Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
THE FORMULATION MAKES THE BEE POISON: CO-FORMULANTS AND POLLINATORS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1000811
Grant No.
2014-67013-21591
Cumulative Award Amt.
$369,639.00
Proposal No.
2013-02605
Multistate No.
(N/A)
Project Start Date
Dec 1, 2013
Project End Date
Jun 30, 2017
Grant Year
2014
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
We are presently focused on study of the role of pesticides and their co-formulants in honey bee and overall pollinator decline. Insufficient research has been done to extend beyond the readily available bee acute toxicity information for individual pesticide active ingredients into an assessment of the consequence of total chemical loads and exposures that incorporate co-formulants and adjuvants, and the resulting agrochemical interactions. Our recent work indicates that honey bees are unusually sensitive to common 'inert' ingredients used in agrochemical formulations and spray tank adjuvants. For the sustainability of honey bees, other pollinators and beneficial insects, it is vitally important that co-formulants be disclosed and their effects investigated. There is a new tendency to market pesticide blends containing multiple classes of insecticides or fungicides, or their mixed combinations without any further ecotoxicology testing. Many insecticide and fungicide combinations are utilized for seed treatments and in crop pest control that can result in hive residues that synergistically combine by themselves or with miticides used to control Varroa to poison honey bees. These blends usually require proprietary adjuvants to achieve high efficacy and broadly control many pests. The impact of these synergistic blends on non-targets including bees cannot be fully understood without knowing the identity of proprietary inerts and adequate testing on the appropriate species. The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Our results indicate that organosilicone surfactants are among the most toxic adjuvants, both sublethally and acutely, to adult honey bees. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to many spray adjuvants as well. These landmark studies should help demonstrate the need to disclose more information on inerts in agrochemicals, since it appears that 'the formulation and not just the dose makes the poison.' 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. We anticipate that if 'inerts' are influencing pesticide levels and general hive stress, formulation recommendations can be optimized for use in bee foraging areas.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2115220115035%
2113010115035%
2112410115030%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops; 3010 - Honey bees; 5220 - Pesticides;

