Honey Bee Responses to Blueberry Fungicides and Varroa Miticides While Used in NJ Blueberry Pollination Services

Progress report for GNE20-226

Project Type: Graduate Student
Funds awarded in 2020: $15,000.00
Projected End Date: 10/31/2023
Grant Recipient: Rutgers, The State University of New Jersey
Region: Northeast
State: New Jersey
Graduate Student:
Faculty Advisor:
Dean Polk
Rutgers University
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Project Information

Summary:

Beekeepers are experiencing over $1 million in annual losses when pollinating NJ blueberries. Fungicide use over the last decade has increased during bloom which correlates with colony declines. Therefore, investigations on the effects of fungicides are needed.

Initial work has shown that bees may prefer fungicide laced food sources, while other research has shown bee attractiveness to some fungicides. As part of my dissertation, I found 36 pesticides returned to the hive in pollen, including numerous fungicides. New papers point to the negative impact of fungicides on different bee species, including honey bees. My initial tests showed that some high fungicide rates caused larval mortality. Therefore, we need to determine the influence of blueberry fungicide use on foraging behavior as well as impacts on brood development.

My objectives include: 1) determining the influence of commonly used blueberry fungicides on honey bee foraging behavior, 2) determining if this behavior influences residues being introduced to the colony, and 3) determining the impacts of in-hive residues on brood development.

Research components include: Field studies that record honey bee forager visitations in number and consumption rate as influenced by plain syrup vs syrup with various fungicides; pollen and nectar analyses for pesticide residues in hives, and in vitro feeding tests with fungicide amended diet to examine resulting effects on brood and maturation.

Outreach will include extension newsletters, web-based blogs, and grower winter and spring update meetings. I will work with extension faculty to update the pollinator protection chapter in the NJ Blueberry Production Guide.

Project Objectives:

This project extends the work being done on an existing SARE grant, now coming to a conclusion in its 3rd and final season, but having opened additional questions relative to bee behavior and fungicide toxicity. The proposed work is designed to address those questions not addressed in the original SARE project.

 

Objective 1. Determine the influence of commonly used blueberry fungicides on honey bee foraging behavior.

 

Objective 2. Determine fungicide levels in honey bee hives, and when they are most likely to enter the hive in relation to fungicide spray programs.

 

Objective 3. Determine if there is any impact on honey bee brood development and resulting colony health by fungicide levels found in the hive as influenced by objectives 1 and 2.

Introduction:

The purpose of this project is to identify the behavioral and developmental effects of fungicide use on honey bee colonies used for blueberry pollination. I will be examining the impacts of fungicide use during bloom on bee forager behavior, resulting hive contamination, and effects on brood development. Declines in brood development during and shortly after pollination services have left beekeepers with severe colony losses resulting in up to 90% hive mortality. Thus, threatening the sustainability of commercialized blueberry pollination. The NJ blueberry industry is valued between $65-70 million annually, and is produced on just over 9,000 acres in the southern NJ Pinelands. NJ blueberry growers contract with NJ and regional beekeepers to bring in about 18,000 colonies each year for pollination. 

Hives with greater pesticide exposure show higher rates of death. In some cases multiple pesticides may show additive or even synergistic mortality effects to bees. Most older literature has focused on pesticide effects on adult bees, and most of this was done with insecticides. Only recently have researchers started to look at larval responses to pesticides, with fungicides being the newest frontier. Since fungicides are heavily used in blueberries during the pollination period, several areas need to be investigated.

I will be examining the behavior of adult foragers to commonly used fungicides, and the resulting residue levels brought back to the hive. Additionally, I will be looking at the developmental effects of fungicide contaminants.

By conducting larval bioassays with fungicide and miticide laced diets, I will identify pesticide combinations that pose risks to bee development. The identification of any negative impacts of fungicides can be used to support mitigation strategies and improve recommendations by Rutgers research and extension faculty. My findings will be presented at apicultural and agricultural meetings across the Eastern states. Such findings will improve honey bee health, impact pest management practices, and reduce beekeeper losses and reduce production costs for fruit growers.

