Determination of Pesticide Co-Formulants and Adjuvants in Honey Bee Related Matrices by LC-ESI-MS

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1 NACRW-FPRW St. Pete Beach, FL July 22, 2014 Determination of Pesticide Co-Formulants and Adjuvants in Honey Bee Related Matrices by LC-ESI-MS Chris Mullin, Jing Chen, Julia Fine, Maryann Frazier, and Jim Frazier Department of Entomology, Penn State University, University Park, PA Julia Stephanie Jim Sara Chris Dan Ryan Jing Wanyi Tim Maryann

2 Understanding the Total Chemical Burden on Pollinators is Needed Co-Formulants, Adjuvants and their Degradates Veterinary Medicines Antibiotics/ Miticides Pesticide Active Ingredients and their Degradates Natural Toxicants in Food and Water Volatiles and Persistent Organic Pollutants Total Chemical Load or Body Burden Major Health Stressor Endogenous Toxicants Hormones/ Effectors/Defenses Detoxification Immunoresponse Disease Endocrine Disruption Behavioral Impairments Photo by M. T. Frazier

3 Honey Bee Products Are An Excellent Indicator of Environmental Pesticides 1-Methyl pyrrolidinone 1-Naphthol (carbaryl) 3-Hydroxycarbofuran 4,4-DiBrbenzophenone Acephate Acetamiprid Aldicarb sulfone Aldicarb sulfoxide Allethrin Amicarbazone Atrazine Azinphos methyl Azoxystrobin Bendiocarb Bifenazate Bifenthrin Boscalid Captan Carbaryl Carbendazim (benomyl) Carbofuran Carbofuran, 3-hydroxy Carfentrazone ethyl Chlorfenapyr Chlorfenvinphos Chlorferone (coumaphos) Chlorothalonil Chlorpyrifos Chlorpyrifos methyl Clothianidin Coumaphos Coumaphos oxon Cyfluthrin Cyhalothrin Cypermethrin Cyphenothrin Cyprodinil DDD-p,p' DDE-p,p' DDT-p,p' Deltamethrin Diazinon Dichlorobenzene-para Dicofol Dieldrin Difenoconazole Diflubenzuron Dimethomorph Diphenamid Diphenylamine DMA (amitraz) DMPF (amitraz) Endosulfan I Endosulfan II Endosulfan sulfate Esfenvalerate Ethion Ethofumesate Etoxazole Famoxadone Fenamidone Fenbuconazole Fenhexamid Fenoxaprop-ethyl Fenpropathrin Fenpyroximate Fipronil Flonicamid Fluoxastrobin Fluridone Flutolanil Fluvalinate Heptachlor Heptachlor epoxide Hexachlorobenzene Hexythiazox Hydroxychlorothalonil Imidacloprid Imidacloprid olefin Imidacloprid, 5-hydroxy Imidacloprid urea Indoxacarb Iprodione Lindane Malathion Metalaxyl Methamidophos Methidathion Methomyl Methoxyfenozide Metolachlor Metribuzin MGK-326 Myclobutanil Norflurazon Oxamyl Oxyfluorfen Parathion methyl Pendimethalin Permethrin Phenothrin Phosalone Phosmet Piperonyl butoxide Pirimiphos methyl Potasan (coumaphos) Prallethrin Pronamide Propanil Propiconazole Pyraclostrobin Pyrethrins Pyridaben Pyrimethanil Pyriproxyfen Quintozene = PCNB Sethoxydim Simazine Spirodiclofen Spiromesifen Sulfoxaflor Tebuconazole Tebufenozide Tebuthiuron Tefluthrin Tetradifon Tetramethrin Thiabendazole Thiacloprid Thiamethoxam THPI (captan) Thymol Triadimefon Tribufos = DEF Trifloxystrobin Trifluralin Triticonazole Vinclozolin

4 Multiresidue Pesticide Analysis on US Beehive Samples Up to 39 different pesticides per sample, 6+ average Only 10% of over 1500 samples incl. controls lacked detections Found 138 different pesticides and metabolites 18 organophosphates, 16 pyrethroids, 10 carbamates, 9 neonicotinoids, 7 chlorinated cyclodienes, 5 organochlorines, 4 insect growth regulators, 32 fungicides, 17 herbicides, 3 synergists, 2 formamidines, 15 misc. No individual pesticide amount correlates with recent bee declines Are generic formulation inerts that co-occur across classes of pesticide involved? Frazier, M. et al Amer. Bee J. 148(6): Mullin et al PLoS ONE 5(4): e9754.

