Final Report for LNC09-316

Project Type: Research and Education
Funds awarded in 2009: $175,000.00
Projected End Date: 12/31/2010
Region: North Central
State: Minnesota
Project Coordinator:
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Project Information

Summary:
Imidacloprid residue in flowers and effects on colony health and foraging of bees

This study determined the effects of higher amounts of imidacloprid aplied to soil on residue in canola pollen, and the effects of imidacloprid in sugar syrup on honey bee and bumblebee colony health.

Introduction:

Imidacloprid residue in flowers and effects colony health and foraging of bees

Since 1990 the neonicotinyl insecticides, imidacloprid, thiamethoxam, clothianidin, and dinotefuran, were implicated in the decline of bees as they accumulate in pollen and nectar, are systemic, and are expressed for years from a single application (Doering 2004, 2005, EPA 2007, Blacquiere et al. 2012). Neonicotinyls are applied in various ways (seed treatments, soil drenches, foliar sprays, irrigation systems, tree injections) on agricultural and landscape plants. Most genetically modified crops (corn, canola, and soybeans) use seed treatments of imidacloprid (Gaucho), clothianidin (Poncho), or thiamethoxam (Crusier) (Farm Press 2003). The annual market for neonicotinyl insecticides is in the billions of dollars due to their low mammalian toxicity, systemic nature, and extended efficacy (Aliouane et al. 2009). In the U.S., at least 143 million acres of the total 442 million acres of cropland are treated with over 2 million pounds of imidacloprid, clothianidin, and thiamethoxam (Pilatic 2012). In 2009 in Minnesota, where most crops use seed treatments, corn, soybeans, potatoes and canola used 46,766 pounds of imidacloprid and 19,347 pounds of clothianidin (MDA 2012). Most research focused on the effects of <10 ppb of imidacloprid that was found in nectar and pollen of neonicotinyl-seed treated crops, such as canola, cotton, corn, soybean, and sunflower, on bee foraging, memory, and colony health. However, new residue data found 50-5,000 ppb in flowers of landscape plants and some flowering crops.

Residue levels of neonicotinoids in pollen and nectar differ depending on application method in crops and landscapes. Gaucho, an imidacloprid seed treatment of ?1.0 mg AI/seed, depending on the crop (Bonmatin et al. 2005, Girolami et al. 2009), resulted in 4.4-7.6 ppb imidacloprid residue in canola pollen, 3 ppb in sunflower pollen, and 3.3 ppb in maize pollen (Scott-Dupree and Spivak 2001, Bonmatin et al. 2005, EFSA 2012).

Landscape applications of imidacloprid result in much higher levels of residue in nectar and pollen. A homeowners’ formulation of imidacloprid, Bayer Advanced Tree and Shrub, or professional Marathon 1% G permits 270-300 mg AI to be applied to a 3 gallon pot, resulting in a 400 times higher application rate compared to Gaucho treated corn of 0.675 mg AI/seed. Doering et al. (2005) found 1,038–2,816 ppb in Cornus spp., dogwood flowers, at 17 months after application. A soil injection around Eucalyptus trees resulted in 660 ppb imidacloprid in nectar (Paine et al. 2011).

Field and cage studies that exposed bees to higher amounts of neonicotinyl-treated sugar syrup have been repeatedly shown to reduce colony health and bee foraging. Foraging was reduced at 10 ppb imidacloprid for B. terrestris (Mommaerts et al. 2010, Gill et al. 2012) and 30 ppb imidacloprid for B. impatiens (Morandin and Winston 2003). Honeybee foraging was reduced at 15 ppb imidacloprid (Schenider et al. 2012), 5 ppb clothianidin (Schenider et al. 2012), and 67 ppb thiamethoxam (Henry et al. 2012).