Research

Materials and methods:

Behavioral tests for honey bee forager response to fungicides

The formulated fungicide products were mixed in sucrose syrup and compared to pure sucrose syrup alone. I used tunnel enclosures in a manner similar to that of Liao et al., 2017. I trained 5-framed hives with sister queens, consisting of approximately 3,000 honey bees (one per tunnel) to feed on 2 separate but identical sugar syrup feeders. Feeders consisted of red (non-see-through) inverted plastic containers with approximately 50 holes (1 mm in diameter) evenly dispersed just below the lid of each container (syrup dispenser). They were placed upon 2 separate cinder blocks oriented in a straight line on one side of the enclosure (opposite side of the hive). After two days of successful foraging at each feeder, I began the experiment. Each feeder was filled with the appropriate diet and weighed before and after each run to determine consumption rate. An experimental run consisted of 30-minute visual observations where the number of foragers visiting each feeder was recorded. 2 runs were conducted each day per hive and continued for a total of 5 days. Appropriate diet was replaced and measured as needed. On the 5th day, 4th and 5th instar larvae were visually inspected for adverse effects of treatment and used for comparison in behavior response analysis. Each experiment was repeated 4 times (over 4 weeks); to ensure there was no bias to feeder location, the feeders were placed in a new location each time. The formulated fungicides that were tested included ziram, azoxystrobin (Abound), metconazole (Quash), cyprodinil & fludioxonil (Switch), and boscalid & pyraclostrobin (Pristine). These tests included the 5 most commonly used FRAC groups: 3-DMIs, 7-SDHIs, 11-Qols, 9-Anilino-Pyrimidines, and M03-dithiocarbamates. Data on consumption rate and larval mortality was analyzed using analysis of variance between the 5 fungicide treatments.

This study was improved in 2022 using potted blueberry bushes plants and the same 5 fungicides. There were two treatment groups, 1) Day Spray; bushes sprayed immediately prior to visual observation and 2) Night Spray; bushes sprayed 12 hours prior to visual observation. Each colony was presented with two new bushes each day, just prior to beginning a 30-minute visual observation, in individual flight tunnels. One of these bushes were sprayed with fungicide and the other was sprayed with water according to their treatment (sprayed at night or during the day). This study was repeated for 5 consecutive days using the same colony in each treatment and fungicide group (each colony was only used for 1 week and was in the same treatment group using the same fungicide). At the end of each trial (5 days), new colonies replaced the original colonies and were given 2 days to forage from bushes that hadn't been sprayed. Trials repeated for 3 consecutive weeks (15 total visual observations per treatment per fungicide; 150 total observations). 

Determination of fungicide levels as reflected by honey bee foraging behavior.

Based on the response of foragers to fungicides, nucleus hives will be placed in the centers of commercial fields and monitored for fungicide residues in pollen, bee bread, and nectar. I will coordinate residue sampling with commercial grower participants so that a total residue load in the colony’s food stores can be calculated. I have previously determined various residues that are present during the middle of bloom. However, this ‘1 shot’ sampling does not give us information over time, or enable a correlation with fresh fungicide applications with the developmental stage of the bee. I will collaborate with 4 blueberry growers who each have individual farm sites of over 200 acres. Four nucleus hives will be placed in the centers of each of these 4 farms so that the foraging ability of the bees is mostly restricted to those fields, and any fungicide residues found will be reflective of that growers’ spray programs. At each farm site, 2 hives will be fitted with pollen traps to prevent the entry of foraged pollen. Each of these 2 hives (treatment hives) on each farm will be provided substitute pollen patties (Mann Lake) as a protein source. The other 2 hives (standard) on each farm will not have pollen traps and will be freely exposed to all pollen and nectar being returned to the hive. This gives an experimental design with 2 treatments and 4 replicates. After each fungicide application (8 to 24 hours, depending on application method), pollen will be sampled along with an internal hive sampling of nectar from 1 of the 2 pollen trapped hives. This will be done every 48 hours for 7 days, or until the next application, upon which time the process will start over. The other pollen trapped hive will not be sampled, but all 4 hives will be examined for parameters of hive health, and pollen+ bee bread samples taken every 7 days. These measurements include hive weight, number of frames with brood, percent brood coverage, egg and queen presence, and disease incidence. At the end of pollination (~3.5-4 weeks), hives will get a final sampling for hive health and residue samples. Data on hive parameters and residues will be analyzed using analysis of variance.

