Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
HONEY BEE TOXIC INTERACTIONS WITH FORMULATION "INERTS" AND PESTICIDE RESIDUES FREQUENTLY FOUND IN U.S. APIARIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0224252
Grant No.
2011-67013-30137
Cumulative Award Amt.
$297,000.00
Proposal No.
2010-03705
Multistate No.
(N/A)
Project Start Date
Mar 1, 2011
Project End Date
Feb 28, 2015
Grant Year
2011
Program Code
[A1111]- Plant Health and Production and Plant Products: Insects and Nematodes
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Entomology
Non Technical Summary
No single stress factor has been identified as the causal agent for honey bee Colony Collapse Disorder (CCD). This work will address if another major crop pollutant that co-occurs across classes of pesticides, 'inerts,' may alter toxicity of frequently found pesticides in honey bee foods, and indicate if new pesticide registration requirements should be implemented to insure pollinator safety in agricultural landscapes. Agrochemical formulations usually contain inerts at higher amounts than the active ingredients, and these penetrating enhancers, surfactants and adjuvants have not been evaluated in regard to bee exposures and toxicities. Inerts often enhance the pesticidal efficacy as well as inadvertent non-target effects of the active ingredient after application. Given the synergistic nature of certain chemicals, we are concerned that active ingredients may effect honey bees differently depending upon the inert ingredients in a particular product formulation or in certain combinations found in field use patterns. The fact that some inert ingredients may indeed be more toxic than active ingredients such as in formulations of the herbicide glyphosate makes this issue especially important for the health of honey bees and other pollinators. We have found unprecedented levels of miticides and agricultural pesticides in honey bee colonies from across the US and one Canadian province. While these samples were not part of a full-scale landscape or grower-level survey, the data contained here is the largest sampling of pesticide residues in N. American bee colonies or worldwide to date. With an average of 7 pesticides in a pollen sample, the potential for multiple pesticide interactions affecting bee health seems likely. While exposure to many of these neurotoxicants elicits acute and sublethal reductions in honey bee fitness, the enhancing effects of these active ingredients in combination with 'inerts' and their direct association with CCD or declining bee health remains to be determined. There is a new tendency to market pesticide blends including seed treatments that contain multiple classes of insecticides or fungicides, or their mixed combinations. These blends usually require proprietary adjuvants to achieve high efficacy and broadly control many pests. The impact of these potentially synergistic blends on non-targets including bees cannot be fully understood without knowing the identity of proprietary inerts. To address if these major crop pollutants alter toxicity of frequently found pesticides in honey bee foods, we will identify and quantify key formulation ingredients in the hive, test their short- and long-term toxic effects on brood and adult honey bees in lab and field, and communicate the results of our study to US beekeepers, growers, extension specialists and other researchers. Inert ingredients which are potentially hazardous need to be redefined by EPA in terms other than inert. For the sustainability of honey bees, other pollinators and beneficial insects, it is vitally important that these inert compounds be disclosed and their effects investigated.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113099113010%
2113099115010%
2113099102010%
2115220113015%
2115220115015%
2115220102010%
2112410113010%
2112410115010%
2112410102010%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3099 - Bees, honey, and other pollinators, general; 2410 - Cross-commodity research--multiple crops; 5220 - Pesticides;

Field Of Science
1130 - Entomology and acarology; 1020 - Physiology; 1150 - Toxicology;
Goals / Objectives
We have found 121 different pesticides and metabolites up to 214 ppm within 887 wax, pollen, honey bee and associated hive samples. No individual pesticide level correlates with recent bee declines. High numbers of active ingredients and great qualitative and quantitative diversity of residues suggest that more generic formulation 'inerts' that co-occur across classes of pesticides may be involved. Formulations usually contain inerts at higher amounts than active ingredients, and these penetrating enhancers, surfactants and adjuvants can be more toxic on non-targets than the active ingredients. Impacts of 'inerts' in pollen and nectar alone or in combination with coincident pesticide residues on honey bee survival and behavior are unknown. To test consequences of formulation inerts on bee health, we will (1) identify and quantify key formulation inerts or adjuvants and major co-occuring pesticides and metabolites in pollen, nectar, bees, brood and other hive matrices, (2) conduct oral and contact toxicity tests of identified inerts and combinations with respective pesticides on brood and adult honey bees, (3) determine chronic and sublethal effects of major pesticides and coincidental inerts on bees in lab and field and (4) disseminate the information generated by this study to US beekeepers, growers, extension specialists and other researchers. The follow outcomes are expected. Inerts common to multiple pesticide formulations will be identified and found at ppm levels in hive samples. Formulation inerts at environmental-exposure levels will significantly enhance by 2-fold or more the acute or sublethal toxicities of the active ingredient residues. Inerts alone will be orally and topically toxic to bees, but greater impacts will be found on combination with single pesticides or their blends. Higher doses of inerts will increase pesticide levels which will result in minimizing locomotor alterations, and potential disruption of tropholaxis and hygienic behaviors, comb and food choices, and potentially overall colony functioning. For the field study, inerts will increase pesticide residues and will result in a significant decrease in bee colony growth, productivity and health compared to both control groups. A timely communication of conclusive research results that provide practical treatment options will be made to those most impacted by honey bee and other pollinator decline. We anticipate that if the inerts are mitigating pesticide levels and general hive stress, recommendations can be made for formulations to be used in bee foraging areas. New operational methods for both the foundation production industry and for bee keepers, both large and small scale, will result with greater bee health being sustained. The impact of synergistic pesticidal blends on bees, which depend on plant nectars and pollens readily contaminated by toxicants, cannot be fully understood without identification and risk assessment of inert residues and their agrochemical interactions.