Project Objectives:
Imidacloprid in canola pollen and soil and effects on bee health

Our research objectives were:
Objective 1. Determine thru residue analysis how much imidacloprid (Admire Pro) is translocated to pollen of canola from a soil treatment and seed treatment. We studied 8 treatments: 1)non-seed treated canola , 2)imidacloprid-seed treated canola, 3)clothianidin seed-treated canola, 4)controls, 5)1X label rate applied to soil (4mg/sgft), 6)2X label rate applied to soil (8mg/sgft), 7)20X field rate or 25% landscape rate applied to soil or (80mg), and 8)40X field rate or 50% landscape rate applied to soil (160mg).

Objective 2. Determine the effects imidacloprid in sugar syrup on the behavior and colony health of bumble bees in a greenhouse at 10, 20, 50, and 100 ppb imidacloprid and clothianidin.

Objective 3. Determine the effects imidacloprid in sugar syrup on the behavior and colony health of honeybees on the St. Paul campus of the University of Minnesota at 0, 50, 100, and 200 ppb imidacloprid.

Objective 4. Implement research results through an outreach website and write publications on landscape management to increase foraging habitat for bees, nectar plants available for bees and, effects of insecticides on bees. See CUES website on Pollinator Conservation http://www.entomology.umn.edu/cues/pollinators/index.html

Research

Materials and methods:
Residue in canola pollen

Our data demonstrated that imidacloprid or clothianidin is not found in canola pollen from imidacloprid- or clothianidin-seed treatments and 1X imidacloprid label rate (4mg/sgft) of soil drench treatments planted in a previously non-treated plot. However, the greater the amount of imidacloprid applied to the soil, the more is found in canola pollen. The higher landscape rates resulted in much higher levels in canola pollen compared to crop rates. In addition, non-treated plants that were grown on a previously treated plot had imidacloprid residue in the canola pollen. Also, when 2 crops were sequentially planted on one plot, the residue in canola pollen was much higher in the canola pollen from the second planting than in the first planting. Other research supports our findings and demonstrated that 10% of the active ingredient is absorbed by the crop, while the remaining imidacloprid is bound to organic material in the soil and can be picked up by the next plants grown on the site (Goulson 2013).

Seed-treated canola and non-seed treated canola was grown on 2 replicate plots to which was applied an imidacloprid (Admire Pro) drench to the soil after planting. The residue in the soil and in canola pollen was determined. Our data showed on previously non-treated soil that neither imidacloprid-, clothianidin-, nor untreated-seed resulted in imidacloprid in canola pollen in 2 replicate plantings (April 5 2010 and May 19 2010). Single imidacloprid soil drenches on previously non-treated soil for 2 replicate plots resulted in: 0X =0 ppb, 1X (4mg/sgft) =0 and 0 ppb, 2X (8 mg/sgft) =14 and 7 ppb, 20X (80 mg/sgft) =461 ppb and 15 ppb, and 40X (160 mg/sgft) =2071 ppb and 341 ppb.

The April 5 2010 plot was subjected to a second imidacloprid application on July 2. Two imidacloprid applications (April 5 and July 2) resulted in higher residue in canola pollen (compared to one treatment in the same plot of April 5): 0X =0 ppb, 1X=313 ppb (0 on April 5), 2X=179 ppb(14 on April 5), 20X=342 ppb (461 on April 5) and 40X=3860 ppb(2072 on April 5).

Consequently, imidacloprid remains in the soil after the first crop is harvested and is translocated from the soil to the canola pollen in the second planting. We measured the residue of imidacloprid in the soil after two plantings on the same plot (April 5 and July 2). We found that the twice treated plots showed higher soil residues: 0X =0ppb, 1X=1552 ppb, 2X=743 ppb, 80X=1816 ppb and 160X=9727 ppb

In addition, we determined the residue in canola pollen of a second crop (July 29) that was not imidacloprid-treated but grown on a previously treated May 19 plot. We found that the 3 seed- treated treatments and the control showed no residue in the pollen, but the second planting translocated imidacloprid from the soil to the pollen: 0X =0ppb, 1X=5ppb, 2X=8 ppb, 80X=24 ppb and 160X=162 ppb. We found that the twice planted plot that was only treated at the first planting showed high soil residues: 0X =1316 ppb, 1X=199 ppb, 2X=257 ppb, 80X=517 ppb and 160X=3913 ppb.

