Quantitative Determination of Trisiloxane Surfactants in Beehive

Jul 15, 2013 - Department of Entomology, Center for Pollinator Research, The Pennsylvania State University, University Park, Pennsylvania 16802,...
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Quantitative Determination of Trisiloxane Surfactants in Beehive Environments Based on Liquid Chromatography Coupled to Mass Spectrometry Jing Chen and Christopher A. Mullin* Department of Entomology, Center for Pollinator Research, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: Organosilicone surfactants are increasingly being applied to agricultural agro-ecosystems as spray adjuvants, and were recently shown to impact the learning ability of honey bees. Here we developed a method for analyzing three trisiloxane surfactants (single polyethoxylate (EO) chain and end-capped with methyl, acetyl, or hydroxyl groups; TSS-CH3, TSS-COCH3, or TSS-H) in beehive matrices based on liquid chromatography coupled to mass spectrometry (LC−MS) and the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach from less than 2 g of honey, pollen, or beeswax. Recoveries for each oligomer (2−13 EO) were between 66 and 112% in all matrices. Average method detection limits (MDL) were 0.53, 0.60, 0.56 ng/g in honey, 0.63, 0.81, 0.78 ng/g in pollen, and 0.51, 0.69, 0.63 ng/g in beeswax. Five honey, 10 pollen, and 10 beeswax samples were analyzed. Trisiloxane surfactants were detected in every beeswax and 60% of the pollen samples. Total trisiloxane surfactant concentrations were up to 390 and 39 ng/g in wax and pollen. The described method is proved suitable for analyzing trisiloxane surfactants in beehive samples. The presence of trisiloxane surfactants in North American beehives calls for renewed effort to investigate the consequence of these adjuvants to bee health and the ongoing global bee decline.

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INTRODUCTION Major honey bee colony lost continues to be reported since 2006.1,2 Since bee and other pollinators contribute more than 200 billion dollars to the global economy,3 the continuing loss of honey bees has led to unprecedented worldwide research efforts.2 At present, multiple factors such as pathogens, parasites, malnutrition, and pesticide exposure are considered to play major roles in the global diminishing of bees.4,5 Worker bees are exposed to pesticides when they gather nectar and pollen from flowers in agro-ecosystems. Environmental pesticides, or hive contaminants from other treatments for bee pests and disease, have long been suspected as a potential cause of honey bee declines.6 However, no correlation was found between any single agrochemical detection and colony collapse.5,7,8 The great qualitative and quantitative diversity of pesticide residues suggest that more generic formulation ‘inerts’ that co-occur across classes of pesticides may be involved. Agrochemical formulations usually contain inerts at higher amounts than the active ingredients, and they are largely assumed to be biologically safe, and are usually not included in risk assessment trials for nontarget organisms like bees required for pesticide registration in the United States.9 Given the synergistic nature of certain chemicals, the active ingredients may affect honey bees differently depending upon the inert ingredients in a formulation or in field use combinations.10−12 © 2013 American Chemical Society

Our previous investigation of the impact of three major classes of agricultural spray adjuvants, including organosilicone surfactants, nonionic surfactants, and crop oil concentrates, on the learning ability of honey bees demonstrated that only the organosilicone surfactants at 20 μg per bee consistently impaired the proboscis extension reflex (PER) for sucrose in response to a cinnamon odor.13 The fact that “inert” organosilicone surfactants substantially inhibited at sublethal levels a behavior essential for successful food acquisition has important implications for the health of honey bees and other pollinators. Organosilicone surfactants are increasingly applied within agro-ecosystems as spray adjuvants due to their super spreading and penetrating abilities.14,15 The chemical structure of an organosilicone surfactant consists of a fully methylated siloxane “backbone” as the hydrophobic part, and a polyethoxylate “tail” as the hydrophilic part (Figure 1).14 The molecular structure is further complicated by variance in the values of w, x, m, and n chains, which are generally not revealed by manufacturers. For utility as spray adjuvants, the organosilicone surfactant must be Received: Revised: Accepted: Published: 9317