Field Of Science
1150 - Toxicology;
Goals / Objectives
Modern pesticide formulations, particularly when multiple active ingredients are blended, require proprietary adjuvants and 'inerts' to achieve high efficacy for targeted pests. Recently we have shown that honey bees are unusually sensitive to organosilicone spray adjuvants and other co-formulants used in agrochemicals, and we now endeavor to analyze their presence and fate in bee ecosystems. Knowing relevant environmental levels of adjuvants and inerts would allow improved risk assessment of total chemical loads and exposures for bee pollinators and other non-targets species. To address under Program Area Priority Plant-Associated Insects and Nematodes (A1111) the hypothesis that co-formulants and adjuvants generally increase the toxicity of pesticide active ingredients and other toxicants to adult and immature bees, we will (1) identify common 'inerts' in agrochemicals and other environmental chemicals used frequently around honey bees or in their preferred foraging areas, (2) develop analytical methods, particularly using LC-MS, to monitor and determine the fate of pesticide formulation and adjuvant ingredients within bee ecosystems, (3) determine acute and sub-lethal effects of pesticides, their formulation ingredients, important metabolites and relevant combinations on bee physiological and behavioral systems, and (4) facilitate integration and communication of results to USA beekeepers, growers, pesticide regulators, the agrochemical industry and the research community.
Project Methods
We will continue to focus on recent formulation technologies, including organosilicone surfactants and solvents like N-methyl-2-pyrrolidone (NMP), of unknown bee ecotoxicity, and to investigate the possibility of recent bee declines being associated with these 'inerts'. We will determine appropriate formulation blanks to use in characterizing the impacts of major pesticide active ingredients associated with bee decline. Impacts of co-formulants and their degradates, individually and corporately at sub-lethal levels, on key honey bee behaviors/physiology including memory and learning will be investigated. Extracts of bees and hive matrices will be analyzed for parent pesticides, metabolites, and formulation inerts using a modified QuEChERS method and LC/MS-MS. Toxic or sublethal effects on honey bees of pesticide and inert combinations relative to formulation controls, including interference with associative learning, will be determined by direct feeding or incorporation in artificial nectar or uncontaminated pollen or wax, or by topical application of extracts to bees or brood. Colony-level impacts of formulation ingredients will be determined in field experiments. Frequently found co-formulants in pesticides and spray tank adjuvants will be characterized and their identity confirmed. Hive samples of stored pollen, comb wax, nectar and bees or field floral samples with known or suspected high levels of frequently occurring fungicides, insecticides and other pesticides will be analyzed for active ingredients and inerts on our LCMS-2020 at primarily the > 5 ppb limit of detection (LOD). Portions of priority samples will be preserved and sent to the USDA-AMS-NSL in Gastonia for follow-up residue analysis at a more sensitive 1 ppb LOD. Remaining portions of each sample will be used in toxicity and behavioral studies. After identification of key inert ingredients in agrochemicals used frequently around bees, we will develop an appropriate sensitive method for their analysis, similar to a recent methods developed in our lab for analyzing three trisiloxane surfactants and nonylphenol polyethoxylates. We will use these analytical methods to study the environmental fate of trisiloxane, nonyl- and octylphenol surfactants and other key inerts, including their degradates, in and around beehives. Metabolism of free or formulated inerts and pesticides within bee bioassays (including excreta) or in pollen, wax, nectar and other matrices will be addressed through analysis over time of residues relative to the treatment or dose using the appropriate LC/MS-MS method based on chromatographic, spectral and mass transition comparisons with authentic standards. To assess potential toxicity or other negative impacts of formulation components, inerts alone or in combination with active ingredients will be fed at dose levels detected in hive samples in artificial nectar, royal jelly diet or pollen-substitute cakes to adult bees, queens, drones, and brood, or topically applied, and other factors such as bee behavior and colony longevity evaluated. 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. Chronic feeding of bioactive formulation ingredients and combinations will also be conducted. Altered behaviors will be investigated further through proboscis extension reflex (PER) bioassays. A tier approach will be used where significant impacts at the larval and adult toxicity bioassay and sublethal PER levels will proceed into semi-field (nuke) or field level studies when priority effects are observed. A field study will be conducted for each of the three years. Hives will be treated with formulants and pesticides in a pollen-substitute diet (oral) or by spraying the wax comb (contact). After treatment we will assess queen egg laying rate, larval development, longevity, Varroa mite levels, hypopharyngeal gland development and other desired endpoints. We will disseminate the information generated by this study through regional and national websites in addition to appropriate professional publication and meeting venues.

