MS is available and using the same dual-extraction method. Overall, the method developed in the current study was shown to be more or as sensitive and accurate as other methods used specifically for either OPs and pyrethroids or neonicotinoids with the additional advantage of having to extract only one sample of 200 mL for the three classes of insecticides. Application to Field-Collected Samples Water samples were collected from a corn field near Taylorville, Illinois, throughout the corn growing season in 2013. A matrix spike and matrix spike duplicate were extracted with each batch of field samples and consisted of a field sample from the same batch divided into two samples and spiked at a final concentration of 250 ng L-1.

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Runoff water often contained TEF (pyrethroid) with the highest concentration measured at 169 ng L-1, which occurred shortly after application. Recoveries for the matrix spike and matrix spike duplicate averaged 81 % with an SD of 11 %. Soil infiltration water and groundwater rarely contained detectable concentrations of TEF, and recoveries of the matrix spike and matrix spike duplicate averaged ±SD 79 ± 5 and 81 ± 8 %, respectively. CLO was consistently detected in all three matrices at all time points throughout the corn-growing season. Highest concentrations in runoff water, soil infiltration water, and groundwater were 1595, 524, and 96 ng L-1, respectively. The matrix spike and matrix spike duplicate average recoveries ±SD for runoff water, soil infiltration water, and groundwater were 77 ± 13, 85 ± 9, and 80 ± 8 %, respectively. It should be noted that the blanks did not contain detectable concentrations of our internal standard PCB 153; therefore, quantification was achievable. An isotopically labeled PCB153 should be used in

Arch Environ Contam Toxicol Table 6 MDLs for the analytes from different aqueous matrices spiked at concentrations close to detection limits (between 0.1 and 80 ng L-1) using the optimized method Deionized water MDLs (ng L-1) DCBP (surrogate) Phostebupirim

–

Lake water Recoveries (%) 85 ± 6

MDLs (ng L-1)

River water Recoveries (%)

–

74 ± 13

MDLs (ng L-1) –

Recoveries (%)

Recoveries at 100 ng L-1 (%)

93 ± 6

93 ± 2

11.68

99 ± 12

15.51

111 ± 18

6.73

100 ± 8

103 ± 8

Chlorpyrifos

0.64

71 ± 12

0.62

70 ± 13

0.32

71 ± 5

95 ± 6

Tefluthrin

0.23

70 ± 14

0.17

74 ± 13

0.17

81 ± 10

89 ± 5

Bifenthrin

3.00

113 ± 10

2.87

71 ± 5

3.58

113 ± 11

112 ± 4

Fenpropathrin

0.10

90 ± 13

0.14

143 ± 25

0.10

115 ± 14

114 ± 4

k-Cyhalothrin

0.17

67 ± 7

0.34

54 ± 19

0.19

65 ± 8

105 ± 3

Permethrin

15.38

100 ± 5

27.18

109 ± 10

24.70

94 ± 8

86 ± 3

Cyfluthrin

0.71

74 ± 6

1.10

90 ± 11

0.91

84 ± 7

91 ± 2

Cypermethrin Esfenvalerate

2.05 0.17

80 ± 6 78 ± 4

3.48 0.33

102 ± 10 88 ± 10

3.42 0.20

101 ± 11 71 ± 5

87 ± 2 90 ± 2 110 ± 15

Thiamethoxam

4.02

57 ± 5

10.58

77 ± 13

11.03

108 ± 16

Clothianidin

12.30

103 ± 17

5.00

87 ± 5

7.68

109 ± 10

Imidacloprid

4.32

89 ± 6

8.01

105 ± 10

2.57

112 ± 3

104 ± 6

Acetamiprid

5.12

90 ± 6

11.09

104 ± 15

3.88

98 ± 5

103 ± 6

Thiacloprid

8.15

90 ± 11

10.06

53 ± 12

7.84

52 ± 10

80 ± 5

98 ± 24

MDLs were not calculated for DCBP (surrogate), but recoveries are reported. Additional analyte recoveries are presented for lake water spiked at 100 ng L-1 (n = 4)

cases when this PCB congener is present in the samples. Based on these results, the utility of this method was deemed acceptable.

Acknowledgments The authors thank Marjorie Brooks for the use of the TOC analyzer; Rebecca Kelley, Amanda Rothert for help in developing the method, and Mary Janello for collecting groundwater. Funding for this project was provided by the Howard G. Buffett Foundation. Use of a company or product name does not imply approval or recommendation of the product by Southern Illinois University or the Howard G. Buffett Foundation.