This research demonstrates that imidacloprid is translocated from soil to pollen above 2X agricultural soil rates and imidacloprid remains in the soil and is picked up by the second planting of a crop. Consequently, crops and volunteer flowering plants grown in the same plot will have higher imidacloprid levels each consecutive year. Landscape rates are much higher (300 mg/sgft) compared to agricultural rates (4mg/sgft) and result in much higher imidacloprid levels in canola pollen.

Imidacloprid in sugar syrup and effects on bumblebees

In 2011-2012, in an 11-week greenhouse study, caged queenright colonies of Bombus impatiens Cresson, were fed 0, 10, 20, 50 and 100 ppb imidacloprid or clothianidin in sugar syrup. Neonicotinyl treatments used in this study ranged from the highest amount found in seed-treatments (10 ppb) to levels found in landscape plants (20-100 ppb). Our highest concentration of 100 ppb was below the estimated oral LC50 for honey bees of 185 ppb (CA EPA 2009) or 192 ppb (Bayer, Fischer and Chalmers 2007). Both of these neonicotinyls had similar toxicity, as expected by their similar acute oral LD50s: for imidacloprid 4-40 ng/bee for honey bees (Decourtye and Devillers 2011,EFSA 2012) and 2 ng/bee for bumblebees (Van der Steen 2008) and for clothianidin 22 ng/bee for honey bees (Iwasa et al. 2004, EFSA, 2012).

Our study demonstrates that 20 ppb imidacloprid or clothianidin fed to queenright colonies of B. impatiens for 11 weeks increased queen mortality (Figure 1) and reduced colony consumption (Figure 2), colony weight (Figure 3), wax syrup pots that were added (Figure 4), and male production (Figure 5). Neither neonicotinyl decreased worker and queen production. Starting at 6 weeks, queen mortality was significantly higher in 50-100 ppb and by 11 weeks in 20-100 ppb imidacloprid- and clothinaidin-treated colonies. As queens started to die at week 6, workers in 20-100 ppb treatments produced fewer males, but did continue to invest in new queen (gyne) production

In 0 ppb treatments, bees secreted wax and added it to the colony nest structure to make new sugar syrup pots, gathered sugar syrup from small containers in the flight box, and filled the wax pots with sugar syrup, thereby increasing the number of stored syrup pots, the weight of the syrup wax pots, and the entire colony weight. In higher neonicotinyl treatments, the number of wax syrup pots (50-100 ppb imidacloprid, 10-100 ppb clothianidin) and the total colony weight (10-100 ppb imidacloprid, 20-100 ppb clothianidin) were reduced. For both imidacloprid and clothianidin, the residue in wax syrup pots for 20-100 ppb was 50-100% less residue than the concentration in the syrup the bees were consuming (Table 1), indicating that syrup was not being returned to the pots in higher treatments. In this study, lethargic behavior of flight box and nest bees was correlated to reduced foraging and colony consumption. In higher neonicotinyl treatments, nest bees emptied the storage pots present prior to treatment and did not re-fill old pots. Queen and nest bees fed on sugar syrup and Bee Happy stored prior to the start of the experiment. This is further supported by the reduction in colony consumption at 10-100 ppb. We speculate that nest bees that went into foraging boxes to collect neonicotinyl-treated sugar syrup were impaired as a result of ingesting and detoxifying the insecticides, fed less, and returned less syrup to the colony. We argue that queen mortality at 20-100 ppb was related to lack of incoming syrup and the resulting lack of storage of this syrup in wax pots. Since neonicotinyls in this and other studies were shown to reduce food consumption and foraging, wild bumblebee colonies that depend on workers to forage will be negatively affected by chronic exposure to imidacloprid starting at 10- 20 ppb