Progress 12/01/13 to 06/30/17

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. D. 2016. The toxicodynamics of two 'inert' agrochemicals and their impacts on honey bees. Department of Entomology, University of Nebraska, Lincoln, NE. (Invited) Mullin C., J. Fine*, R. Reynolds, J. Chen and M. Frazier. 2016. The formulation makes the bee poison: Co-formulants and pollinators. NIFA Project Director meeting, 25th International Congress of Entomology, Orlando, FL. (Invited) Reynolds, R. D. 2017. Save the bees! Methods for supporting pollinators from the field to the floret. Beaver Valley Area Beekeeping Association, Baden, PA. (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? We have found adjuvants like organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants and the co-solvent N-methyl-2-pyrrolidone (NMP) at ppm levels in beehive samples. Adjuvants like organosilicone surfactants or the solvent NMP alone are orally and topically toxic to bees, but greater impacts are found on combination with other stressors including pesticides and viruses. An organosilicone surfactant adjuvant frequently used during almond pollination, when combined at a field-relevant concentration with prevalent bee viruses, causes synergistic mortality in honey bee larvae. Formulations are generally more toxic than respective active ingredients, particularly fungicides, by up to 26,000-fold. We have found 100% of co-formulants analyzed for in beehive samples, while only 70% of pesticide active ingredients searched for have been detected. In chronic feeding assays, the common agrochemical inert formulant, NMP is at least 20 times more toxic to honey bee larvae than to adults, but the underlying cause of this difference is unknown. In other taxa, NMP is primarily detoxified via a cytochrome P450 mediated pathway. Using a previously described LC-MS method, putative cytochrome P450 metabolites of NMP were identified and quantified in adults and larvae following chronic exposure to 200 ppm of NMP. Oxidized NMP metabolites were identified in bees following the feeding assay, suggesting that cytochrome P450 mediated metabolism is a major route of detoxification in larvae and adults. Major differences were observed between adult and larval metabolism. N-methyl-succinimide was detected in honey bee larvae. This metabolite was not observed in adults, but a compound with an identical mass/charge ratio was detected. Ionization patterns detected using quantitative time of flight mass spectrometry are suggestive of a positional isomer. A higher percentage of the administered NMP was recovered as the parent compound in larval honey bees than in adults, indicating that adults are able to metabolize a higher percentage of the administered NMP to its more polar metabolites when it is administered in food. The observed differences in toxic effects and metabolite profiles between adults and larvae were further explored using a spectrofluorometric method to compare general cytochrome P450 enzyme activity by monitoring the transformation of a 7-ethoxycoumarin substrate. Higher microsomal levels of 7-ethoxycoumarin-O-deethylase activity in adult honey bee fat bodies suggests that the higher percentage of unmetabolized NMP in larvae relative to adults may be due to lower cytochrome P450 activity in fat body. Taken together, the results presented here suggest that larvae may be less able to detoxify xenobiotics absorbed through the cuticle and midgut than adults, and these findings will help to inform future risk assessment. We also examined the hypothesis that the organophosphate alternative, Rimon® 0.83EC, can have consequences to honey bee health by combining newly acquired field residue data, laboratory bioassays, and colony level feeding studies. Following label rate applications of Rimon® 0.83EC to apple trees, average residue concentrations of the active ingredient, novaluron, were found to be 3.38 ppm in pollen. Residues of the major co-formulant in Rimon® 0.83EC, NMP, were below the limit of detection in the field, but a greenhouse study found that NMP can persist in pollen for up to 7 days with average concentrations of 69.3 ppm. Concurrent larval rearing studies found novaluron and NMP to be toxic to developing honey bees at doses as low as 100 ppb and 100 ppm respectively. Nucleus colony feeding studies found that chronic exposure to Rimon® 0.83EC at doses as low as 200 ppm (18.6 ppm novaluron) can result in interruptions to brood production that can last for up to 2 weeks after the feeding period. Taken together, these data indicate the use of Rimon® 0.83EC on blooming flowers is a significant threat to honey bee reproduction, and suggest the need for more strict and clear usage guidelines. Organosilicone surfactants are the most potent adjuvants available for formulating and applying agricultural pesticides and fertilizers, household cleaning and personal care products, dental impressions and medicines. Risk assessment of pesticides, drugs or personal care products that takes into account only active ingredients without the other formulation ingredients and spray adjuvants commonly used in their application will miss important toxicity outcomes detrimental to non-target species including pollinators and humans. Over a billion pounds of organosilicones from all uses are released into USA environments, making this a major component of the chemical landscape to which bees and humans are exposed. These methyl silicones, like most "inerts", are generally recognized as safe, have no mandated tolerances, and their residues are largely unmonitored. Lack of their public disclosure and adequate analytical methods constrains evaluation of their risk. Organosilicone surfactants, the most super-spreading and -penetrating adjuvants available, at relevant exposure levels impair honey bee learning, are acutely toxic, and in combination with bee viruses cause synergistic mortality. Methyl silicone surfactants need to be regulated as a separate class of "inerts" from the more common methyl silicones. In turn, impacts of OSS exposures on humans need to be evaluated. Methylated silicones in their great diversity, probably represent the single most ubiquitous environmental class of global synthetic pollutants. Do honey bees, a model environmental indicator organism, forewarn of hidden risks to humans of ubiquitous silicone exposures?