Project Methods
Pesticide formulations associated with active ingredients frequently detected in the hive samples and often used within the foraging arena of honey bees will be analyzed by LC-MS. Major inerts including spreaders, stickers, penetration enhancers, solubilizers, and emulsifiers in pesticide formulations will be tentatively identified by selective ion monitoring. Hive samples of bee bread, nectar and wax will be extracted and analyzed for inerts along with residues of active ingredients using a modified QuEChERS method. Identity will be confirmed by the USDA-AMS-NSL in Gastonia, NC, which routinely analyzes samples for an average of 171 pesticides and toxic metabolites per analysis using GC/MS and/or LC/MS-MS. Toxic or sublethal effects on honey bees, including interference with associative learning and immune system suppression, of pesticide and inert combinations relative to formulation controls, will be determined by direct feeding or incorporation in artificial nectar or uncontaminated pollen, or by topical application of extracts to worker bees or brood. Mortality and other toxicity symptoms as well as altered behaviors will be scored over the course of the bioassay, and regressed relative to pesticide treatment dosages. Potential metabolism of inerts and parent compounds within bioassays will be addressed through analysis over time of bee or excreted residues. Chronic feeding of bioactive formulation ingredients and combinations will also be conducted. In addition to feeding studies with adults, a series of different individual and combinations of pesticides and inerts will be incorporated into the brood food and daily observations made during larval development to assess survival, malformations, and food consumption. Resulting adult bees will be evaluated for abnormal hypopharyngeal gland development as well as associative learning. Test bees will also be fed formulation-treated and control pollen and nectar containing known amounts of inerts and active ingredients, and their behaviors recorded on a single frame bioassay chamber in the lab. Potential behavioral alterations resulting from neural disruption will be sought, including altered tropholaxis and hygienic behaviors, grouping on the comb, rate and coordination of locomotor activity, antennation of bees and substrate, and choice of treated or nontreated comb for clustering. Test bees will be fed various sublethal doses of inerts/pesticides singly and in combinations prior to proboscis extension reflex (PER) tests and the resulting impacts on associative learning task responses will be determined. Colony-level impacts of formulation ingredients will be determined in semi-field experiments for each of the three years, where two pesticides formulations and their active ingredients (alone) will be evaluated each year for longevity, precocious foraging behavior hypopharyngeal gland development and associative learning using PER. We will disseminate the information generated by this study through regional MAAREC and national Honey Bee CAP E-Extension websites in addition to appropriate professional publication and meeting venues.

Progress 03/01/11 to 02/28/15

Outputs
Target Audience: The impact of systemic pesticides, seed treatments, formulation additives, and other pesticides and their combinations on non-target species, and their role in honey bee and other pollinator health are of global consequence to food security and future crop protection strategies. Major stakeholders include the research community, USDA, EPA, PDA, pesticide regulators, agrochemical industry, growers, and beekeepers and their associated organizations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have presented and participated in numerous conferences, extension workshops, seminars and professional meetings on the potential role of pesticides and their inerts, co-formulants or adjuvants in pollinator decline, some of which follow: Fine, J., C. Mullin and J. Chen. 2014. Determination of N-methyl-2-pyrrolidone and metabolites in honey bees by LC-ESI-MS. Entomological Society of America National Meeting, Portland, OR. (Poster) Frazier, J. 2015. Dying bees: Harbingers of the end times or opportunity buzzing. American Beekeeping Federation North American Conference, Anaheim, CA. (Invited) Frazier, M. 2014. Factors impacting bee health. Pacific Northwest Treatment-Free Beekeeping Conference, Medford, OR. (Invited) Frazier, M. 2014. Nature or nurse. Eastern Apicultural Society Annual Meeting, Eastern Kentucky University, Richmond, KY. (Invited) Frazier, M. 2014. Safe haven for honey bees: Ag vs. urban environments. Marin County Beekeepers' Association, Marin County, CA. (Invited) Frazier, M. 2014. Thinking inside and outside of the [bee] box. 7th Annual University of Florida Bee College, UF Whitney Marine Lab, Saint Augustine, FL. (Invited) Frazier, J. and M. Frazier. 2014. Bringing back the bees - Penn State research on the road. Echo Lake Aquarium and Science Center, Burlington, VT. (Invited) Frazier, M., J. Frazier and C. Mullin. 2014. Toxic house: Pesticide exposure and impacts on honey bee (Apis mellifera) colonies used for commercial pollination. In: Buzz-kills: The Genomics and Ecology of Stress in Pollinators Symposium, Entomological Society of America National Meeting, Portland, OR. (Invited) Frazier, M., C. Mullin and J. Frazier. 2014. Can there be honey still for tea? NY Metro Bee Conference, New York, NY. (Invited) Mullin, C., *M. Frazier. 2014. What influences bee health? How do these factors interact? Pesticides (inerts). In: Pollinator and Pollination In-Service for Extension Educators, Penn State University, University Park, PA. (Invited) Mullin C.A. 2015. Formulation ingredients are poisonous to pollinators and are largely unstudied. DuPont Crop Protection, Stine-Haskell Research Center, Newark, DE. (Invited) Reynolds, R. and M. Frazier. 2014. Pesticide conundrum: Pesticides, neonicotinoids and pollinators. Western PA Fall Greenhouse Meeting, Wexford, PA. Nov. 6, 2014. (Invited) Reynolds, R. and M. Frazier. 2015. Pollinators and pesticides. Mid-Atlantic Fruit and Vegetable Convention, Hershey, PA. Jan. 27, 2015. (Invited) How have the results been disseminated to communities of interest? We have delivered numerous presentations on the potential role of pesticides and co-formulants in pollinator decline at local, state, regional and national beekeeping conferences, professional, industrial and governmental meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Honey bees are sensitive to widespread co-formulants used in agrochemicals, and evaluation of the role of these 'inerts' in pollinator decline has only begun. Effects include learning impairment for adult bees and oral toxicity for larvae and adults. Billions of pounds of formulation ingredients from all uses are released into US environments, making this an important component of the chemical landscape to which bees are exposed. Most inerts are generally recognized as safe, have no mandated tolerances, and their residues are unmonitored. Lack of disclosure of co-formulants in major products, and no or inadequate methods developed for their analysis, prevents the risk evaluation of total agrochemical load for bees. Most studies to document pesticide effects on terrestrial non-targets like honey bee are performed without the formulation or other relevant spray adjuvant components used to environmentally apply the toxicant. Inerts have been found at ppm levels in hive samples. Formulation inerts at environmental-exposure levels significantly enhance the acute toxicities of the active ingredient residues. Inerts alone are orally and topically toxic to bees, but greater impacts are found on combination with single pesticides or their blends. We have found that honeybees are 'poisoned' by widespread co-formulants used in agrochemicals, particularly organosilicone surfactants and the co-solvent N-methyl-2-pyrrolidone (NMP). We have found 100% of co-formulants analyzed for in beehive samples, while only 70% of pesticide active ingredients searched for have been detected. Formulations are generally more toxic than respective active ingredients, particularly fungicides, by up to 26,000-fold based on published literature. By employing this knowledge, we can begin to optimize formulation recommendations for use in bee foraging areas. Honey bees reveal that the formulation and not just the dose makes the poison. Why most formulations are more toxic to bees than respective active ingredients and how pesticides interact to causepollinator decline cannot be answered without understanding the prevailing environmental chemical background to which bees are exposed. Modern pesticide formulations and seed treatments, particularly when multiple active ingredients are blended, require proprietary