 

Fungicide impact on honey bee brood development

Using the fungicides found to be attractive, if any in objective 1, and those fungicides found at the highest residue levels in objective 2, I will examine those fungicides alone and in combination with miticides used by beekeepers to treat varroa mites, in in-vitro feeding tests. Each residue will be tested in its pure active ingredient form and by formulated product. The concentrations of each will be tested on a gradient starting with the highest concentration found from objective 2 in both pollen and nectar. Additional tested rates will be cut in half in serial divisions, resulting in a minimum of 5 tested dietary concentrations. A control of pure diet, and an acetone control (solvent) will be included. Laboratory procedures will be adapted from Schmehl et al., 2016.  I will establish eight 5-framed honey bee hives (nucleus hives) with newly mated sister queens. Inside each nucleus hive there will be a caged frame on empty drawn comb, this frame will be swapped out every week (four days prior to larvae extraction) to ensure all larvae will be of the exact age. 1st instar larvae will be grafted to 48 well-plates and fed fungicide and miticide laced diets with concentrations corresponding to pollen residue samples with 12 larvae per treatment and each treatment will be replicated 4 times. Larvae will be fed daily until the fifth day where they will be then transported to a pupation incubator (which serves as capped cells) until adult emergence. Dosing of fungicide active ingredients will occur only on day 1-3. Mortality will be recorded at 24, 48, 72, 96, and 120 experimental hours and on the 21st day after adult emergence. All grafting, feeding, and data collection will be conducted inside a positive laminar flow hood which will be sterilized for 30-minutes before and after each procedure via UV light and by wiping down with 10% bleach.  Subjects which complete pupation and achieve adult stage will be measured (head dimensions, length and width and weight of body) and compared across treatments for additional sublethal effects. Data on larval mortality and adult measurements will be analyzed using analysis of variance between rate treatments and probit analysis for mortality rates vs pesticide concentration.

Research results and discussion:

The 2021 forager behavioral study resulted in all foragers showing preference to dispensers with pure sugar syrup (lower visits and consumption rates to sugar syrup laced with fungicides). These results did not support grower reports of bees landing on puddles of freshly sprayed fungicide to forage from. When the study was improved in 2022, using potted blueberry bushes which were sprayed with fungicides, results varied. While foragers did not show a preference between a bush sprayed with water and a bush sprayed with either Quash, Pristine, or Switch, foragers showed a strong preference to in the treatments where bushes were sprayed with either Ziram or Abound. Ziram had higher forager visits than the bush sprayed with water, suggesting that it attracts foragers. Abound had lower forager visits than the bush sprayed with water, suggesting it acts as a repellent. 

Larval toxicity work is still under investigation. I'm currently working on establishing the baseline of LD50's for all 5 fungicide active ingredients (Abound; Azoxystrobin, Quash; Metconazole, Pristine; Boscalid & Pyraclostrobin, Switch; Cyprodinil & Fludioxonil, and Ziram; Zinc Dimethyldithiocarbamate) and 1 miticide active ingredient (Apivar; Amitraz). Once this is established, synergistic work will begin using Amitraz in conjunction with each of the active ingredients found in the above-mentioned fungicides. So far, Ziram has shown to be the most toxic when fed to honey bee larvae, it exceeds the LC 25 at 3 ppm and the LC50 at 24 ppm. 

Fungicide residues have been found in all pollen samples collected during blueberry bloom. This data will be applied to a Pollen Hazard Risk Assessment using LD50's to determine the risk which each fungicide poses to honey bees. Due to the lack of honey production during blueberry pollination, collecting samples of honey for residual analysis may not be possible, at this time. 

Research conclusions:

Ziram had the highest mortality rate out of all 5 tested fungicides when fed to larvae, followed by Pristine, Switch, Abound, and Quash. 

Ziram had the highest visitation rate of foragers when applied to potted blueberry bushes. 

Abound had the lowest visitation rate of foragers when applied to potted blueberry bushes. 

Based on these results, Ziram should not be used when bees are present because it attracts foragers and kills larvae. 

Participation Summary
6 Farmers participating in research

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

Outreach activities will be divided into 1) Extension based to blueberry growers and to commercial and hobbyist beekeepers; and 2) To professional journals and the commercial beekeeping industry.

Extension based information to growers and beekeepers: I will present my work at 2 annual blueberry grower meeting reaching at least 70 growers and 1 statewide NJ Beekeepers Association (NJBA) meeting each year reaching about 150 beekeepers (total 3 per year), and will contribute 1 article annually to the online NJBA newsletter. This online newsletter reaches over 1,000 NJ beekeepers, many of whom have already donated to my research efforts. These meetings will include the blueberry session at the Atlantic City NJ Agricultural Convention each January, the annual Blueberry Open House in Hammonton, NJ in late February, and twilight fruit grower update meetings in Hammonton during the spring season. I will work with commercial beekeepers throughout the year, communicate by email and phone, make annual reports available to them and hold 1 informal meeting per year, likely during the winter. I will contribute efforts to the Rutgers Blueberry Production Guide, so that bee safety information is updated.