Conclusions The optimized method provided good accuracy and reproducibility for the simultaneous extraction of OP, pyrethroid, and neonicotinoid insecticides from the same water sample. This method was highly applicable to environmental samples at trace concentrations as shown by the MDLs (in the ng L-1 range), the satisfactory recoveries, and the minimal effect of the matrix, even challenging ones, on sensitivity and quantification. The current method was a great improvement of existing procedures because it combined extraction of several classes of insecticides usually performed separately without affecting performance. One simple extraction procedure will help scientists determine the fate and transport of these insecticides in aqueous environments, and thus the risks involved in their use, while saving time and money in solvent and cartridge costs by performing a single extraction. This method was also applied to field-collected samples resulting in acceptable recoveries, thus providing valuable information on the environmental fate of these insecticides.

References Alder L, Greulich K, Kempe G, Vieth B (2006) Residue analysis of 500 high priority pesticides: better by GC–MS or LC–MS/MS? Mass Spectrom Rev 25:838–865 Amelin VG, Bol’shakov DS, Tretiakov AV (2012) Identification and determination of synthetic pyrethroids, chlorpyriphos, and neonicotinoids in water by gas and liquid chromatography. J Anal Chem 67:354–359 Belden JB, Lydy MJ (2006) Joint toxicity of chlorpyrifos and esfenvalerate to fathead minnows and midge larvae. Environ Toxicol Chem 25:623–629 Bonwick GA, Sun C, Abdul-Latif P et al (1995) Determination of permethrin and cyfluthrin in water and sediment by gas chromatography-mass spectrometry operated in the negative chemical ionization mode. J Chromatogr A 707:293–302 Chandrasena D, DiFonzo C, Byrne A (2011) An aphid-dip bioassay to evaluate susceptibility of soybean aphid (Hemiptera: Aphididae) to pyrethroid, organophosphate, and neonicotinoid insecticides. J Econ Entomol 104:1357–1363 Ensminger MP, Budd R, Kelley KC, Goh KS (2013) Pesticide occurrence and aquatic benchmark exceedances in urban surface

123

Arch Environ Contam Toxicol waters and sediments in three urban areas of California, USA, 2008–2011. Environ Monit Assess 185:3697–3710 Gamon M, Lleo C, Ten A, Mocholi F (2001) Multiresidue determination of pesticides in fruit and vegetables by gas chromatography/tandem mass spectrometry. J AOAC Int 84:1209–1216 Guardino X, Obiols J, Rosell MG et al (1998) Determination of chlorpyrifos in air, leaves and soil from a greenhouse by gaschromatography with nitrogen–phosphorus detection, high-performance liquid chromatography and capillary electrophoresis. J Chromatogr A 823:91–96 Hawker DW, Connell DW (1988) Octanol-water partition coefficients of polychlorinated biphenyl congeners. Environ Sci Technol 22:382–387 Hladik ML, Calhoun DL (2012) Analysis of the herbicide diuron, three diuron degradates, and six neonicotinoid insecticides in water—Method details and application to two Georgia streams: US Geological Survey scientific investigations report 2012–5206 Kamel A (2010) Refined methodology for the determination of neonicotinoid pesticides and their metabolites in honey bees and bee products by liquid chromatography-tandem mass spectrometry (LC-MS/MS). J Agric Food Chem 58:5926–5931 Kamel A, Byrne C, Vigo C et al (2009) Oxidation of selected organophosphate pesticides during chlorination of simulated drinking water. Water Res 43:522–534 Lo´pez Flores J, Molina Dı´az A, Ferna´ndez de Co´rdova ML (2007) Development of a photochemically induced fluorescence-based optosensor for the determination of imidacloprid in peppers and environmental waters. Talanta 72:991–997 Lydy M, Belden J, Wheelock C et al (2004) Challenges in regulating pesticide mixtures. Ecol Soc 9(6):1. http://www.ecologyandsoci ety.org/vol9/iss6/art1 Main AR, Headley JV, Peru KM et al (2014) Widespread use and frequent detection of neonicotinoid insecticides in wetlands of Canada’s Prairie Pothole Region. PLoS One 26:e92821 Mohan C, Kumar Y, Madan J, Saxena N (2010) Multiresidue analysis of neonicotinoids by solid-phase extraction technique using high-performance liquid chromatography. Environ Monit Assess 165:573–576 Onstad DW, Mitchell PD, Hurley TM et al (2011) Seeds of change: corn seed mixtures for resistance management and integrated pest management. J Econ Entomol 104:343–352 Pucarevic´ M, Bursic´ V, Pankovic´ D et al (2013) Supercritical fluid extraction of tebupirimphos residues in sugar beet. J Anim Plant Sci 23:277–280 Seccia S, Fidente P, Barbini DA, Morrica P (2005) Multiresidue determination of nicotinoid insecticide residues in drinking water by liquid chromatography with electrospray ionization mass spectrometry. Anal Chim Acta 553:21–26 Siegfried BD (1993) Comparative toxicity of pyrethroid insecticides to terrestrial and aquatic insects. Environ Toxicol Chem 12:1683–1689 Tomizawa M, Casida JE (2005) Neonicotinoid insecticide toxicology: mechanisms of selective action. Ann Rev Pharmacol Toxicol 45:247–268 United States Environmental Protection Agency (2000) Methods for measuring the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates, 2nd edn. USEPA, Washington