adjuvants and inert ingredients to achieve high efficacy for targeted pests. Although we have found over 130 different pesticides and metabolites in beehive samples, no individual pesticide or amount correlates with recent bee declines. Formulations usually contain inerts at higher amounts than active ingredients, and these penetrating enhancers, surfactants and adjuvants can be more toxic on non-targets than the active ingredients. Organosilicone surfactants are increasingly being applied to agricultural agro-ecosystems as spray adjuvants, while nonylphenol and octylphenol ethoxylates are major toxicants in agrochemical formulations used around beehives. A liquid chromatography-mass spectrometry (LC-MS) method for analysis of organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants in beehive matrices was developed. A combined liquid-liquid extraction and solid phase extraction method was used. Less than 2 grams of honey, pollen or wax were extracted using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach. Identification and quantification were accomplished employing liquid chromatography coupled to electrospray ionization mass spectrometry. Trisiloxane surfactants were detected in every beeswax and 60% of the pollen samples. Much higher nonylphenol polyethoxylate residues levels were found in wax followed by pollen than in honey, while octylphenol polyethoxylate residues were 10 times lower in pollen and wax. A significant positive correlation was found between total concentrations of nonylphenol polyethoxylates and pesticide residues. We have also shown that chronic dietary exposure to a fungicide chlorothalonil, pesticide mixtures, and a formulation solvent have the potential to impact honey bee populations, and warrant further investigation. Most notable are the chronic larval toxicities of the fungicide chlorothalonil and its synergistic combinations with frequently used in-hive miticides, and the unexpected high toxicity of the formulation ingredient NMP. Trisiloxane surfactants (TSS) have been associated with honey bee learning impairment and the on-going global bee decline. A LC-MS strategy for identification of TSS from agrochemical adjuvants and pollinator-related matrices was developed. The strategy incorporates chromatographic retention behavior, isotope ratio, reference to a compiled database of accurate masses, and TSS hydrolysis when necessary. Using this analytical strategy, three TSS were identified for the first time from almond flowers of a commercial orchard. The three major purified TSS components in popularly used spray tank adjuvants were also identified, and their structures confirmed by nuclear magnetic resonance spectroscopy. With these monitoring tools, we can now assess the agricultural residues and potential risks of major TSS contaminants to important non-target species such as honey bee and other essential pollinators. We have found much higher toxicity to honey bees fed directly on related organosilicone surfactants in 50% sucrose, with oral LC50s for pure commercial trisiloxane surfactants ranging to below 10 ppm, and significant mortality down to 100 ppb. Some 'inert' candidates for future risk assessment for pollinators include the organosilicone surfactants and the co-solvent NMP.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Mullin, C. A., J. Chen, J. D. Fine, M. T. Frazier and J. L. Frazier. 2015. The formulation makes the honey bee poison. Pestic. Biochem. Physiol. In press. http://dx.doi.org/10.1016/j.pestbp.2014.12.026
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Chen, J. and C. A. Mullin. 2015. Characterization of trisiloxane surfactants from agrochemical adjuvants and pollinator-related matrices using liquid chromatography coupled to mass spectrometry. J. Agric. Food Chem. In press. http://dx.doi.org/10.1021/jf505634x
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Frazier, M., C. Mullin, J. Frazier, S. Ashcraft, T. Leslie, E. Mussen and F. Drummond. 2015. Assessing honey bee (Apis mellifera L.) foraging populations and the potential impact of pesticides on eight U. S. Crops. J. Econ. Entomol. In review.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Mullin, C. A. 2015. Effects of inactive ingredients on bees. Curr. Opin. Insect Sci. In review.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mullin, C. A. 2014. Honey bee as a prime bioindicator of agrochemical pollution. In: Fifty Years of Research and Mentoring: Symposium in Honor of the Life and Career of Professor Fumio Matsumura, AGRO 5, 13th IUPAC International Congress of Pesticide Chemistry and 248th ACS National Meeting, San Francisco, CA. p. 79. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mullin C.A., J. Chen, J. D. Fine, M. T. Frazier and J. L. Frazier. 2014. Determination of pesticide co-formulants and adjuvants in honey bee related matrices by LC-ESI-MS. In: Pesticides and Bees, Analysis Tools and Toxicological Effects Session, 51st NACRW-North American Chemical Residue Workshop, St. Pete Beach, FL. p. 43. (Abstract)


Progress 02/28/13 to 02/27/14

Outputs
Target Audience: The impact of systemic pesticides, seed treatments, formulation additives, and other pesticides and their combinations on non-target species, and their role in honey bee and other pollinator health are of global consequence to food security and future crop protection strategies. Major stakeholders include the USDA, EPA, PDA, and the agrochemical industry, growers and beekeeping organizations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have presented and participated in numerous conferences, extension workshops, seminars and professional meetings on the potential role of pesticides and their inerts, co-formulants or adjuvants in pollinator decline, some of which follow: Biddinger, D. J., C. Mullin, J. L. Robertson, E. Rajotte, M. Vaughan, J. Frazier, N. K. *Joshi, M. Otieno and M. Frazier. 2013. The impact of neonicotinoid insecticides on pollinators in tree fruit IPM programs, Entomological Society of America National Meeting, Austin, TX. Frazier, J. 2014. Pesticides and pollinators: From subtle to substantial. 7th Annual Florida Bee College, UF Whitney Marine Lab, Saint Augustine, FL. (Invited) *Frazier, J., C. Mullin, M. Frazier and W. Zhu. 2013. Pesticide impacts and interactions for migratory beekeepers. American Beekeeping Federation Annual Meeting, Hershey, PA. (Invited) *Frazier, J., C. Mullin and M. Frazier. 2014. Pesticides and pollinators: From subtle to substantial. American Honey Producers Association 45th Annual Meeting, San Antonio, TX. (Invited) Frazier, M. 2013. Pesticide conundrum: Pesticides, neonicotinoids and pollinators; Green Industry, University Park, PA. (Invited) Frazier, M. 2014. Can there be honey still for tea? 7th Annual Florida Bee College, UF Whitney Marine Lab, Saint Augustine, FL. (Invited) Frazier, M. 2014. The pesticide conundrum: Protecting crops and pollinators. Department of Entomology and Nematology, University of California, Davis, CA. (Invited) *Frazier, M., J. Frazier, C. Mullin and D. Biddinger. 2013. Honey bees running the risk: Pesticide exposure during pollination, Great Lakes Fruit, Vegetable and Farm Market Expo; Apple Session, Grand Rapids, MI. (Invited) *Frazier, M., J. Frazier, C. Mullin, W. Zhu, T. Ciarlo and S. Ashcraft. 2014. Pesticide conundrum - Pesticides, neonicotinoids and pollinator protection. Turf and Ornamental Conference, Kutztown, PA. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2014. Can there be honey still for tea? American Honey Producers Association 45th Annual Meeting, San Antonio, TX. (Invited) Mullin, C. A. 2013. Pesticide adjuvants/co-formulants and bee health. In: Pam-Monsanto Honey Bee Health Summit, St. Louis, MO. (Invited) *Mullin, C., J. Chen, W. Zhu, M. Frazier and J. Frazier. 2013. Agrochemical formulant and adjuvant toxicities for honey bees. In: Beyond the LC50: Advancements in Toxicological Research on Pollinators Symposium, Entomological Society of America National Meeting, Austin, TX. (Invited) How have the results been disseminated to communities of interest? We have delivered numerous presentations on pollinator decline, CCD and the potential role of pesticides and co-formulants at local, state, regional and national beekeeping conferences, professional, industrial and governmental meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue to focus on recent formulation technologies, including organosilicone surfactants and solvents like NMP, and to investigate the possibility of recent bee declines being associated with these ‘inerts’. Using our sensitive analytical methods, we will study the environmental fate of organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants in and around beehives, and further analyzed their shorter chain oligomers (EO<3) and other degradates.