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6 Common Formulation Terms Adjuvant, Inert or Co-formulant - an additive (2,000+) used to enhance the performance or aid the stability of formulations (20,000+) of active ingredients (< 1000) Examples - surfactant, penetrant enhancer, activator, spreader, sticker, wetting agent, buffer, antifoaming agent, drift retardant, etc. Formulation ingredients Spray tank adjuvants h"p:// work- cask- tractor- sprays- a- chemical- treatment- in- the- orchard- of- peach- with- pink- flowers.html

7 Problem of Non-Disclosure of Formulation Ingredients Roundup WeatherMAX MSDS Info Typical Component CAS No. % by weight Potassium salt of glyphosate Other ingredients Trade secret 51.2 Landmaster II Herbicide MSDS Info More Recent Component CAS No. % by weight Isopropylamine salt of glyphosate Isopropylamine salt of 2,4-D Ethoxylated tallowamine Trade secret N-Methyl-2-pyrrolidone Water

8 Problem of Non-Disclosed Information on Formulation Chemicals CAS Number Organosilicone Polyethoxylate Surfactants* References & Patents Sources Chemical Structure EPA Regulated HPV (lb/yr) Yes Yes withheld Yes Yes withheld Yes Yes 41, No Yes 26, No Yes No No Yes 263, No Yes No No Yes No No Yes No Yes Yes No Yes Yes No No No No Yes Yes No Tallow & Fatty Amine Polyethoxylate Surfactants* CAS Number References & Patents Sources Chemical Structure EPA Regulated HPV (lb/yr) Yes Yes 10,000, Yes Yes No Yes Yes No Yes Yes withheld Yes Yes No Yes No No No Yes 6,413, No Yes withheld No Yes 25, No Yes 10,000, No Yes 50,000, No Yes 10,000, No Yes 1,999,276 Plus 43 other CAS identified chemicals; others? No Plus 32 other CAS identified chemicals; others? No *From SciFinder Scholar (CAS) and EPA Chemical Data Reporting *From SciFinder Scholar (CAS) and EPA Chemical Data Reporting

9 US PESTICIDE ACTIVE RELATIVE TO INERT INGREDIENTS: NATIONAL PRODUCTION AND USE PATTERNS U.S. Production (thousands of lbs.)* Chemical Class Organosilicones (13 of > 560 chemicals) inert - defoaming > 496,080 Nonylphenol polyethoxylates # inert - surfactant 50, ,000 Glyphosate herbicide 250,000 Nonylphenols inert - surfactant 220,000 1-Methyl-2-pyrrolidinone (NMP) inert - solvent 184,703 Tallow amine and similar polyethoxylates inert - surfactant > 88,000 Organosilicone surfactants (> 43 chemicals) inert - surfactant withheld Atrazine herbicide 67,000 2,4-D herbicide 35,000 Chlorothalonil fungicide 10,300 Mancozeb, Ziram, Maneb, Metiram, Thiram fungicide - dithiocarbamates 9,600 Chlorpyrifos insecticide - organophosphate 7,000 Pyraclo-,Azoxy-,Trifloxy-,Fluoxa- strobins fungicide 5,800 Captan fungicide 3,400 Clothianidin insecticide - neonicotinoid 1,800 Imidacloprid insecticide - neonicotinoid 1,700 Bifenthrin insecticide - pyrethroid 830 Cyhalothrin insecticide - pyrethroid 650 Fluvalinate-tau insecticide - pyrethroid 0.5 *Soure: US EPA Chemical Data Reporting (2014, primarily 2010 production) or USGS-NAWQA Pesticide Use Maps (2011) #US EPA estimated total use, internal report 2007; cf. 857,000,000 lbs conventional pesticides

10 Adult Honey Bee Toxicity of Taktic vs Amitraz LD50 = 28 µg/bee Taktic Amitraz Linear (Taktic) Linear (Amitraz) Probit LD50 > 103 µg/bee Formulation 4X More Toxic than Amitraz Log Dose 48 hr oral toxicity of Taktic or its active ingredient amitraz fed in 50% sucrose to adult honey bees Ashcraft et al Bee Culture 140(11):

11 Spray Adjuvant Classes Surfactant activity - extent to which a surfactant (soap, detergent, emulsifier) reduces surface tension of water LOW Crop Oil Concentrates Nonionic Surfactants HIGH Organosilicone Surfactants Research QuesFons Are spray adjuvants harmful to honey bees? If so, is one class more harmful than others?