Our data provide strong support that chronic exposure to imidacloprid or clothianidin above 20 ppb significantly reduced colony health (lower colony weight, less wax pots added, and higher queen mortality) as a result of decreased worker foraging (consumption, movement, and storage of syrup). Sublethal chronic effects on foraging were found in 12 research papers discussed above. Neonicotinyl insecticides are neurotoxins that affect vision, olfaction, learning, and memory (Gauthie 2010, Tome et al. 2012) and bind to mushroom bodies in bee brains (Tome 2012) which are particularly large in social bees compared. Bees fed 13 ppb or 23 ppb imidacloprid were less likely to form long-term memory and had reduced learning (Willamson and Wright 2013) and at 24 ppb imidacloprid performed fewer communicative waggle dances (Eiri and Nieh 2012). The ubiquitous use of neonicotinyl insecticides on crops and landscape plants throughout the season will lead to chronic sublethal and lethal effects on worker foraging and colony health. Social bee colonies, such as bumblebees and honey bees, rely on division of labor and need foragers to return nectar to the hive for the queen and brood. Native, annual bee colonies or bumblebee queens in spring and fall are even more vulnerable to neonicotinyl insecticides since the solitary queens can be impaired when foraging. Since most studies show reduction in foraging behavior below 10 ppb and residues in crop and landscape flowers are higher than 10 ppb, bees are likely to be experiencing chronic, sublethal doses with consequences on queen and colony health. The 20 year research focus on residue levels below 10 ppb of neonicotinyl insecticides found in nectar and pollen of seed-treated crops (corn, canola, and sunflower) has made us preoccupied with the lowest rates used in agriculture. Much higher rates and chronic exposure at low doses will lead to colony failure thru reduced navigation and foraging.

Imidacloprid in sugar syrup and effects on honey bees

In 2011, honeybee colonies (n=10 colonies/trt) were provided 0, 50, 100, 200 ppb imidacloprid in sugar syrup for 15 weeks and colonies were assessed five times: June 8, July 6, August 3, August 31, and September 21 for 16 parameters of colony health: frames of bees, open brood, sealed brood, total brood, pollen stores, missing cell count, brood pattern, returning pollen foragers, percent returning foragers, sugar syrup consumption in 48 hrs and 1 week, dead bee counts, Varroa numbers, Nosema numbers, virus (distorted wing virus(DWV), black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV)) and queen mortality.

Frames of bees (Figure 1) was not affected by imidacloprid treatment. Only sealed (Figure 2) and total brood (Figure 3), pollen stores (Figure 4), and returning foragers (Figure 6) were reduced in 200 ppb imidacloprid compared to 0, 50, 100 ppb treatments. Nosema numbers (Figure 5) and DWV (Figure 9) were highest in late summer in 200 ppb treatments. However, imidacloprid in stored nectar was around 33 % less in all treatments indicating mixing of nectar with nectar from foraging in the field (Table 1). The levels of imidacloprid found in nectar are below LD50 of 185-192 ppb found in studies (CA EPA 2009, Bayer, Fischer and Chalmers 2007) and should not kill bees on ingestion.

There was no correlation of treatment with dead bee counts and queen replacement (Figure 8) was higher in 100 but not 200 ppb treatments from only August 26 – September 22, not the entire experimental period, indicating a nontoxic effect of imidacloprid. Reduction in returning foragers, pollen stores, and colony consumption (Figure 7) indicate a sublethal effect of chronic exposure of imidacloprid on bee foraging. These data support accruing scientific data on the sublethal effects of imidacloprid on bee foraging. In addition, DWV was higher in 50, 100, and 200 ppb treatments compared to controls and BQV was higher in only 100 ppb treatments. One phorid fly, Apocephalus borealis, was found in the 200 ppb imidacloprid treatment. Microarray analyses of larvae and adult phorids and honey bees from phorid-infected hives revealed that bees are often infected with deformed wing virus and Nosema ceranae (Core et al. 2012). A metagenomic analysis of showed that honey bees from CCD-positive colonies had four pathogens: two viruses and two species of microsporidia, Nosema spp. (Cox-Foster et al. 2007).

Research results and discussion:
Imidacloprid is translocated to pollen and alters colony health of bees

These data support accruing scientific data on the sublethal effects of imidacloprid on bee foraging.