Impacts
What was accomplished under these goals? Modern pesticide formulations, particularly when multiple active ingredients are blended, require proprietary adjuvants and ‘inerts’ to achieve high efficacy for targeted pests. Although numerous pesticides have been found in beehive samples, no individual pesticide amount correlates with recent bee declines. Formulations usually contain inerts at higher amounts than active ingredients, and these penetrating enhancers, surfactants and adjuvants can be more toxic on non-targets than the active ingredients. Organosilicone surfactants are increasingly being applied to agricultural agro-ecosystems as spray adjuvants, while nonylphenol and octylphenol ethoxylates are major toxicants in agrochemical formulations used around beehives. A LC-MS method for analysis of organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants in beehive matrices was developed. Nonylphenol more than organosiloxane and octylphenol polyethoxylates were found in wax samples, while pollen and particularly honey residues were lower. The high wax residues may accumulate from multiple-year exposures. A significant positive correlation was found between total concentrations of nonylphenol polyethoxylates and pesticide residues. We have also shown that chronic dietary exposure to a fungicide chlorothalonil, pesticide mixtures, and a formulation solvent have the potential to impact honey bee populations, and warrant further investigation. Most notable are the chronic larval toxicities of the fungicide chlorothalonil and its synergistic combinations with frequently used in-hive miticides, and the unexpected high toxicity of the formulation ingredient N-methyl-2-pyrrolidone (NMP). Honey bees are unusually sensitive to organosilicone surfactants, nonylphenol polyethoxylates and the solvent NMP, widespread co-formulants used in agrochemicals and frequent pollutants within the beehive. Effects include learning impairment for adult bees and chronic toxicity in larval feeding bioassays. Multi-billion pounds of synthetic organic chemicals used and released into US environments are formulation ingredients like NMP, generally recognized as safe, having no mandated tolerances, and whose residues remain largely unmonitored. These “inerts” overwhelm the chemical burden from active pesticide, drug and personal care ingredients with which they are formulated. Monitoring methods are needed for major adjuvant residues so risks of formulation additives and their pesticide synergisms for pollinators can be assessed. Organosiloxane surfactants and nonyl- and octyl-phenol polyethoxylates are widely used as nonionic surfactants around honey bee hives or in their foraging areas as spray adjuvants or additives in agrochemical formulations. Methods for analysis of organosiloxane, nonylphenol (NP) and octylphenol (OP) polyethoxylate surfactants in beehive matrices were developed. A combined liquid-liquid extraction and solid phase extraction method was used. Less than 2 grams of honey, pollen or wax were extracted using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach. Identification and quantification were accomplished employing liquid chromatography coupled to electrospray ionization mass spectrometry. For the three trisiloxane surfactants (single polyethoxylate (EO) chain and end-capped with methyl, acetyl or hydroxyl groups; TSS-CH3, TSS-COCH3, or TSS-H) respectively, recoveries for each oligomer (2-13 EOn) were between 66 -112% in all matrices. Average method detection limits were below 1 part per billion (ppb) in honey, pollen, and beeswax. Five honey, 10 pollen and 10 beeswax samples were collected and 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. For analysis of NP(EO)3-13 and OP(EO)3-13 oligomers in bee hive matrices, recoveries for each oligomer in all matrices are between 75-111% at three spiked concentrations. Similarly, this method proved to be rapid, precise and sensitive, with method detection limits below 1 ppb. NP(EO)n was detected in every hive sample with concentrations ranging from 26 ppb to 10,239 ppb. Much higher NP(EO)n residues levels were found in wax followed by pollen than in honey. OP(EO)n concentrations on average were more than 10 times lower in pollen and wax. A larval rearing method was adapted to assess the chronic oral toxicity to honey bee larvae of the four most common pesticides detected in pollen and wax - fluvalinate, coumaphos, chlorothalonil, and chloropyrifos - tested alone and in all combinations. All pesticides at hive-residue levels triggered a significant increase in larval mortality compared to untreated larvae by over two fold, with a strong increase after 3 days of exposure. Among these four pesticides, honey bee larvae were most sensitive to chlorothalonil compared to adults. Synergistic toxicity was observed in the binary mixture of chlorothalonil with fluvalinate at the concentrations of 34 mg/L and 3 mg/L, respectively; whereas, when diluted by 10 fold, the interaction switched to antagonism. Chlorothalonil at 34 mg/L was also found to synergize the miticide coumaphos at 8 mg/L. The addition of coumaphos significantly reduced the toxicity of the fluvalinate and chlorothalonil mixture, the only significant non-additive effect in all tested ternary mixtures. We also tested the common ‘inert’ ingredient NMP at seven concentrations, and documented its high toxicity to larval bees at all concentrations tested down to 0.01% in their diet. Given the critical sensitivity of larvae to chlorothalonil and NMP and their potential complex interactions with other pesticides, the impacts of fungicides and these formulation ingredients on colony survival and development need further investigation. We are in the process of identifying common inerts in pesticide formulations used frequently around honey bees or in their preferred foraging areas. Presently the fate and toxicity of formulation and spray adjuvants in modern agrochemical technologies, and their potential movement into honey/pollen are unknown. We are determining acute and sub-lethal effects of pesticides, their formulation ingredients, important metabolites and selected combinations on bee physiological and behavioral systems. More recently we are investigating the impacts of inerts or co-formulants found in hives alone or in combination with coincident pesticide residues on honey bee survival and behavior. To achieve this, we have developed analytical methods, particularly using LC-MS, to monitor and determine the fate of pest control chemicals and their formulants within bee ecosystems. By first identifying and quantifying key formulation inerts or adjuvants and major co-occuring pesticides and metabolites in pollen, nectar, bees, brood and other hive matrices, we can then test identified inerts and combinations with pesticides for their short- and long-term toxic effects on brood and adult honey bees in the lab and field. These methods demonstrate a probable wide occurrence of substantial amounts of alkylphenol ethoxylates and organosiloxane surfactants in US beehives, and calls for renewed effort to investigate the consequence of these adjuvants to bee health and the ongoing global bee decline. The impact of systemic pesticides, seed treatments, formulation additives, and other pesticides and their combinations on non-target species, and their role in honey bee and other pollinator health are of global consequence to food security and future crop protection strategies. Why most formulations are more toxic to bees than respective active ingredients and how pesticides interact to causepollinator decline cannot be answered without understanding the prevailing environmental chemical background to which bees are exposed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Chen, J. and C. A. Mullin. 