12 Adjuvant Summary - PER Test 4/4 Organosilicones impair learning 1/3 Nonionics 0/3 Crop Oil Conconcentrates 20 μg per bee Impairment occurs during acquisition Must be ingested Movement to CNS? Learning Impairment Surfactant Activity Pesticide spray adjuvants interfere with the learning abilities of honey bees Ciarlo et al PLoS ONE 7(7) e40848.

13 LC-MS of Pesticide Formulations UV Absorbance Chromatograms NMP NMP A.I. A.I. POEs Formulation A Organosilicones Positive Ion Chromatograms Formulation B

14 LC-ESI-MS of 250 ppm Silwet L-77 on Shimadzu LCMS-2020 Positive Ion Chromatogram using Shim-Pak XR-ODS 2 x 100 mm column Organosilicone Surfactants Used on CA Almonds in 2009* Adjuvant Lbs Applied DYNE-AMIC SYL-TAC PHASE SYLGARD KINETIC BREAK-THRU HI-WETT FREEWAY SILWET L *CA Dept. of Pesticide Regulation J. Chen & Mullin Env. Sci. Tech. es Organosilicone surfactants presently regulated based on toxicology of classical inert silicones, although their mode of action, adsorption and penetration capacity are very different.!

15 LC-MS Analysis of Trisiloxane Surfactants in Beehives Chen, J. & C. A. Mullin Environ. Sci. Technol. 47: honey pollen beeswax sample wax 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).

16 Polyethoxylate Surfactant Composition of Top Used Organosilicone Adjuvants on California Almonds in 2009 NP(EO) n +OP(EO) n Jing Chen (2012)

17 LC-MS Analysis of Alkylphenol Ethoxylates in Beehives Chen, J. & C. A. Mullin Food Chem. 158: Total ion chromatograms of NP(EO) n and OP(EO) n spiked in solvent (A), honey (B), pollen (C) and wax (D). I.S. = internal standard.

18 Monitored Ions for Major Ethoxylate Surfactants in Beehives Monitored Ammonium Adducts for Trisiloxane Surfactants oligomer TSS- TSS-CO TSS-H m/z [M+NH 4 ] + 2 EO m/z [M+NH 4 ] + 3 EO m/z [M+NH 4 ] + 4 EO EO EO EO EO EO EO EO EO Monitored Mass Ions for NP(EO) 3-13 and OP(EO) 3-13 oligomer m/z [M+NH 4 ] + oligomer m/z [M+NH 4 ] + NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) NP(EO) OP(EO) EO Exact mass used; Si m+2/m isotope abundance ratio diagnostic Chen, J. & C. A. Mullin Environ. Sci. Technol. 47: Exact mass used Chen, J. & C. A. Mullin Food Chem. 158:

19 Total Concentrations (ppb) of Trisiloxane Capped with Methyl (R= ), Hydroxyl (R=H), Acetyl (R=C(O) ), and of NP(EO) n and OP(EO) n in Pollens, Waxes and Honeys H 3 C Si O Si O Si O Si w C 3 H 6 O x w=0-1 x=1-2 n=0-15 m=1-3 R=, OH, CC(O) C 2 H 4 O C 3 H 6 O R n m C m H 2m+1 O n OH m=9 nonylphenol polyethoxylate (NP(EO) n ) m=8 octylphenol polyethoxylate (OP(EO) n ) J. Chen & Mullin Env. Sci. Tech. es

20 Field Relevant Concentrations H 3 C Si O Si O Si O Si w C 3 H 6 O x w=0-1 x=1-2 n=0-15 m=1-3 R=, OH, CC(O) C 2 H 4 O C 3 H 6 O R n m Survival rates of adult honey bees fed on 10 ppm TSS-CO, TSS- or TSS-H mixed in their sucrose/water (w/w, 50/50) diet for 10 days. J. Chen & C. Mullin. ACS Poster Estimated Environmental Concentrations (EPA or EFSA Tier 1 models) in pollen or nectar after spraying 1% (10,000 ppm) of organosilicone surfactant tank adjuvant = ppm (worst case?) D. Fischer. PSU Pollinator Conf