Our research found:
Objective 1. Imidacloprid residue in canola pollen.
Our data demonstrated that imidacloprid or clothianidin is not found in canola pollen from imidacloprid- or clothianidin-seed treatments and 1X imidacloprid label rate (4mg/sgft) of soil drench treatments planted in a previously non-treated plot. However, the greater the amount of imidacloprid applied to the soil, the more is found in canola pollen. The higher landscape rates resulted in much higher levels in canola pollen compared to crop rates. In addition, non-treated plants that were grown on a previously treated plot had imidacloprid residue in the canola pollen. Also, when 2 crops were sequentially planted on one plot, the residue in canola pollen was much higher in the canola pollen from the second planting than in the first planting. Other research supports our findings and demonstrated that 10% of the active ingredient is absorbed by the crop, while the remaining imidacloprid is bound to organic material in the soil and can be picked up by the next plants grown on the site (Goulson 2013).

Objective 2. Imidacloprid effects on bumblebees.
These data support accruing scientific data on the sublethal effects of imidacloprid on bee foraging. Our study demonstrates that 20 ppb imidacloprid or clothianidin fed to queenright colonies of B. impatiens for 11 weeks increased queen mortality and reduced colony consumption, colony weight, wax syrup pots that were added, and male production. Neither neonicotinyl decreased worker and queen production. Starting at 6 weeks, queen mortality was significantly higher in 50-100 ppb and by 11 weeks in 20-100 ppb imidacloprid- and clothinaidin-treated colonies. As queens started to die at week 6, workers in 20-100 ppb treatments produced fewer males, but did continue to invest in new queen (gyne) production.

Since neonicotinyls in this and other studies were shown to reduce food consumption and foraging, wild bumblebee colonies that depend on workers to forage will be negatively affected by chronic exposure to imidacloprid starting at 10- 20 ppb.

Objective 3. Imidacloprid effects on honey bees.
These data support accruing scientific data on the sublethal effects of imidacloprid on bee foraging. Frames of bees was not affected by imidacloprid treatment. Only sealed and total brood, pollen stores, and returning foragers were reduced in 200 ppb imidacloprid compared to 0, 50, 100 ppb treatments. Nosema numbers and DWV were highest in late summer in 200 ppb treatments. However, imidacloprid in stored nectar was 33% less for all treatments indicating mixing of nectar with nectar from foraging in the field. The levels of imidacloprid found in nectar are below LD50 of 185-192 ppb found in studies (CA EPA 2009, Bayer, Fischer and Chalmers 2007) and should not kill bees on ingestion.

Objective 4. Outreach program. See CUES website on Pollinator Conservation http://www.entomology.umn.edu/cues/pollinators/index.html

Research conclusions:
Pollinator conservation website and materials

Outreach results
A new pollinator conservation site was posted on the CUES website that contains 11 sections on education and research: value of bees, types of bees, bees and pesticides, colony collapse disorder, conservation and habitat, plants for pollinators, bumble bee conservation, US EPA and pollinators, European Union and pollinators, online 5 part workshop for Master Gardeners, Krischik research on bees, beneficial insects, and butterflies. We developed a bulletin on ready to use consumer insecticides and their toxicity to bees, poster on best bee plants, and a pollinator conservation bulletin with agricultural and consumer insecticides and effects on bees See CUES website on Pollinator Conservation http://www.entomology.umn.edu/cues/pollinators/index.html

The PI discussed her research results with Steve Ellis and Jeff Anderson, two Minnesota beekeepers and presented talks at the annual meeting of the Minnesota Honey Producers. The funds on the grant supported a Master Student who worked on the bumble bees and is graduating in August 2013. Numerous talks were provide on the research: Wild Ones Native Plant Society, Minneapolis; Fruit and Vegetables Conference, St. Cloud; MN Rose Society, Minneapolis; Anoka County Master Gardeners Meeting, Maplewood; MN State Fair Bee keepers, St. Paul; MN Honey Producers summer meeting, Minnesota Green Expo, MNLA (MN Nursery and Landscape Association)

Economic Analysis

Loss of bees is a disaster for seeds and fruits

Honey bees, bumblebees, and other native bees pollinate 30% of the plants that produce the vegetables, fruits, and nuts that we consume (Klein et al. 2007). According to the Department of Agriculture (USDA), more than 100 crops in North America require pollinators. Pollination contributes approximately $15 billion worth of additional crop yields (Morse and Calderone 2000), and wild bees contribute substantially to crop production (McGregor 1976, Garibaldi et al. 2013).Bees are needed for pollination and we need to understand the impacts of systemic insecticides on bee colony health and foraging.