March 8, 2013 June 25, 2013 July 15, 2013 July 15, 2013 dx.doi.org/10.1021/es4010619 | Environ. Sci. Technol. 2013, 47, 9317−9323

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MATERIALS AND METHODS

Chemicals. The following commercial organosilicone adjuvants were used as sources for the trisiloxane surfactants analyzed here: Dyne-Amic, Kinetic HV and Silwet L-77 from Helena (Colliervill, TX), Sylgard 309 from Dow-Corning (Midland, MI), Syl-Tac from Willbur-Ellis (San Francisco, CA), and Silkin from Agriliance LLC (Inver Grove Heights, MN). The LC-MS mobile phase was comprised of acetonitrile (HPLC grade), methanol (HPLC grade), and formic acid (98%) from EMD Chemical Inc. (Gibbstown, NJ); and ammonium formate from Alfa Aesar (Pittsburgh, PA). Reagents used for trisiloxane surfactant extraction were acetonitrile from EMD Chemical Inc., sodium acetate (99%, anhydrous) from Sigma-Aldrich (St. Louis, MO), magnesium sulfate (MgSO4, anhydrous) and ENVIRO Clean 2 mL dispersive solid-phase extraction (d-SPE) tubes (containing 150 mg MgSO4, 150 mg primary secondary amine (PSA), and 50 mg end-capped C18) from UCT (Bristol, PA). 4-n-butylchalcone oxide was synthesized from condensation of p-butylbenzaldehyde (Eastman, Kingsport, TE) and acetophenone (Aldrich) followed by basic hydrogen peroxide treatment using routine methods.20 Water used throughout was purified by a MilliDI system (Millipore, MA). LC-MS Conditions. Liquid chromatography coupled to mass spectrometry was performed on an LC-MS 2020 system (Shimadzu, Japan). Trisiloxane surfactant oligomers were separated on a Shimadzu Shim-pack XR-ODS column (100 mm × 2.0 mm, 2.2 μm particle). The column temperature was maintained at 50 °C. The binary mobile phase included (A) water and (B) acetonitrile/methanol (90/10, v/v), both buffered with 2 mM ammonium formate and 0.01% formic acid. The flow rate was set to 0.35 mL min−1. The 30 min gradient started at 20% B, kept for 0.25 min, and linearly changed to 50% B at 3 min, and then to 73% B at 8.5 min, to 77% B at 20 min, and 100% B at 22 min and kept till 26 min. After that, the mobile phase was set back to 20% B for a 4 min re-equilibrium. MS was performed using electrospray ionization in the positive mode (ESI+). ESI source parameters were: nebulization gas flow 1.5 L min−1, drying gas flow 15 L min−1, heat block temperature 300 °C, disolvation line (DL) temperature

Figure 1. General chemical structure of organosilicone surfactants adapted from reference;14 typically R = methyl, acetyl or hydroxyl group; the values of w, x, n, m are explained in the text.

water-soluble, and the most commonly used commercialized agrichemical organosilicone surfactants are trisiloxane-based derivatives,16 where w = 0, x = 1, naverage = 6−8, and m = 0 (Figure 1). Less frequently polypropoxylate units (m > 0) or additional dimethylsiloxy groups (w > 0) are incorporated into the respective “tail” and “backbone” to optimize the desired surfactancy. Chemical components of the first commercial organosilicone surfactant adjuvant Silwet L-77 were characterized using liquid chromatography coupled to mass spectrometry (LC-MS).17 The major component found was a trisiloxane with a single polyethoxylate (EO) chain that was methyl capped, and smaller amounts of other trisiloxanes, methyl polyethoxylates, polyethylene glycols, and other synthetic and hydrolytic byproducts were also identified. A detection method for this silicone polyether copolymer was also introduced in the same paper,17 but so far, no quantitative analytical method for trisiloxane surfactant oligomers has been reported. Here, a method for the analysis of trisiloxane surfactants in beehive samples is described. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method18,19 was employed to extract three frequently used trisiloxane surfactants from honey, pollen, and beeswax. The quantitative determination for each polyethoxylate oligomer (2−13 EO) was accomplished based on LC-MS. The analytical method was validated by establishing the recovery, precision, and method detection limit (MDL). The method was applied to 5 honey, 10 pollen, and 10 beeswax samples, in order to demonstrate the contamination of beerelated environments by trisiloxane surfactants.