2013. Quantitative determination of trisiloxane surfactants in beehive environments based on liquid chromatography coupled to mass spectrometry. Environ. Sci. Technol. 47: 9317-9323.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen, J. and C. A. Mullin. 2014. Determination of nonylphenol ethoxylate and octylphenol ethoxylate surfactants in beehive samples by high performance liquid chromatography coupled to mass spectrometry. Food Chem. 158: 473-479. http://dx.doi.org/10.1016/j.foodchem.2014.03.004
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Chen, J. and C. A. Mullin. 2013. Identification of organosilicone and alkylphenol polyethoxylate surfactants in beehive environments by liquid chromatography-mass spectrometry. In: 2nd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 91. (Abstract)
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhu, W., D. R. Schmehl, C. A. Mullin and J. L. Frazier. 2014. Four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae. PLoS ONE 9(1): e77547.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Biddinger, D., C. Mullin, J. Robertson, E. Rajotte, M. Vaughan, J. Frazier, N. Joshi, M. Otieno and M. Frazier. 2013. The impact of neonicotinoid insecticides on pollinators in tree fruit IPM programs. In: 2nd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 69. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Chen, J. and C. A. Mullin. 2013. Analysis of organosilicone surfactants and their degradation products in beehive environments by liquid chromatography-mass spectrometry. In: General Poster, Sci-Mix Session, AGFD 118, 246th ACS National Meeting, Indianapolis, IN. Cornucopia - Fall 2013: p. 51. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhu, W., T. Reluga, C. Mullin and J. Frazier. 2013. A new stage-structured model of honey bee colony population dynamics assessing impacts of pesticides and other stressors. In: 2nd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 108. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Mullin, C. A., J. Chen, W. Zhu, M. T. Frazier and J. L. Frazier. 2013. The formulation makes the bee poison. In: American Bee Research Conference, Hershey, PA. Amer. Bee J. 153(3): 307. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Mullin, C., J. Chen, W. Zhu, M. Frazier, and J. Frazier. 2013. Colony-level burden of environmental pollutants for honey bees. In: 2nd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 48. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Mullin C.A., J. Chen, W-Y. Zhu, M. T. Frazier and J. L. Frazier. 2013. Agrochemical formulant toxicities for honey bees. In: Pollinators and Pesticides Symposium, AGRO 146, 246th ACS National Meeting, Indianapolis, IN. Picogram 84: p. 124. (Abstract)


Progress 02/29/12 to 02/27/13

Outputs
Target Audience: The impact of systemic pesticides, seed treatments, formulation additives, and other pesticides and their combinations on non-target species, and their role in honey bee and other pollinator health are of global consequence to food security and future crop protection strategies. Major stakeholders include the USDA, EPA, PDA, and the agrochemical industry, growers and beekeeping organizations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? *Chen, J. and C. A. Mullin. 2012. Identification and quantification of organosiloxane and nonylphenol polyethoxylate surfactants in honey bee hive samples by LC-ESI-MS. In: Protection of Agricultural Productivity, Public Health and the Environment Poster Session, AGRO 205, 244th ACS National Meeting, Philadelphia, PA. (poster) Chen, J. and *C. A. Mullin. 2012. Identification and quantification of organosiloxane and alkylphenol polyethoxylate surfactants in honey bee samples by LC-ESI-MS. In: NRI/AFRI Pest and Beneficial Insect in Plant Systems Awardee Conference, Oak Ridge, TN. (poster) *Chen, J., J. L. Frazier , M. T. Frazier, C. A. Mullin. 2012. Identification of organosiloxane surfactants in agrochemical spray adjuvants using liquid chromatography coupled to mass spectrometry. In: American Bee Research Conference, Riverdale, MD. (poster) *Frazier, J. L , C. A. Mullin, and M. T. Frazier. 2012. The sub-lethal impacts of pesticides on honey bees: the importance for beekeepers. American Beekeeping Federation, LasVegas, NV. (invited) *Mullin C.A. 2012. Impacts of modern pesticide formulation technologies on pollinator health. School of Forest Resources Seminar Series, Penn State, University Park, PA. (invited) *Mullin, C. A. 2012. The formulation makes the poison: Inert ingredients and honey bees. In: Center for Pollinator Research Symposium, Penn State, University Park, PA. (invited) *Mullin, C. A. 2012. Pesticides and honey bees – Risks and benefits. In: 2012 Northeast Pesticide Certification and Training Workshop, Saratoga Springs, NY. (invited) *Mullin, C. A. 2012. BBSRC Trans-Atlantic Scientific Exchange and Workshop, Issues on Bee Health and Sustainability, National Bee Unit, York, UK. (invited) *Mullin C.A., J. Chen, T. J. Ciarlo, W-Y. Zhu, M. T. Frazier, and J. L. Frazier. 2012. Impacts of modern pesticide formulation technologies on honey bee health. In: Fate and Exposure of Urban Applied Pesticides in the Context of Human and Ecological Risk Assessments Symposium, AGRO 282, 244th ACS National Meeting, Philadelphia, PA. (invited) *Mullin, C. A., T. J. Ciarlo, W. Zhu and J. Chen. 2012. Pesticide formulation adjuvants may impact honey bee health. In: American Bee Research Conference, Riverdale, MD. *Mullin, C. A., T. J. Ciarlo, J. Chen, W. Zhu, M.T. Frazier and J. L. Frazier. 2012. Formulation makes the poison: Inerts and pollinators. In: A Global Perspective of Bee Decline - Situation, Strategies and Successes Symposium, Entomological Society of America National Meeting, Knoxville, TN. (invited) *Zhu, W., T. Reluga and J. Frazier. 2012. A stage–structured model of honey bee colony population dynamics assessing impacts of pesticides and other stressors. The 5th European Conference of Apidology EURBEE 5, Halle an der Saale, Germany. (invited) *Zhu, W., T. Reluga, C. Mullin and J. Frazier. 