In 2007, there were 49.5% fewer managed honey bees (Apis mellifera) colonies in North America than in 1961 (van Engelsdorp and Meixner 2010). Managed honey bee colony mortality has been estimated to be 30% since 2007 (USDA 2011, van Engelsdorp et al. 2013). Colony stressors include habitat loss, nutrient deficiencies, Nosema pathogens (Higes et al. 2008, 2009), viruses (Cox-Foster et al. 2007), Varroa mites (van Engelsdorp et al. 2013), pesticide exposure (Johnson et al. 2010, Frazier et al. 2008, 2011), interactions between Nosema and imidacloprid (Alaux et al. 2010, Petits et al. 2012), and Nosema and fipronil (Vidau et al. 2011, Aufauvre et al. 2012). Additionally, North American bumblebee species Bombus occidentalis B. pensylvanicus and B. affinus are in decline. These species had significantly higher N. bombi loads and lower genetic diversity compared to healthy populations (Winter et al. 2006, Cameron et al. 2010). A combination of factors is most likely to contribute to bee losses (Fraiser et al. 2011, Blacquiere et al. 2012).

Farmer Adoption

Beekeepers Steve Ellis and Jeff Anderson participated in the September 2011 honeybee colony assessment. Throughout the project the research was discussed with these beekeepers and data were presented at the annual meeting on the MN Honey Producers from 2010-1012. These bee keepers are using this information and other research to request EPA policy changes on imidacloprid and other neonicotinyl insecticides due to residue levels and effects on bees.

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:
Publications and outreach

From this research we will develop peer reviewed publications and outreach educational materials.
1. Effects of seed treatments and soil applications of imidacloprid on residue in canola pollen and soil. submit Oct 2013
2. Effects of imidacloprid in sugar syrup on bumblebee colony health. submit July 2013
3. Effects of imidacloprid in sugar syrup on honey bee colony health. submit Septemebr 2013
4. Pollinator conservation website. http://www.entomology.umn.edu/cues/pollinators/index.html done
5. Pollinator conservation online workshop for master gardeners at pollinator conservation website. done on website
6. Pollinator conservation poster.done on website
7. Pollinator conservation brochure. done on website
8. Bees and pesticides brochure. done on website

Project Outcomes

Recommendations:

Areas needing additional study

Future research: levels of imidacloprid residue in landscape plants and crops and field tests on effects on bumblebees

One of the major deficits in knowledge is how much neonicotinyl insecticide is found in pollen and nectar of neonicotinyl–treated plants in the field, besides seed-treated crops. A canola seed is covered with 0.11 mg active imidacloprid (neonicotinyl chemical) that results in 7.6 ppb imidacloprid pollen. In urban landscapes, where bees forage for pollen and nectar, a soil surface application of imidacloprid can be applied to a native plant (300 mg) and basswood tree (67 g) from which basswood honey is produced We calculate that a 609,000 times greater amount of imidacloprid is applied to basswood trees compared to a canola seed. We do not know how much imidacloprid accumulates in pollen and nectar from these applications in the landscape and field.

Future objectives are: 1. Determine imidacloprid residue in pollen and nectar of basswood trees from an imidacloprid soil and trunk injection; 2. Determine imidacloprid residue in pollen and nectar of native flowers, squash, and blueberry from soil applied imidacloprid; 3. Determine the imidacloprid residue in native plants around imidacloprid-treated crops; and 4. The impacts of these imidacloprid residues need to be assessed on field colonies on native bumblebee colonies.

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.