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Fine, J. D. 2017. The toxicodynamics of inert and reduced risk agrochemicals and their impacts on honey bees. Ph.D. thesis, Department of Entomology, Penn State University, University Park, PA, 123 pp.
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Fine, J. D. and C. A. Mullin. 2017. Metabolism of N-methyl-2-pyrrolidone in honey bee adults and larvae: Exploring age related differences in toxic effects. Environ. Sci. Technol., In review.
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Fine, J. D., C. A. Mullin, M. T. Frazier and R. D. Reynolds. 2017. The field residues and effects of the insect growth regulator novaluron and its major co-formulant N-methyl-2-pyrrolidone on honey bee reproduction. J. Econ. Entomol., In review.
  • Type: Journal Articles Status: Other Year Published: 2017 Citation: Mullin, C. A., J. Chen and J. D. Fine. 2017. Are organosilicone surfactants safe for bees or humans? Science of the Total Environment, in preparation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mullin, C. A. 2017. Are silicone exposures safe for pollinators or humans? In: One Health, One Planet Symposium, Phipps Conservatory, Pittsburgh, PA. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mullin, C. A. 2017. The effects of agrochemical inerts on pollinators and humans. In: Center for Pollinator Research Mini-Symposium, Penn State University, University Park, PA. (Abstract)


Progress 12/01/15 to 11/30/16

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:A seven month no-cost extension was approved. 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: Frazier, M. 2015. Talking to the press and public about bees and pesticides. PA State Beekeepers Annual Meeting. Lewisburg, PA. (Invited) Mullin, C. 2015. Inactive ingredients and their effects. AgChem Summit 2015: Pollinators and Pesticides, Arlington, VA. (Invited) Fine, J. D. 2016. The toxicodynamics of two 'inert' agrochemicals and their impacts on honey bees. Department of Entomology, University of Nebraska, Lincoln, NE. (Invited) Frazier, M. 2016. IPM approach to pest and disease management. Pest and disease identification. Maui Bee Conference, Hale Akua, Maui, HI. (Invited) Frazier, M. 2016. Managing honey bee maladies by letting them be. West Palm County Beekeepers Association, West Palm Beach, FL. (Invited) Frazier, M. 2016. PA Hosted a field day at Singing Creek Farm. Discussion topics centered around factors impacting honey bee health including pesticides. Women In Agriculture, Mount Union, PA. (Invited) Frazier, M. 2016. BeeDazzled. Eastern Apiculture Society, Stockton University, Galloway NJ. (Invited) Frazier, M. 2016. BeeDazzled. Montgomery County Beekeepers Association, Montgomery County, PA. (Invited) Frazier, M. 2016. Led a day-long workshop on honey bee health and productivity. Discussion topics focused on factors impacting honey bee health including pesticides. PA Women In Agriculture, Warren PA. (Invited) Frazier, M. 2016. Cell phones as a lifeline for Kenyan beekeepers. Susquehanna Beekeepers Association, Montrose PA. (Invited) Mullin C., J. Fine*, R. Reynolds, J. Chen and M. Frazier. 2016. The formulation makes the bee poison: Co-formulants and pollinators. NIFA Project Director meeting, 25th International Congress of Entomology, Orlando, FL. (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. (see above) What do you plan to do during the next reporting period to accomplish the goals?To test the hypothesis that co-formulants and adjuvants generally increase the toxicity of pesticide active ingredients and other toxicants to adult and immature bees, we will continue to focus on recent formulation technologies, including organosilicone surfactants and solvents like N-methyl-2-pyrrolidone (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. Impacts of co-formulants such as NMP and their degradates, individually and corporately at sub-lethal levels, on key honey bee behaviors/physiology including memory and learning will be investigated. Toxic or sublethal effects on honey bees of pesticide and inert combinations including viruses relative to formulation controls, will be determined by direct feeding or incorporation in artificial nectar or uncontaminated pollen or wax, or by topical application of extracts to bees or brood. Colony-level impacts of formulation ingredients will be determined in field experiments. We will disseminate the information generated by this study through regional and national websites in addition to appropriate professional publication and meeting venues.

