Pollinators, including honey bees, are responsible for the successful reproduction of more than 87% of flowering plant species: therefore, they are vital to ecosystem health and agricultural services world-wide [1
]. Agriculture depends strongly on the services of the honey bee, which is responsible for providing more than 90% of commercial pollination services [2
]. Thus, elevated loss rates seen in managed honey bee colonies [3
] threatens the pollination services they provide. A variety of different stressors—including pathogens, parasites, floral resource loss, environmental stresses, and socioeconomic factors—have been suggested as contributing to the elevated levels of bee population decline [4
]. One stressor, exposure to pesticides, has been, and remains, an important focus for research.
Until the early 1980s, the primary, if not exclusive, exposure to pesticides experienced by honey bees, and the source of hive product contamination, was from pesticides applied to agricultural or horticultural landscapes. With the introduction, nearly three decades ago, of the parasitic Varroa
mite (Varroa destructor
) into North America, concerns about pesticide residues in wax, honey, bees, pollen, bee bread, and other honey bee-related matrices expanded to include residues resulting from direct application of pesticides within colonies [14
]. The most frequently reported compounds in honey bee products are acaricides used to control V. destructor
, including coumaphos, fluvalinate, and amitraz; however, many other compounds have been detected, including some at levels that may result in sublethal and lethal outcomes [20
To date, studies of pesticide residue in honey bee colonies have been cross-sectional. Residues have been evaluated in a variety of matrices, including but not exclusive to honey, beebread/pollen, wax, propolis, and bees [15
]. These cross-sectional studies have described contaminants from samples obtained from multiple colonies/hives at a single time during the foraging season, or after the application of an acaricide to colonies. Most have investigated a limited range of pesticide residues, those primarily resulting from the management of honey bee pests [15
]. Some studies used analytical methods capable of detecting of a wide range of pesticide residues found in the foraging environment as well as those used in colony management [18
Mullin et al. [22
], in their cross-sectional study of North American apiaries, detected 121 different herbicides, fungicides and insecticides, along with metabolites, in multiple matrices, e.g., wax, pollen, bee bread, and bees. In 259 wax samples, 87 compounds were detected with a mean of eight detections per sample. One sample contained residues of 39 compounds. The four most frequently detected pesticides in wax were fluvalinate (98.1%), coumaphos (98.1%), coumaphos oxon (89.9%, a metabolite of coumaphos) and chlorpyrifos (63.2%). Of these, fluvalinate, coumaphos, and coumaphos oxon are all compounds used by beekeepers against Varroa
. In 350 pollen samples, a combination of bee bread (320 samples), trapped pollen (28 samples) and anther pollen (2 samples), 98 compounds were detected with a mean of 7.1 detections per sample. One sample contained residues of 31 compounds. The most frequently detected compounds were fluvalinate (88.3%), coumaphos (75.1%), and chlorpyrifos (43.7%), again with fluvalinate and coumaphos likely being introduced as compounds targeting Varroa
Cross-sectional studies like these provide valuable information on bee colonies at a single point in time, or immediately following a single exposure. They are not capable of assessing the impact on bee health from multiple exposures, the cumulative and possibly synergistic effects of multiple sequential or simultaneous exposures, or the effect of multiple residues within a single matrix or spread throughout multiple matrices (e.g., wax, pollen, bee bread, nectar, honey, propolis, and adult bees, within a hive) [8
]. The order of exposure is also important: Johnson et al. [8
] demonstrated that the toxicity of amitraz was not changed when honey bees were exposed to sublethal doses of tau-fluvalinate or coumaphos prior to amitraz exposure, but tau-fluvalinate and coumaphos toxicity increased when bees were treated with amitraz prior to tau-fluvalinate or coumaphos.
This study is the first to sample the same colonies, from the time of their establishment until their death, using a prospective study design. Its objective is to extend the study of honey bee health impacts by assessing the level of pesticide contaminants, over a period of four years, carried to honey bee colonies by foragers in pollen loads. Colonies established in six states were included in the study.
, Table 3
, Table 4
and Table 5
, provide different perspectives on the number of detections in the different matrices (pollen, wax comb, and foundation wax), the number of compounds detected in each matrix, and the years in which each compound was detected. Salient features of the data are described in the subsections that follow.