123

United States Environmental Protection Agency (2007a) Method 614: the determination of organophosphorus pesticides in municipal and industrial wastewater. USEPA, Washington United States Environmental Protection Agency (2007b) Method 1699: pesticides in water, soil, sediment, biosolids, and tissue by HRGC/HRMS. USEPA, Washington United States Environmental Protection Agency (2013) 2006-2007 Pesticide market estimates: Usage | pesticides | US EPA. In: 2006-2007 Pesticide Market Estimates. http://www.epa.gov/ opp00001/pestsales/07pestsales/usage2007.htm. Accessed 13 May 2013 Walker CH (2009) Organic pollutants: an ecotoxicological perspective. CRC Press, Boca Raton Wang D, Weston DP, Lydy MJ (2009) Method development for the analysis of organophosphate and pyrethroid insecticides at low parts per trillion levels in water. Talanta 78:1345–1351 Watanabe E, Miyake S, Baba K et al (2006) Immunoassay for acetamiprid detection: application to residue analysis and comparison with liquid chromatography. Anal Bioanal Chem 386:1441–1448 Watanabe E, Baba K, Eun H (2007) Simultaneous determination of neonicotinoid insecticides in agricultural samples by solid-phase extraction cleanup and liquid chromatography equipped with diode-array detection. J Agric Food Chem 55:3798–3804 Wijngaarden RPV, Brock TC, Van Den Brink PJ (2005) Threshold levels for effects of insecticides in freshwater ecosystems: a review. Ecotoxicology 14:355–380 Wisconsin Department of Natural Resources (1996) Analytical detection limit guidance & laboratory guide for determining method detection limits. PUBL-TS-056-96 Wu J, Lu J, Wilson C et al (2010) Effective liquid–liquid extraction method for analysis of pyrethroid and phenylpyrazole pesticides in emulsion-prone surface water samples. J Chromatogr A 1217:6327–6333 Yalkowsky SH (2010) Handbook of aqueous solubility data, 2nd edn. CRC Press, Boca Raton Ying G-G, Kookana RS (2004) Simultaneous determination of imidacloprid, thiacloprid, and thiamethoxam in soil and water by high-performance liquid chromatography with diode-array detection. J Environ Sci Health B 39:737–746 You J, Lydy MJ (2006) Determination of pyrethroid, organophosphate and organochlorine pesticides in water by headspace solidphase microextraction. Int J Environ Anal Chem 86:381–389 You J, Lydy MJ (2007) A solution for isomerization of pyrethroid insecticides in gas chromatography. J Chromatogr A 1166:181–190 Zhang Z-Y, Yu X-Y, Wang D-L et al (2010) Acute toxicity to zebrafish of two organophosphates and four pyrethroids and their binary mixtures. Pest Manag Sci 66:84–89 Zhang F, Li Y, Yu C, Pan C (2012) Determination of six neonicotinoid insecticides residues in spinach, cucumber, apple and pomelo by QuEChERS method and LC–MS/MS. Bull Environ Contam Toxicol 88:885–890 Zhou Q, Ding Y, Xiao J (2006) Sensitive determination of thiamethoxam, imidacloprid and acetamiprid in environmental water samples with solid-phase extraction packed with multiwalled carbon nanotubes prior to high-performance liquid chromatography. Anal Bioanal Chem 385:1520–1525