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 or inactives' in pollinator decline is only in its formative stages. Effects include learning impairment for adult bees and oral toxicity for larvae and adults. Multi-billion pounds of formulation ingredients from all uses are released into USA 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 adjuvant and formulation ingredients coupled with a lack of adequate analytical methods constrains the assessment of total chemical load and agrochemical exposures on bees. Most studies to document pesticide effects on honey bees are performed without the formulation or other relevant spray adjuvant components used to environmentally apply the toxicant. Formulation composition and not just the dose of active ingredient makes the poison. Adjuvants and co-formulants generally greatly enhance the pesticidal efficacy and inadvertently the non-target effects of the active ingredient. These co-formulants at environmental-exposure levels significantly enhance the acute toxicities of the active ingredient residues. Organosilicone surfactants are the most potent tank adjuvants and super-penetrants available to growers. Based on the California Department of Pesticide Regulation data for agrochemical applications to almonds, there has been increasing use of adjuvants, particularly organosilicone surfactants, during bloom when two-thirds of USA honey bee colonies are present. This database archives every application of a spray tank adjuvant with detail that is unprecedented globally. Increased tank-mixing of these with fungicides and insect growth regulators may be associated with recent USA honey bee declines. Spray tank adjuvants are largely assumed to be biologically inert and are not registered by the EPA, leaving their regulation and monitoring to individual states. Agrochemical risk assessment that takes into account only pesticide active ingredients without the spray adjuvants commonly used in their application will miss important toxicity outcomes detrimental to non-target species including pollinators and humans. Organosilicone surfactants are good stand alone pesticides, toxic to bees, and also present in drug and personal care products, particularly shampoos, and thus represent an important component of the chemical landscape to which pollinators as well as humans are exposed. Adjuvants like organosilicone surfactants or the solvent N-methyl-2-pyrrolidone alone are orally and topically toxic to bees, but greater impacts are found on combination with other stressors including pesticides and viruses. Colony losses following a major pollination event in the United States, almond pollination, have been characterized by brood mortality with specific symptoms, followed by eventual colony loss weeks later. We demonstrated that these symptoms can be produced by chronically exposing brood to both an organosilicone surfactant adjuvant (OSS) commonly used on many agricultural crops including wine grapes, tree nuts and tree fruits and exogenous viral pathogens by simulating a horizontal transmission event. The OSS Sylgard 309 frequently used during almond pollination, when combined at a field-relevant concentration with prevalent bee viruses, causes synergistic mortality in honey bee larvae during the larval-pupal molt. Using Q-PCR techniques to measure gene expression and viral levels in larvae taken just prior to observed mortality at metamorphosis, we found that exposure to the OSS Sylgard 309 and exogenous virus resulted in significantly heightened Black Queen Cell Virus (BQCV) titers and lower expression of a Toll 7-like-receptor associated with autophagic viral defense (Am18w). These results demonstrate that organosilicone spray adjuvants that are considered biologically inert may potentiate viral pathogenicity in honey bee larvae, and guidelines