21 Correlation Between Total Nonylphenol Ethoxylates and Total Pesticide Residues Found in Beehive Samples Spearman s test, r = 0.588, p = Relationship between total NP(EO) concentration (sum of NP(EO) 3-13 ) and total pesticide residues found in beehive samples. Pesticide residue results are from (Mullin, et al., 2010). Chen, J. & C. A. Mullin Environ. Sci. Technol. 47:

22 NMP Toxic to Bee Larvae When Fed in Royal Jelly Diet at Field Rates! 250 ppb NMP Me N O W. Zhu et al PLoS ONE e µg residue detected by LC-MS in surface wash of single apple bud 8 hours after insecticide formulation sprayed at pink stage. J. Fine, 2014; unpubl.

23 Some Pesticide Formulations that Disclose in MSDS the Percentage of Solvent NMP Formulation # Registrant Active Ingredient(s) % NMP Acatak Pour-On Tick Inhibitor Novartis fluazuron Advantix for Dogs Bayer imidacloprid, permethrin Agri-Mek or Ardent 0.15EC Syngenta abamectin 30 Alto 100 SL Fungicide Syngenta cyproconazole 10 Assert Herbicide Nufarm imazamethabenz methyl ester 49 Assure II Herbicide DuPont quizalofop-p-ethyl < 8 Baycor 300, Turf Fungicide Bayer bitertanol 54 Bayfidan 250 EC Fungicide Bayer triadimenol 60 Campaign Herbicide Monsanto isopropylamine glyphosate, 2,4-D 7 Confidor SL 200 Bayer imidacloprid 38 Confidor Supra Bayer imidacloprid, cyfluthrin 87 Crown 225SL Scotts acetamiprid 30 Diamond 0.83EC Makhteshim Agan novaluron Goal 2XL EC Herbicide Dow oxyflurofen 10 Ortho Systemic Insect Killer Ortho acephate < 25 Saprol Fungicide BASF triforine 24 Score Foliar Fungicide Syngenta difenoconazole Sphere Fungicide Bayer cyproconazole, trifloxystrobin 55 Temprano Chemtura abamectin Tilt Xtra Fungicide Syngenta cyproconazole, propiconazole < 20 # Insecticide or miticide unless noted otherwise. N-methyl-2-pyrrolidone; application rate of pesticides range from 0.05 to 100% of formulation diluted in water with an 1% average per product, and rates increasing from insecticide > fungicide > herbicide.

24 Modern Agrochemical Formulants and Bee Ecotoxicity Bulk of synthetic organic chemicals used and released into US environments are formulation ingredients generally recognized as safe, having no mandated tolerances, and whose residues remain unmonitored. Multi-billion pounds of these inerts overwhelm the total chemical burden from the active pesticide, drug and personal care ingredients with which they are formulated. Among these co-formulants are surfactants and solvents of known high ecotoxicity to fish, amphibians, honey bees and other non-target organisms. Their overall role in pollinator decline remains to be determined. Problem of non-disclosure and claiming formulant as a proprietary trade secret, even if it s a high production volume chemical: If you do not know what is used, you will never look for it, and if you do not look for it, you will never find it!

25 THANK YOU COLLABORATORS David J. Biddinger PSU Fruit and Extension Labs, Biglerville, PA Diana L. Cox-Foster PSU Department of Entomology, University Park, PA Jeff Pettis USDA Bee Research Lab, Beltsville, MD Roger Simonds USDA-AMS National Science Lab., Gastonia, NC Dennis vanengelsdorp University of Maryland, College Park, MD FUNDING SOURCES USDA-NIFA Foundational Awards and CAP programs National Honey Board College of Agricultural Sciences, Penn State Haagen-Dazs, Jasper Wyman & Sons, and other Private Donors NC1173 Sustainable Solutions to Problems Affecting Honey Bee Health Project Apis mellifera The Foundation for the Preservation of Honey Bees Beekeeping Associations (Florida State and Tampa Bay)