Table 1. Molecular Weight and Monitored Ammonium Adducts for the Detected Trisiloxane Surfactant (TSS) Oligomersa TSS-CH3

TSS-COCH3

TSS-H

oligomerb

molecular weightc

m/z

molecular weight

m/z

molecular weight

m/z

2 EO 3 EO 4 EO 5 EO 6 EO 7 EO 8 EO 9 EO 10 EO 11 EO 12 EO 13 EO

382.20 426.33 470.26 514.28 558.31 602.33 646.36 690.39 734.41 778.44 822.46 866.49

400.3 444.3 488.3 532.3 576.4 620.4 664.4 708.4 752.4 796.5 840.5 884.5

454.22 498.26 542.28 586.31 630.33 674.36 718.39 762.41 806.44 850.46 894.49

472.3 516.3 560.3 604.3 648.4 692.4 736.4 780.5 824.5 868.5 912.5

456.24 500.27 544.29 588.32 632.34 676.37 720.40 764.42 808.45 852.48

474.3 518.3 562.3 606.3 650.4 694.4 738.4 782.4 826.5 870.5

a

Chemical structures are shown in Figure 1; TSS-CH3 (w = 0, x = 1, m = 0, R = methyl), TSS-COCH3 (w = 0, x = 1, m = 0, R = acetyl), TSS-H (w = 0, x = 1, m = 0, R = hydroxyl). bEO (ethoxylate) oligomers increasing from 2 EO units representing n = 2 in Figure 1. cMolecular weight is exact molecular mass. 9318