2012. A stage-structured model of honey bee colony population dynamics assessing impacts of pesticides and other stressors. In: American Bee Research Conference, Riverdale, MD. Pesticide Conundrum; Understanding the impacts of Pesticides on Pollinators. The British Beekeepers Association, UK, April 2013 Pesticide Conundrum; Understanding the impacts of Pesticides on Pollinators. American Beekeeping Federation, Hershey PA, January 2013 Pesticide Conundrum; The Challenge of Protecting Crops, People and Pollinators. Vanishing of the Bees Conference, Yakima WA, October 2012 Disappearing Bees: An Update on the Search for Prime Suspects. PA House Agriculture Committee, York PA, August 2012 Update on Issues Related to Pesticides and Pollinator Protection. Eastern Apiculture Society, Burlington VT, August 2012 Honey Bee Conservation and the Challenge of Pesticides. The Women’s Agriculture Network, Rodale Institute, June 2012 Risky Business of Pesticides and Protecting Pollinators. Empire State Beekeepers Association, NY, November 2011 How have the results been disseminated to communities of interest? We have delivered numerous presentations on pollinator decline, CCD and the potential role of pesticides and co-formulants at local, state, regional and national beekeeping conferences. What do you plan to do during the next reporting period to accomplish the goals? We will continue to focus on recent formulation technologies, including organosilicone surfactants and solvents like NMP, and to investigate the possibility of recent bee declines being associated with these ‘inerts’.

Impacts
What was accomplished under these goals? Modern pesticide formulations, particularly when multiple active ingredients are blended, require proprietary adjuvants and ‘inerts’ to achieve high efficacy for targeted pests. Although numerous pesticides have been found in beehive samples, no individual pesticide amount correlates with recent bee declines. Formulations usually contain inerts at higher amounts than active ingredients, and these penetrating enhancers, surfactants and adjuvants can be more toxic on non-targets than the active ingredients. For example, we found that the miticide formulation Taktic® was four times more orally toxic to adult honey bees than the respective active ingredient amitraz. Honey bee learning was impaired after ingestion of 20 µg of any of the four tested organosilicone adjuvants. A LC-MS method for analysis of organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants in bee hive matrices was developed. We have shown that chronic dietary exposure to a fungicide, pesticide mixtures, and a formulation solvent have the potential to impact honey bee populations, and warrant further investigation. Impacts of 'inerts' in pollen and nectar alone or in combination with coincident pesticide residues on honey bee survival and behavior are largely unknown. An improved, automated version of the proboscis extension reflex assay with a high degree of trial-to-trial reproducibility was used to measure the olfactory learning ability of honey bees treated orally with sub-lethal doses of the most widely used spray adjuvants on almonds in the Central Valley of California. Three different adjuvant classes (nonionic surfactants, crop oil concentrates, and organosilicone surfactants) were investigated. Learning was impaired after ingestion of 20 µg of any of the four tested organosilicone adjuvants, indicating harmful effects on honey bees caused by agrochemicals previously believed to be innocuous. Organosilicones were more active than the nonionic adjuvants, while the crop oil concentrates were inactive. Monitoring methods are needed for major adjuvant residues so risks of formulation additives and their pesticide synergisms for pollinators can be assessed. Organosiloxane, nonyl- and octyl-phenol polyethoxylates are widely used as nonionic surfactants around honey bee hives or in their foraging areas as spray adjuvants or additives in agrochemical formulations. A method for analysis of organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants in bee hive matrices was developed. A combined liquid-liquid extraction and solid phase extraction method was used. Less than 2 grams of honey, pollen or wax were extracted using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach. Identification and quantification were accomplished employing liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS). Nonylphenol more than organosiloxane and octylphenol polyethoxylates were found in wax samples, while pollen and particularly honey residues were lower. A larval rearing method was adapted to assess the chronic oral toxicity to honey bee larvae of the four most common pesticides detected in pollen and wax - fluvalinate, coumaphos, chlorothalonil, and chloropyrifos - tested alone and in all combinations. All pesticides at hive-residue levels triggered a significant increase in larval mortality compared to untreated larvae by over two fold, with a strong increase after 3 days of exposure. Among these four pesticides, honey bee larvae were most sensitive to chlorothalonil compared to adults. Synergistic toxicity was observed in the binary mixture of chlorothalonil with fluvalinate at the concentrations of 34 mg/L and 3 mg/L, respectively; whereas, when diluted by 10 fold, the interaction switched to antagonism. Chlorothalonil at 34 mg/L was also found to synergize the miticide coumaphos at 8 mg/L. The addition of coumaphos significantly reduced the toxicity of the fluvalinate and chlorothalonil mixture, the only significant effect in all tested ternary mixtures. We also tested the common ‘inert’ ingredient N-methyl-2-pyrrolidone (NMP) at seven concentrations, and documented its high toxicity to larval bees. NMP was more orally toxic to larvae than adult honey bees. Honey bees are highly-susceptible to crop protection chemicals and serve as an excellent bioindicator of environmental quality. Our focus is on study of the role of pesticides in the honey bee and overall pollinator decline. Colony Collapse Disorder (CCD) is estimated to have produced a loss of about one third of all honey bee colonies in the U.S. during each of the last six winters from 2006-12. Bee disappearances threaten the production of nuts, berries, fruits, vegetables and seeds, where their pollination is responsible for over $15 billion in added crop value. Systemic pesticide uses, particularly neonicotinoids, have greatly increased recently through transgenic seed treatments and other crop, ornamental, turf and structural applications. Studies have focused on action of a single pesticide. Given the synergistic nature of certain combinations, we are concerned that active ingredients may affect honey bees differently depending upon inert ingredients in a particular product formulation or on active ingredient mixtures that dominate field use. There is a new tendency to market pesticide blends including seed treatments that contain multiple classes of insecticides or fungicides, or their mixed