for OSS use may be warranted. To further our goal to determine acute and sub-lethal effects of relevant pesticide combinations on honey bees, we analyzed residues from live in-hive bees, stored pollen, and wax in migratory colonies over time and compared exposure to colony health. Pollination environments subjected colonies to increased pesticide exposure compared to honey-production locations and holding yards. We found clear links between an increase in the total number of products in comb wax and colony mortality, specifically fungicides. Hazard quotient (HQ) scores, an estimate of total pesticide exposure risk, in beebread from March-September were higher in colonies that died over the course of the study, with the HQ score of fungicides being notably predictive. The fungicide chlorothalonil was particularly prevalent in beebread and wax samples, and its presence was associated with increased colony mortality. The occurrence of queen events, which had previously been associated with an increased risk of mortality, was predicted by the HQ in wax and total number of products contributing 50+ to a samples HQ score.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mullin, C. A., J. D. Fine, R. D. Reynolds and M. T. Frazier. 2016. Toxicological risks of agrochemical spray adjuvants: Organosilicone surfactants may not be safe. Frontiers in Public Health 4:1-8. Article 92. doi:10.3389/fpubh.2016.00092.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Traynor, K. S., J. S. Pettis, D. R. Tarpy, C. A. Mullin, J. L. Frazier, M. Frazier and D. vanEngelsdorp. 2016. In-hive pesticide exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States. Scientific Reports 6:33207.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Fine, J. D., D. L. Cox-Foster and C. A. Mullin. 2017. An inert pesticide adjuvant synergizes viral pathogenicity and mortality in honey bee larvae. Scientific Reports 7:40499.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Biddinger, D., S. Shugrue, N. Joshi, E. Rajotte, C. Mullin, N. Phan, J. Fine and J. Belsky. 2016. Determining field realistic exposure levels of systemic insecticides to pollinators in apple orchards. In: 3rd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 106. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Fine, J., D. Cox-Foster and C. Mullin. 2016. Organosilicone surfactant adjuvant and viral pathogens cause synergistic mortality in honey bee larvae. In: 3rd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 92. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Fine, J. D. and C. A. Mullin. 2016. Comparative study of the detoxification of the pesticide inert N-methyl-2-pyrrolidone in Apis mellifera adults and larvae. In: Pollinators: Agrochemicals, Behavior & Disease symposium, AGRO 151, 252nd ACS National Meeting, Philadelphia, PA. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Fine, J., C. Mullin and D. Cox-Foster. 2016. Chronic exposure to an agricultural spray adjuvant and honey bee pathogen causes synergistic mortality in larval honey bees (Apis mellifera). In: 25th International Congress of Entomology, Orlando, FL. Abstract 4010, doi: 10.1603/ICE.2016.114653.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Frazier, M., C. Mullin, R. Reynolds and J. Fine. 2016. Insect growth regulator impacts on honey bee development. In: 3rd International Conference on Pollinator Biology, Health and Policy, Center for Pollinator Research, Penn State University, University Park, PA. p. 94. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mullin C. A., J. D. Fine, R. D. Reynolds, J. Chen and M. T. Frazier. 2016. Toxicological risks of agrochemical spray adjuvants and other inactive ingredients to bees. In: Pollinators: Agrochemicals, Behavior & Disease symposium, AGRO 152, 252nd ACS National Meeting, Philadelphia, PA. (Abstract)