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250 °C, and interface bias voltage 4500 V. Ammonium adducts of each oligomer were monitored (Table 1). Identification and Purification of Trisiloxane Surfactants. Based on our LC-MS analysis of the top used commercial organosilicone spray adjuvants on California almonds21 (including Dyne-Amic, Kinetic HV, Silkin, Silwet L-77, Sylgard 309, and Syl-Tac), three classes of oligomers with polyethoxylate units were frequently detected. Three fractions were purified using a LC-18 SPE cartridge (500 mg/6 mL, Supelco, Sigma-Aldrich), and were identified as methoxy(polyethyleneoxy)propyl-heptamethyl trisiloxane (TSS-CH3), acetoxy(polyethyleneoxy)propyl-heptamethyl trisiloxane (TSSCOCH3) and hydroxy(polyethyleneoxy)propyl-heptamethyl trisiloxane (TSS-H). Their structures are shown in Figure 1, where w = 0, x = 1, m = 0; R = methyl, acetyl and hydroxyl, respectively. Structure identifications were validated by proton nuclear magnetic resonance (1H NMR) and heteronuclear multiple quantum coherence (HMQC) spectra (600 MHz, 14.1T Bruker AV-III-600). Sample Collection and Preparation. Beehive samples (5 honey, 10 pollen, and 10 beeswax samples) were collected as before7 from seven U.S. states, including Arizona, California, Iowa, Michigan, Minnesota, Pennsylvania, and Vermont, and stored at −20 °C until analysis. The extraction method was developed based on the original QuEChERS method,18,19 which is now the most widely used extraction method in pesticide analysis. TSS-CH3, TSS-COCH3 and TSS-H oligomers were analyzed from 2 ± 0.1 g honey, 1 ± 0.1 g pollen, or 1 ± 0.1 g beeswax. Samples were weighed into a 50 mL polypropylene centrifuge tube, whereas beeswax was previously homogenized with liquid nitrogen to reduce the particle size. Each sample was dissolved or dispersed into 2 mL water, and then mixed with 2 mL acetonitrile, 66.7 μL internal standard (IS) stock solution (75 μg/g 4-n-butylchalcone oxide) and vortexed for 30 s. Two g magnesium sulfate and 0.5 g sodium acetate were added and the plastic tube was immediately vortexed for 30 s. After placing on ice for 10 min, the tube was centrifuged (10 min at 5443 rcf). The upper layer comprised the initial extract, and 1 mL was transferred to the d-SPE tube and vortexed 15 s for cleanup. When the d-SPE tube cooled to room temperature, it was centrifuged for 10 min at 3830 rcf and 25 °C, the supernatant was transferred to autosampler vial and 10 μL was injected into the LC-MS system. The final extracts were equivalent to 1 g/mL for honey and 0.5 g/mL for pollen and beeswax. Method Validation. Quantitative determination was performed for each individual trisiloxane surfactant oligomer including TSS-CH3 with 2−13 EO, TSS-COCH3 with 3−13 EO and TSS-H with 4−13 EO. Linearity was evaluated in the range of 20 ng/g-1000 ng/g for the sum and each of the trisiloxane surfactant oligomers found. The standard was dissolved in water, and each 1 mL standard solution was prepared by the QuEChERS method as described above (adding 1 mL water and 2 mL acetonitrile here). Area ratios of analyte/internal standard were plotted as functions of the oligomer concentrations. Concentration for each oligomer was normalized according to the oligomer percentage shown in Figure 2. For example, the concentration of TSS-CH3 with a 6 EO side chain was normalized to the range 3.7 ng/g to 184.7 ng/g, representing 19.03% of the total. Concentrations in the linear range used in detector calibration for other trisiloxane surfactant oligomers are summarized in Supporting Information (SI) Table S1.

Figure 2. Percentage distribution of individual oligomers (total =100%) for purified TSS-CH3 (A), TSS-H (B) and TSS-COCH3 (C); “n” values are the number of ethoxylate units.

Recoveries were evaluated by spiking standard into honey, pollen and beeswax at three concentrations, each in triplicate. Spiking concentrations for each oligomer are summarized in SI Table S2. Six replicate samples were analyzed to study the precision of the final method by calculating the relative standard deviation (RSD) of each oligomer. Instrumental detection limit (IDL) was calculated according to a signal/noise ratio of 3, by diluting the standard solutions with acetonitrile. MDL was obtained by spiking standards into blank matrices and using a signal/noise ratio of 3. Blank honey and pollen were produced in our control bee hives and were considered as “clean”. Blank beeswax was obtained from Kenya, where pesticides are seldom used around beehive and foraging areas.

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RESULTS AND DISCUSSION Chemical Structures of Trisiloxane Surfactants Identified from Popular Spray Adjuvants. Commercially available organosilicone adjuvants are usually mixtures of trisiloxanes and their synthetic percursors (polyethylene glycol and its derivatives) and byproducts.17 Exact compositions are trade-secrets and generally undisclosed by manufacturers. Based on pounds of usage on California almonds (primary global site for honey bee pollination) compiled from the California Pesticide Information Portal,21 top used organosilicone surfactant adjuvants (Dyne-Amic, Kinetic HV, Silkin, Silwet L-77, Sylgard 309, and Syl-Tac) were selected and analyzed by LC-MS. 9319

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Figure 3. LC-MS detection of TSS-CH3 oligomers in solvent, peak values 2−13 represent the number of 2−13 EO (A); TICs and SIMs of 4−7 EO of TSS-CH3 oligomers (total 100 ng/g) spiked in honey (B), pollen (C), and beeswax (D), TIC represents total ion chromatogram; and the detection of TSS-CH3 oligomers (4−7 EO) in a real beeswax no. 2 (E).