combinations. These blends usually require proprietary co-formulants and adjuvants to achieve high efficacy and broadly control many pests. We are in the process of identifying common inerts in pesticide formulations used frequently around honey bees or in their preferred foraging areas. Presently the fate and toxicity of formulation and spray adjuvants in modern agrochemical technologies, and their potential movement into honey/pollen are unknown. We are determining acute and sub-lethal effects of pesticides, their formulation ingredients, important metabolites and selected combinations on bee physiological and behavioral systems. More recently we are investigating the impacts of inerts or co-formulants found in hives alone or in combination with coincident pesticide residues on honey bee survival and behavior. To achieve this, we have developed analytical methods, particularly using LC-MS, to monitor and determine the fate of pest control chemicals and their formulants within bee ecosystems. By first identifying and quantifying key formulation inerts or adjuvants and major co-occuring pesticides and metabolites in pollen, nectar, bees, brood and other hive matrices, we can then test identified inerts and combinations with pesticides for their short- and long-term toxic effects on brood and adult honey bees in the lab and field. The impact of systemic pesticides, seed treatments, formulation additives, and other pesticides and their combinations on non-target species, and their role in honey bee and other pollinator health are of global consequence to food security and future crop protection strategies. Practical outcomes include developing both selective pest control strategies and regulatory processes that assure safety for pollinators and products from the hive.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Ashcraft, S., C. Mullin, M. Frazier, and J. Frazier. 2012. Managed Pollinator CAP Coordinated Agricultural Project. From the front Lines - The war against Varroa. Bee Culture 140(11): 25-29.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Ciarlo T.J., C. A. Mullin, J. L. Frazier, and D. R. Schmehl. 2012. Learning impairment in honey bees caused by agricultural spray adjuvants. PLoS ONE 7(7): e40848.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Zhu, W. 2013. Assessing impacts of pesticides and other stressors on honey bee colony health. Ph. D. thesis, Entomology and Operations Research, The Pennsylvania State University, University Park, PA. 170pp.
  • Type: Book Chapters Status: Published Year Published: 2012 Citation: Johnson, R. M., M. D. Ellis, C. A. Mullin, and M. Frazier. 2012. Pesticides and honey bee toxicity in the United States. In Honey Bee Colony Health - Challenges and Sustainable Solutions (D. Sammataro, and J. A. Yoder, Eds.), pp. 145-160. CRC Press, Boca Raton, FL.
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Chen, J. and C. A. Mullin. 2013. Quantitative determination of trisiloxane surfactants in beehive environments based on liquid chromatography coupled to mass spectrometry. Environ. Sci. Technol.
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Zhu, W., D. R. Schmehl, C. A. Mullin and J. L. Frazier. 2013. Four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae. PLoS ONE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Chen, J. and C. A. Mullin. 2012. Identification and quantification of organosiloxane and nonylphenol polyethoxylate surfactants in honey bee hive samples by LC-ESI-MS. In: Protection of Agricultural Productivity, Public Health and the Environment Poster Session, AGRO 205, 244th ACS National Meeting, Philadelphia, PA. Picogram 82: 125. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Chen, J. and C. A. Mullin. 2012. Identification and quantification of organosiloxane and alkylphenol polyethoxylate surfactants in honey bee samples by LC-ESI-MS. In: NRI/AFRI Pest and Beneficial Insect in Plant Systems Awardee Conference, Oak Ridge, TN. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Mullin C.A., J. Chen, T. J. Ciarlo, W-Y. Zhu, M. T. Frazier, and J. L. Frazier. 2012. Impacts of modern pesticide formulation technologies on honey bee health. In: Fate and Exposure of Urban Applied Pesticides in the Context of Human and Ecological Risk Assessments Symposium, AGRO 282, 244th ACS National Meeting, Philadelphia, PA. Picogram 82:140. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Zhu, W., T. Reluga, C. Mullin and J. Frazier. 2012. A stage-structured model of honey bee colony population dynamics assessing impacts of pesticides and other stressors. In: American Bee Research Conference, Riverdale, MD. Amer. Bee J. 152(4): 407-408. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Mullin, C. A., J. Chen, W. Zhu, M. T. Frazier and J. L. Frazier. 2013. The formulation makes the bee poison. In: American Bee Research Conference, Hershey, PA. Amer. Bee J. 153(3): 307. (Abstract)


Progress 03/01/11 to 02/28/12

Outputs
OUTPUTS: Modern pesticide formulations and seed treatments, particularly when multiple active ingredients are blended, require proprietary adjuvants and inert ingredients to achieve high efficacy for targeted pests. Although we have found over 130 different pesticides and metabolites in beehive samples, some at high amounts and frequency, no individual pesticide or amount correlates with recent bee declines. We are examining if more generic formulation inerts that co-occur across classes of pesticides used around hives or where bees forage may be involved. The formulations Bravo (fungicide) and Taktic (miticide) were four times more toxic to adult honey bees orally than the respective active ingredients, chlorothalonil and amitraz. Since the formulation is enhancing the poison, we focused on new technologies including organosilicone surfactants and co-solvents like N-methylpyrrolidone (NMP) which have not been evaluated for bee ecotoxicity. Nonionic surfactants like Silwet L-77 and Triton X-100 were highly lethal to adult honey bees at 1% in artificial nectar. NMP exhibited greater oral toxicity to honey bee larvae than to adults, and 1% NMP can kill all larvae in 24 hours following ingestion. Use of novel microemulsion formulations of agrochemicals are often dependent on this and newer organosilicone adjuvants such as Silwet L-77. An improved, automated version of the proboscis extension reflex (PER) assay with a high degree of trial-to-trial reproducibility was used to measure the olfactory learning ability of honey bees treated orally with sublethal doses of the most widely used spray adjuvants on almonds in the Central Valley of California. Three different adjuvant classes (nonionic surfactants, crop oil concentrates, and organosilicone surfactants) were investigated. Organosilicones including Silwet L-77 at or above 5 micrograms/bee impaired learning more than the nonionic adjuvants at 20 micrograms/bee, while the crop oil concentrates were inactive. Ingestion was required for the tested adjuvant