Progress 12/01/14 to 11/30/15

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: *Frazier, J., C. Mullin and M. Frazier. 2015. Dying bees: Harbingers of the end times or opportunity buzzing. Ornamentals meeting, Hendersonville, NC. (Invited) *Frazier, J., C. Mullin and M. Frazier. 2015. Dying bees: Harbingers of the end times or opportunity buzzing. Minnesota State Beekeepers. (Invited) *Frazier, J., C. Mullin and M. Frazier. 2015. Dying bees: Harbingers of the end times or opportunity buzzing. California State Beekeepers. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Factors that influence bee health: Focus on pesticides. Upper Valley Sierra Club. So. Royalton, VT. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Colony losses puzzle: The pesticide piece. Colorado State Beekeepers. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Bees, or not to bee: Factors that influence bee health: Pesticides. York County Beekeepers Association, York, PA. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Improving your odds; Factors impacting overwintering success. Montgomery County Beekeepers Association, Silver Spring, MD. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Colony losses puzzle: The pesticide piece. New Zealand National Beekeepers Association. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Toxic house: pesticide exposure and impacts on honey bee colonies. Texas State Beekeeper Meeting, Rogers, TX. (Invited) *Frazier, M., C. Mullin and J. Frazier. 2015. Hazardous foraging: honey bee exposure to pesticides on eight crops. Penn State University Ornamentals and Pollinators Workshop, University Park, PA. (Invited) *Mullin, C., J. Fine, M. Frazier and R. Reynolds. 2015. Do pesticide co-formulants and adjuvants impact bee health? In: Colony Collapse Disorder Eight Years Later Symposium, Entomological Society of America National Meeting, Minneapolis, MN. (Invited) Mullin, C. 2015. The formulation makes the poison. Paul A. Dahm memorial lecture, Dept. of Entomology, Iowa State University, Ames, IA. (Invited) Mullin, C. 2015. Formulation ingredients are poisonous to pollinators and are largely unstudied. DuPont Crop Protection, Newark, DE. (Invited) *Reynolds, R. and M. Frazier. 2015. Pollinators and pesticides. Mid-Atlantic Fruit and Vegetable Convention, Hershey, PA. Jan. 27, 2015. *Reynolds, R. and M. Frazier. 2015. Bringing back the bees. NY Southern Tier Beekeepers, Binghamton, NY. Oct. 17, 2015. 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. (see above) 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. Impacts of co-formulants such as NMP and their degradates, individually and corporately at sub-lethal levels, on key honey bee behaviors/physiology including memory and learning will be investigated. Toxic or sublethal effects on honey bees of pesticide and inert combinations including viruses relative to formulation controls, will be determined by direct feeding or incorporation in artificial nectar or uncontaminated pollen or wax, or by topical application of extracts to bees or brood. Colony-level impacts of formulation ingredients will be determined in field experiments. We will disseminate the information generated by this study through regional and national websites in addition to appropriate professional publication and meeting venues.

Impacts
What was accomplished under these goals? 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). Effects include learning impairment for adult bees and chronic toxicity in larval feeding bioassays. Lack of disclosure of formulation ingredients in major products and spray tank adjuvants and lack of adequate methods for their analysis constrain the assessment of total chemical load and agrochemical exposures on bees. Since trisiloxane surfactants have been associated with honey bee learning impairment and the on-going global bee decline, we developed a liquid chromatography-mass spectrometry (LC-MS) strategy for their identification from agrochemical adjuvants and pollinator-related matrices. The strategy incorporates chromatographic retention behavior, isotope ratio, reference to a compiled database of accurate masses, and their hydrolysis when necessary. Using this analytical strategy, three trisiloxane surfactants were identified for the first time from almond flowers of a commercial orchard. The three major purified trisiloxane surfactants 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 organosilicone surfactant contaminants to important non-target species such as honey bee and other essential pollinators. 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. Most studies to document pesticide effects on honey bees are performed without the formulation or other relevant spray adjuvant components used to environmentally apply the toxicant. By using LC-MS and the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach, we have found spray adjuvant surfactants at mg/kg levels in hive samples that significantly correlated with lower total concentrations of pesticide active ingredient residues in the same sample. This provides an opportunity of predicting pesticide exposures via the more abundant residues of the adjuvant component used in their application. 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 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. Honey bees reveal that the formulation and not just the dose makes the poison. By employing this knowledge, we can begin to optimize formulation recommendations for use in bee foraging areas. During 2009-2010 we assessed changes in the field force populations of nine to ten colonies at one location per crop on each of eight crops by counting departing foragers leaving colonies at regular intervals during the respective crop blooming periods. The number of frames of adult bees was counted before and after bloom period. For pesticide analysis we collected dead and dying bees near the hives, returning foragers, crop flowers, trapped pollen, and corn-flowers associated with the cotton crop. The number of departing foragers changed over time in all crops except almonds; general patterns in foraging activity included: declines (cotton), noticeable peaks and declines (alfalfa, blueberries, cotton, corn, and pumpkins), and increases (apples and cantaloupes). The number of adult bee frames increased or remained stable in all crops except alfalfa and cotton. A total of 53 different pesticide residues were identified in samples collected across eight crops. Hazard quotients were calculated for the combined residues for all crop-associated samples and separately for samples of dead and dying bees. A decrease in the number of departing foragers in cotton was one of the most substantial crop-associated impacts and presented the highest pesticide risk estimated by a summed pesticide residue hazard quotient.