For each adjuvant, mass ions with m/z 44 duplicate units were frequently observed, and assigned to the polyethoxylate chain. The most frequent oligomers containing a polyethoxylate chain found in our samples were nonylphenol ethoxylates, octylphenol ethoxylates, trisiloxane surfactants, and polyethylene glycols and their derivatives. Polyethylene glycols and primarily their monomethyl ethers seen here had relatively low starting molecular weights (e.g., m/z 212 + 44n and m/z 226 + 44n, respectively for glycols and methyl ethers as ammonium adducts), and eluted at the beginning of LC gradient. Trisiloxane surfactants were distinguished from other nonionic surfactants mainly based on the isotopic abundance ratio of I[M+2]/I[M]. Due to three silicon atoms existing in trisiloxane surfactants, its I[M+2]/I[M] ratio are >10%, whereas I[M+2]/I[M] ratio of compounds without silicon atom are about 4%. Details of the identification will be discussed elsewhere. As a result, the three most frequently detected trisiloxane compounds in the analyzed adjuvants were identified as TSSCH3, TSS-COCH3, and TSS-H (Figure 1, where w = 0, x = 1, m = 0; R = methyl, acetyl and hydroxyl, respectively). The structure identifications were validated by 1H NMR and HMQC spectra (J. Chen et al., publication in preparation). Quantitative determinations were focused on these three dominant trisiloxane surfactants. Distributions of Trisiloxane Oligomers. Since pure trisiloxane surfactant standards were not commercially available, TSS-CH3, TSS-H, and TSS-COCH3 were purified on SPE cartridges to remove synthetic precursors (polyethylene glycols and derivatives) and their byproducts. According to the LC-MS analysis, purities of the fractions are more than 96.9% for TSSCH 3 , 95.0% for TSS-H and 97.6% for TSS-COCH 3 respectively, and qualified as standards for subsequent quantitative analysis. Representative oligomer distributions of the purified standards are shown in Figure 2. Oligomers ranged from 2 to 13 EO groups in TSS-CH3 (Figure 1, w = 0, x = 1, m = 0, R = methyl, n = 2−13), 3−13 EO groups in TSS-COCH3

(Figure 1, w = 0, x = 1, m = 0, R = acetyl, n = 3−13), and 4−13 EO groups in TSS-H (Figure 1, w = 0, x = 1, m = 0, R = hydroxyl, n = 4−13). Method Performance. Generally, hydrogen adducts ([M +H]+) of trisiloxane surfactants are not readily detected by ESIMS methods. 17 However, the polyethoxylate chain in trisiloxane surfactants can trap a metal ion, and form a complex such as the [M+NH4]+, [M+Na]+, and [M+K]+ adducts.22 Our experimental results (data not shown) were consistent with Bonnington et al.’s work,17 where in the absence of any salt added in the mobile phase, both [M+Na]+ and [M+K]+ adducts were observed, and no [M+H]+ were detected. Besides, [M+NH4]+ adducts were also detected due to traces of NH4+ ion residues in the LC system. Oligomers with longer polyethoxylate chains show an increased ratio of [M+K]+ adducts, and this was also consistent with results from various investigations.17,23 To simplify the mass spectra and acquire better quantitative results, 2 mM ammonium formate buffer was added to the mobile phase to suppress [M+Na]+, and [M +K]+, and force the [M+NH4]+ to prevail in the spectra. Therefore, all the mass ions of trisiloxane surfactant oligomers monitored were ammonium adducts (Table 1). Through reversed-phase chromatography, trisiloxane surfactants were separated based on their end-capping functional group. The retention times of TSS-H oligomers ranged from 12.8 to 13.0 min, TSS-CH3 ranged from 14.8 to 16.7 min, and TSS-COCH3 ranged from 15.1−16.6 min. For trisiloxane surfactants, the polyethoxylate chain serves as the hydrophilic moiety and the polysiloxane as the lipophilic headgroup (Figure 1). Figure 3A shows that TSS-CH3 oligomers with a longer polyethoxylate chain eluted sooner in LC-MS. Total ion chromatograms of TSS-CH3 oligomers spiked in honey, pollen and beeswax are displayed in Figure 3B−D. Pollen represents the most complex matrix, because of its high content of protein (peptides) and phenolics which may cause interferences with the oligomers after sample extraction. The high content of polar carbohydrates in honey and very nonpolar lipids in beeswax are 9320