to have an effect on learning, as exposure via antennal contact only induced no level of impairment. This is the first demonstration of a honey bee conditioned learning response being decreased by an organosilicone surfactant. Olfactory learning is important for foraging honey bees because it allows them to exploit the most productive floral resources in an area at any given time. Impairment of this learning ability may have serious implications for foraging efficiency at the colony level, as well as potentially many social interactions. Organosilicone spray adjuvants may therefore contribute to the ongoing losses that characterize bee decline. In addition, we have identified utilizing LC-MS common adjuvants in pesticide formulations used around hives or where bees forage. Improved environmental monitoring methods are needed for organosilicone surfactants and other newer adjuvants. The impact on bees of synergistic pesticidal blends which readily contaminate plant nectars and pollens cannot be fully understood without proper identification and risk assessment of formulation additives and other spray adjuvants. PARTICIPANTS: Chris Mullin provides overall project leadership for the residue analysis, HPLC, and toxicity and sublethal behavioral bioassays. Maryann Frazier supervises apiary studies, extension and media communications. Jim Frazier provides overall leadership on behavioral experiments. Jing Chen is our postdoctoral scholar conducting the residue analysis of pesticide formulation additives. Our interdisciplinary research on the role of pesticides on honey bee health is in collaboration with David J. Biddinger of the PSU Fruit and Extension Labs, Biglerville, PA, and Roger Simonds, pesticide residue analytical chemist of the USDA-AMS National Science Laboratory, Gastonia, NC. TARGET AUDIENCES: The impact of systemic pesticides, seed treatments, formulation additives, and other pesticides and their combinations on non-target species, and their role in honey bee and other pollinator health are of global consequence to food security and future crop protection strategies. Major stakeholders include the USDA, EPA, PDA, and the agrochemical industry and beekeeping organizations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our results to date indicate that pesticides are very likely involved with Colony Collapse Disorder (CCD) and that agrochemical interactions with other stressors including mites, diseases and nutrition are likely factors contributing to the decline of honey bee and other pollinator health. CCD is estimated to have produced a loss of about one third of all honey bee colonies in the U.S. during each of the last five winters from 2006-11. Bee disappearances threaten the production of nuts, berries, fruits, vegetables and seeds, where their pollination is responsible for over $15 billion in added crop value. Formulation and spray adjuvants are applied to crops in conjunction with agricultural pesticides in order to boost the efficacy of the active ingredient(s). The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to spray adjuvants as well. Given the synergistic nature of certain chemicals, we are concerned that active ingredients may affect honey bees differently depending upon the inert ingredients in a particular product formulation or in certain combinations found in field use patterns. There is a new tendency to market pesticide blends including seed treatments that contain multiple classes of insecticides or fungicides, or their mixed combinations, that require these newer inerts to achieve high efficacy. Formulations and spray adjuvants usually contain inerts such as co-solvents, penetrating enhancers, surfactants, wetters etc. at higher total amounts than active ingredients. The fact that some inert ingredients may indeed be more toxic than active ingredients such as in formulations of the herbicide glyphosate makes this issue especially important for the health of honey bees and other pollinators. We are in the process of identifying common inerts in pesticide formulations used frequently around honey bees or in their preferred foraging areas. Presently the fate and toxicity of formulation and spray adjuvants in modern agrochemical technologies, and their potential movement into honey/pollen are unknown. We will also test their short- and long-term toxic effects on brood and adult honey bees in lab and field. We will disseminate the information generated by this study through regional MAAREC and national Honey Bee CAP E-Extension websites in addition to appropriate professional publication and meeting venues. For the sustainability of honey bees, other pollinators and beneficial insects, it is vitally important that these inert compounds be disclosed and their effects investigated. Inert ingredients which are potentially hazardous need to be redefined by EPA in terms other than inert.

Publications

  • Bahn, D. G. 2011. Qualitative analysis of effects of formulation additives on metabolism of chlorothalonil in honeybees. Department of Entomology. Honors Thesis. The Pennsylvania State University Schreyer Honors College, University Park, PA. 45 pp.
  • Ciarlo, T. J. 2011. Learning impairment in honey bees caused by agricultural spray adjuvants. M.S. Thesis. The Pennsylvania State University, University Park, PA. 71pp.
  • Ciarlo T. J., C. A. Mullin, J. L. Frazier, and D. R. Schmehl. 2012. Learning impairment in honey bees caused by agricultural spray adjuvants. PLoS One. (Pending).
  • Mullin, C. A., T. J. Ciarlo, J. Chen, W. Zhu, M. T. Frazier, and J. L. Frazier. 2012. Pesticide formulation adjuvants and their health impacts on pollinators and other non-target species. Pest Manag Sci. (Pending).
  • Ciarlo, T. J., C. A. Mullin, and J. L. Frazier. 2011. Pesticide adjuvants and inert ingredients may impair foraging behavior in honey bees (Apis mellifera). Entomological Society of America Eastern Branch 82nd Annual Meeting, Harrisburg, PA. (Poster Abstract #9) p. 37.
  • Mullin, C. A., T. J. Ciarlo, W. Zhu, M. T. Frazier, and J. L. Frazier. 2011. Analyzing pesticide formulation adjuvants to assess their impact on pollinator health. Analytical Challenges for Crop Protection Products Symposium, AGRO 10, 242nd ACS National Meeting, Denver, CO. (Abstract). Picogram 80:82.
  • Mullin, C. A., J. L. Frazier, M. T. Frazier, and T. J. Ciarlo. 2011. A primer on inerts and honey bees. American Bee Research Conference. Galveston, TX. Amer. Bee J. (Abstract). 151(5):513.
  • Chen, J., J. L. Frazier, M. T. Frazier, and C. A. Mullin. 2012. Identification of organosiloxane surfactants in agrochemical spray adjuvants using liquid chromatography coupled to mass spectrometry. American Bee Research Conference. Riverdale, MD. Amer. Bee J. (Poster Abstract). 152(5).
  • Mullin, C. A., T. J. Ciarlo, W. Zhu, and J. Chen. 2012. Pesticide formulation adjuvants may impact honey bee health. American Bee Research Conference. Riverdale, MD. Amer. Bee J. (Abstract). 152(5).