Professional journals and commercial beekeeping industry: I will target articles for the American Bee Journal, and on a more scientific level, The Journal of Apiculture Research. I will present my work at the Eastern Branch of the Entomological Society of America (ESA), as well as the National ESA meetings, and at the regional Cumberland-Shenandoah Fruit Workers Meeting (95-100 extension, researchers, industry and graduate students from the mid-Atlantic to New England states).

Project Outcomes

44 Farmers reporting change in knowledge, attitudes, skills and/or awareness
11 Farmers changed or adopted a practice
5 Grants applied for that built upon this project
5 Grants received that built upon this project
$377,000.00 Dollar amount of grants received that built upon this project
1 New working collaboration
Project outcomes:

A direct outcome of my project is an ordered pollinator-risk ranking of the top five fungicides used during New Jersey blueberry bloom. I've found one fungicide, Ziram, in particular which exceeds the LC50 (Lethal Concentration which kills 50% of subjects) when fed to larvae at realistic concentrations. That same fungicide was also found to attract foragers. While, Abound is significantly less toxic to larvae and acts as a repellant to foragers. With this knowledge, I have and will continue to discuss the implications of these results with growers and beekeepers alike at conferences, during seminars, at meetings, and with one-on-one discussions between growers, beekeepers, and other extension agents in my field. 

As growers implement this new understanding, beehive rental prices will stabilize and beekeepers will experience less colony losses due to blueberry pollination. This will improve the economic sustainability of pollination services for both beekeepers and growers while bringing the two industries closer together with a better understanding of bee health, crop demands, and pollinator safety. As the communication/science gap is closed, this will restore and improve grower and beekeeper relationships as they will work together to ensure the safety of their bees and the success of their crops. 

A survey of 44 growers reported an improvement in their knowledge and understanding of pollinator health and safety. At least 6 growers and 5 beekeepers have changed their practices based on the results of this project. Of which, some growers now spray exclusively at night (approximately 500 blueberry acres), another grower has reduced his fungicide applications from 3 to 1 while beehives are present (>200 acres of blueberries), and all 6 growers have stopped using Ziram (the most toxic fungicide) when beehives are present. Beekeepers no longer use the miticide, Apivar, which was found to have significantly negative synergistic impacts on honey bee larvae when applied in conjunction with DMI fungicides.

This work has led to the collaboration with other blueberry scientists who seek to improve the recently (10-15 years) decreasing blueberry yields via improving soil health. I hypothesize that by improving soil health, we will improve the nutrient availability in blueberry pollen and thus increase blueberry and honey yields in New Jersey blueberry crops. Upon the completion of my PhD program, I would like to repeat this work in cranberry bogs which have also reported recent declines in New Jersey crop yields. 

Knowledge Gained:

I gained skills in data collection and analysis, communicating science with industries (growers, beekeepers, and pesticide companies), and am beginning to learn how factsheets can be used as a form of extension when scientific results are difficult (or complex) to communicate to industries in an effective manner.
This project has inspired me to continue my work in extension because I have had the opportunity to tackle current problems with science, digest the results, design new experiments to learn how the original results might be used, communicate that knowledge with the industries, and then see how those changes can improve our working relationships, environment, and economy. 

I wish to pursue a career in pollinator and fruit IPM extension where I will be in a position to reduce our dependency on environmentally harmful pesticides while improving the economic sustainability of honey and fruit production. My research will continue to investigate the impacts of pesticides on our environment (pollinators, soil health, and yields) and seek to understand how IPM "tools" (forms of pest control) can be used most efficiently to increase yields and improve both human and environmental health.

Assessment of Project Approach and Areas of Further Study:

When I conducted my first forager behavioral study to examine the preference of foragers to plain sugar syrup and sugar syrup laced with fungicides, I found that all 5 fungicides tested acted as a repellent to foragers, meaning foragers preferred to "drink" from plain sugar syrup over fungicide laced syrup. However, this evidence did not support grower reports of bees landing on puddles of freshly sprayed fungicide to "drink". When I improved the experiment in 2022 using freshly sprayed potted blueberry bushes, I found opposing results. While 3 fungicides didn't seem to impact forager visits, 1 fungicide acted as a repellent (Abound), and another acted as an attractant (Ziram). With this knowledge I hope that future efforts in understanding forager preference to pesticides will be conducted on living plants, instead of using sugar syrup in dispensers, as this method was not as reliable. 

When it comes to extension, growers and beekeepers have repeatedly expressed their need for "simple" science. While scientists are accustomed to presenting their research in great detail, this information can often be received as an "overload" of information to the people that comprise the industries that we support. I have learned to keep these extension talks, short and sweet. Additionally, when new practices are being recommended, a simple factsheet will assist in the implementation of sustainable practice changes.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.