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Table 2. Extraction Recoveries (Mean %) of Trisiloxane Surfactants from Spiked Honey, Pollen and Beeswax Samplesa TSS-CH3

a

TSS-COCH3

TSS-H

Oligomer (EO)

honey

pollen

beeswax

honey

pollen

beeswax

honey

pollen

beeswax

2 3 4 5 6 7 8 9 10 11 12 13

94 90 89 88 86 88 86 85 85 84 83 89

95 100 101 105 103 106 104 105 104 104 104 98

103 107 100 108 108 106 109 106 108 100 102 101

74 75 74 76 76 76 74 76 74 74 70

91 101 96 102 106 101 103 98 96 100 100

100 99 104 101 102 105 99 103 97 97 93

80 80 84 78 78 78 79 79 77 77

90 95 95 94 100 98 99 94 88 86

92 102 97 101 100 101 100 102 103 104

recoveries were average values from three spiking concentration, please refer to SI Table S3 for detailed information.

ng/g in honey, 0.63, 0.81, 0.78 ng/g in pollen, and 0.51, 0.69, 0.63 ng/g in beeswax. Based on data of recovery, precision, IDL and MDL, this QuEChERS method demonstrated adequate sensitivity, selectivity, and stability in extracting trisiloxane surfactants from beehive matrices, including honey, pollen, and beeswax. An advantage lies in its versatility to analyze the trisiloxane surfactant inerts along with co-occurring active ingredients remaining as pesticide formulation residues within hives. With a MS/MS operating under multiple reactions monitoring (MRM), the selectivity would be even better. However, the sensitivity described in our work is adequate due to the high and frequent residues of these pollutants within the beehive samples (see below). Sample Analysis of Trisiloxane Surfactants. To validate the method, 5 honey, 10 pollen, and 10 beeswax samples collected from seven U.S. states were analyzed with regard to TSS-CH3 (2−13 EO), TSS-H (4−13 EO), and TSS-COCH3 (3−13 EO) residues. These 25 samples were unrelated and randomly named as honey nos. 1−5, pollen nos. 1−10, and beeswax nos. 1−10. SIMs of TSS-CH3 (4−7 EO) of beeswax no. 2 are shown in Figure 3E. The concentration of each oligomer in these beehive samples are presented in SI Table S4, and summarized in Table 3. Among honey, pollen, and beeswax samples, beeswax was the most trisiloxane surfactant polluted and honey was the cleanest matrix. Every beeswax sample had detectable trisiloxane surfactants (TSS-CH3, TSS-COCH3, or TSS-H) with total concentrations up to 390 ng/g, and 6 out of 10 pollen samples had trisiloxane surfactants with total concentrations up to 39 ng/g (Table 3). No trisiloxane surfactant was found in honey samples. Additionally, the average total trisiloxane surfactant concentration was 116 ng/g in beeswax and 18 ng/g in pollen (Table 3). Trisiloxane surfactants may accumulate and persist from year to year in the lipophilic beeswax, and represent the highest residue in beehive matrices. Based on the frequently detected and high residues of trisiloxane surfactants, and relatively less complex background interferences by QuEChERS extraction, beeswax is considered to be the best beehive matrix to study trisiloxane surfactant residues, compared to honey and pollen. None of the five honey samples had detectable trisiloxane surfactants above the MDL, and in contrast to pollen and beeswax, was the cleanest matrix. Honey contains significant quantities of minor compounds including polyphenols and