Publications

  • Type: Journal Articles Status: Published 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. 63:5120-5125. http://dx.doi.org/10.1021/jf505634x
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Frazier, M., C. Mullin, J. Frazier, S. Ashcraft, T. Leslie, E. Mussen and F. Drummond. 2015. Assessing honey bee (Hymenoptera: Apidae) foraging populations and the potential impact of pesticides on eight U. S. Crops. J. Econ. Entomol. 108:2141-2152. http://dx.doi.org/10.1093/jee/tov195
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Mullin, C. A. 2015. Effects of inactive ingredients on bees. Curr. Opin. Insect Sci. 10:194-200.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Fine, J. D. and C. A. Mullin. 2015. Toxicodynamics of the pesticide inert N-methyl-2-pyrrolidone and its impacts on honey bees. In: Pollinators and Agrochemical symposium, AGRO 208, 250th ACS National Meeting, Boston, MA. (Abstract)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mullin C., J. Chen, J. Fine, R. Reynolds and M. Frazier. 2015. Formulation composition makes the pollinator poison. In: Pollinators and Agrochemical symposium, AGRO 214, 250th ACS National Meeting, Boston, MA. (Abstract)


Progress 12/01/13 to 11/30/14

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 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, 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. 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. The pesticide conundrum: Protecting crops and pollinators. Department of Entomology and Nematology, University of California, Davis, CA. (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., 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? 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. Reynolds, R. and M. Frazier. 2014. Pesticide conundrum: Pesticides, neonicotinoids and pollinators. Western PA Fall Greenhouse Meeting, Wexford, PA. Nov. 6, 2014. 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? 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 of co-formulants such as NMP and their degradates, individually and corporately at sub-lethal levels, on key honey bee behaviors/physiology including memory and learning will be investigated. Toxic or sublethal effects on honey bees of pesticide and inert combinations relative to formulation controls, including interference with associative learning, will be determined by direct feeding or incorporation in artificial nectar or uncontaminated pollen or wax, or by topical application of extracts to bees or brood. Colony-level impacts of formulation ingredients will be determined in field experiments. We will disseminate the information generated by this study through regional and national websites in addition to appropriate professional publication and meeting venues.

Impacts
What was accomplished under these goals? 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. Recently we have shown that honey bees are sensitive to organosilicone surfactants, nonylphenol polyethoxylates and the solvent N-methyl-2-pyrrolidone (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 formulation ingredients like NMP are used and released into US environments. These synthetic organic chemicals are generally recognized as safe, have no mandated tolerances, and residues remain largely unmonitored. In contrast to finding about 70% of the pesticide active ingredients searched for in our pesticide analysis of beehive samples, we have found 100% of the other formulation ingredients targeted for analysis. These "inerts" overwhelm the chemical burden from active pesticide, drug and personal care ingredients with which they are formulated. Honey bees serve as an optimal terrestrial bioindicator to determine if 'the formulation and not just the dose makes the poison.' 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. 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. 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. We are also monitoring NMP and its major degradates in beehive samples. 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.

Publications

  • 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: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Mullin, C. A., J. Chen, Julia 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: 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: 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)