largely removed during the sample preparation. Using selective ion monitoring (SIM) detection, oligomers from all three trisiloxane surfactants were clearly separated from the matrix background. Unlike the very highly lipophilic nonpolyethoxylate linear and cyclic methyl siloxanes,24−26 trisiloxane surfactants are both water- and lipid-soluble. Thus, the three trisiloxane surfactants (TSS-CH3, TSS-H, and TSS-COCH3) were readily extracted with the QuEChERS method,18,19 which is popularly used in pesticide analysis. Extraction recoveries of trisiloxane oligomers from honey, pollen and beeswax were evaluated at three spiked concentrations. At all three concentrations, the recoveries were between 78 and 111% of TSS-CH3 oligomers (2−13 EO), 70− 108% of TSS-H oligomers (4−13 EO), and 66−112% of TSSCOCH3 oligomers (3−13 EO). Average recovery data are shown in Table 2, and detailed recovery data for each oligomer is displayed in SI Table S3. For each spiking concentration, three replicate samples were prepared and the standard deviations (SD) are also shown in SI Table S3. Compared to pollen and beeswax, extraction efficiency was slightly lower in honey because of the much higher water content of honey. The amount of magnesium sulfate may be increased to improve extraction recovery. However, the recovery values from honey were acceptable (Table 2). The reliability of the extraction method was confirmed through analysis of six parallel samples for each matrix, where the %RSD of all trisiloxane oligomers were below 9.9% in honey, pollen and beeswax. Here 2 g honey, 1 g pollen, or 1 g beeswax samples were sufficient for analysis, which is reduced from the 15 g resorted to in the original QuEChERS method for pesticide analysis in fruits and vegetables.18,19 This allowed analysis of less available pollen samples (especially the beebread samples), which are often difficult to obtain in 15 g quantities. Using QuEChERS method for the sample preparation, both pesticide and trisiloxane surfactants can be extracted simultaneously. The IDLs were 0.11 ng/g for TSS-CH3 (12 EO), 0.27 ng/g for TSSH (12 EO), and 0.15 ng/g for TSS-COCH3 (12 EO) based on analyzing the respective standard. To assess matrix effects, MDLs were calculated by spiking standards into matrix blanks, and the MDLs for TSS-CH3 (12 EO) were 0.23, 0.28, 0.23 ng/ g in honey, pollen, and wax. These MDL values are close to instrumental IDL values. MDL values may vary depending on the length of polyethoxylate chain. Average MDLs for TSSCH3, TSS-COCH3, and TSS-H oligomers were 0.53, 0.60, 0.56 9321

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for honey bees.29−33 However, four different organosilicone adjuvants (Dyne-Amic, Syl-Tac, Silwet L-77, and Sylgard 309) were tested for learning impairment in honey bees using the proboscis extension reflex (PER) assay in our previous work.13 The major components of these adjuvants were found to be TSS-CH3, TSS-COCH3, and TSS-H. All the above tank adjuvants strongly reduced honey bee ability to undergo PER in response to a cinnamon odor after 20 μg ingestion, which could lead to inadequate bee foraging and colony collapse. In this study, trisiloxane surfactants were found in several samples with concentration as high as 390 ng/g in beeswax and 22 ng/g in pollen. The presence of trisiloxane surfactants in beehives results from pesticide applications to agricultural fields where honey bees collect nectars and pollens. The beeswax comb is where honey bees are reared and their pollen and honey foods stored. High amounts of trisiloxane surfactants in pollen and beeswax have significant potential hazard to honey bee health. In this study, a LC-MS analytical method has been developed for determination of trisiloxane surfactants, including TSS-CH3, TSS-H, and TSS-COCH3, in beehive related matrices, and the method is proved simple, rapid, and sensitive. By applying the method to 25 beehive samples, beeswax was found the most highly polluted and the most suitable matrix for future trisiloxane surfactant residue study. The high level residue of trisiloxane surfactants in North American beeswax samples calls for attention to impacts of these trisiloxane surfactant compounds on bee health and the ongoing global bee decline. Future work with the described method will include study of the environmental fate of trisiloxane surfactants in and around beehives.

Table 3. Summed Trisiloxane Surfactant Concentrations (ng/g) in Pollen and Beeswax Samples (Refer to SI Table S4) sample

TSS-CH3

TSS-COCH3

TSS-H

sum

beeswax no. 1 beeswax no. 2 beeswax no. 3 beeswax no. 4 beeswax no. 5 beeswax no. 6 beeswax no. 7 beeswax no. 8 beeswax no. 9 beeswax no. 10 pollen no. 1 pollen no. 2 pollen no. 3 pollen no. 4 pollen no. 5 pollen no. 6 pollen no. 7 pollen no. 8 pollen no. 9 pollen no. 10

129 59

210

14

352 59 60 153 390 12 22 16 37 57

60 153

11

390 12 11 16

23

9 18 19 11 22 8

14 47

9

20

9 18 39 11 22 8

flavonoids, enzymes, organic acids, Maillard reaction products, furanoic aldehydes and acids, amino acids, minerals, and watersoluble vitamins,27 and honey is an acidic matrix.28 The silicon−oxygen bonds in the backbone of trisiloxane are susceptible to hydrolytic cleavage under acidic or alkaline conditions.15 We hypothesize that trisiloxane surfactants may undergo significant hydrolysis in honey. In comparing the three kinds of trisiloxane surfactants, TSSCH3 was the most frequently found, detected in 50% of beeswax samples and in 60% of pollens with “mean concentrations” at 75 ng/g in beeswax and 14 ng/g in pollen. Though TSS-COCH3 was only detected in beeswax, its residue was up to 390 ng/g and constituted the highest concentration observed. Only 10% of pollens and 30% of beeswax samples had detectable TSS-H residues, and the mean concentrations were also relatively low. These results may reflect the relative amounts of different trisiloxane surfactants applied in agrochemical spray formulations. Much more TSS-CH3 and TSS-COCH3 were used than TSS-H on almond orchards, based on the LC-MS analysis of top used organosilicone adjuvants compiled from the CalPIP database21 (data not shown). As trisiloxane surfactants are used both as spray tank adjuvants and in agrochemical formulations, honey bees may be exposed to both active ingredients (pesticides) and trisiloxane surfactants when foraging. For some of our pollen and beeswax samples, over 176 kinds of pesticides or their metabolites were also analyzed.7 However, total pesticide concentration was not found correlated with total trisiloxane surfactant concentration (data not shown). This may be possibly due to contrasting degradation dynamics between pesticides and trisiloxane surfactants in beehives, or that use patterns of these spray tank adjuvants and specific pesticide formulations around beehives greatly differ. To date, most research involving impacts of pesticides on pollinators has been with active ingredient (especially the neonicotinoid) residues in bee eco-systems and their toxicities

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ASSOCIATED CONTENT

S Supporting Information *

Normalized concentrations for each trisiloxane oligomer used in linearity and recovery experiments are displayed in supplementary Tables S1 and S2. Detailed recovery values of each oligomer are displayed in supplementary Table S3. TSSCH3, TSS-COCH3 and TSS-H concentrations of each oligomer in beehive samples are presented in supplementary Table S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (814) 865-2435; fax (814) 865-3048; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Maryann T. Frazier, Stephanie E. Mellott, and Sara A. Ashcraft for providing the beehive samples used here and to the USDA-NIFA-AFRI Foundational Award program (no. 2011-67013-30137) for funding this work.

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REFERENCES

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