3.1. Pollen and Wax Pesticide Residues—General Findings
Viewed as a whole, 716 compound (pesticides, metabolites and/or synergists) detections occurred in the 168 pooled pollen samples and 993 detections occurred in the 142 wax comb samples. These 1709 detections were of 91 different compounds. Of the 91 compounds detected, 42% (38) were insecticides, 32% (29) were fungicides, 25% (23) were herbicides, and one was a synergist.
Of these 91 different compounds detected, 79 pesticides and metabolites were detected in the pollen samples, while 58 pesticides, metabolites and/or synergists were detected in wax.
Also, of the 91 compounds detected, 32 were found only in pollen, 12 were found only in wax, and 47 were found in both wax and pollen. If a pesticide or metabolite was found only in one matrix (pollen or wax), the pesticide or metabolite was significantly more likely to be found only in pollen rather than in wax (χ2(1) = 11.5; p < 0.001).
, Table 3
, Table 4
and Table 5
show all detected compounds, the matrix (pollen, wax comb, or foundation wax) in which the detections were made, and how many detections were made of each compound in each matrix. Table 2
organizes this information by pesticide and includes each pesticide’s oral and contact LD50
, matrix and years of detection, mean concentration and range and mode of action (MOA) [40
]. Table 3
organizes the residues in pollen by year. Table 4
organizes the wax comb residues by year and Table 5
describes the foundation wax residues.
3.2. Predictive Power of Pollen Samples from a Single Colony to an Apiary
Twenty pesticides were detected in the 20 unpooled pollen samples obtained from the five WA colonies. The mean (SD) number of residues detected per unpooled pollen sample was 6 (1.7); no sample had fewer than three detections. Dimethoate was detected in all samples. Acephate, carbaryl, chlorpyrifos, dithiopyr, epiconazole, and fluvalinate were each detected in a single sample.
The predictive power, defined in Section 2.4.2
, of a pollen sample from one colony to indicate the presence of a pesticide in other colonies in the same apiary was 2.75: i.e., the presence (absence) of a pesticide in a pooled sample was a good predictor of the presence (absence) of the compound throughout the apiary.
3.3. Fungicides in Pollen, Wax Comb and Foundation Wax
Of the 1709 compound detections in pollen and wax, the number of fungicide and their metabolites detections in pollen, wax comb, and wax foundation were 192, 262, 24, respectively. These fungicide detections were of 29 different fungicides. Of these 29 fungicides, 24 (83% of all fungicides found) were found in pollen and 20 (69%) were found in wax. Also of these 29 fungicides, 9 (31%) were found only in pollen samples, while five (17%) were found only in the wax samples.
summarizes the assay results for pesticides in pollen. Focusing on the 24 fungicides detected in the pollen samples, nine (38%) were detected in only a single year, while four (17%) were detected in all years. The fungicides carbendazim, azoxystrobin, and propiconazole-1 were the most frequently detected compounds in pollen: 48 (28% of the 168 samples), 41 (24%), and 32 (19%), respectively. The greatest number of fungicides detected in any one year was in 2011, when 17 different fungicides were detected. Also in 2011, the highest concentration of any single fungicide, carbendazim (548 ppb), was observed.
summarizes the assay results for pesticides in wax comb samples. Focusing on the 14 fungicides detected in the wax comb samples, five (36%) were detected in only a single year while two (14%) were detected in all four years. The fungicides azoxystrobin and pyraclostrobin were the most frequently detected compounds in wax: 56 (39% of the 142 wax comb samples) and 53 (37%), respectively. The greatest number of fungicides detect in any one year was in fall 2010, when 11 different fungicides were detected. The highest concentration of any single fungicide in wax was 137 ppb of carbendazim in the (established Spring 2009)/(sampled Fall 2011) samples of drawn comb.
summarized the results for pesticides in the wax used to coat the plastic foundation. Focusing on the 11 fungicides detected in the foundation wax, seven (64%) were detected only once and one fungicide, boscalid, was detected all five samples. Iprodione was found in the highest concentration—165 ppb.
3.4. Herbicides in Pollen, Wax Comb and Foundation Wax
In the 168 pollen samples, 142 wax comb samples, and 5 wax foundation samples, there were a total of 220, 166, and 8 detections of 23 different herbicides, respectively. Of the 23 detected herbicides, 20 herbicides were detected in pollen samples and 14 were detected in wax (comb and foundation) samples. Also of the 23 detected herbicides, 11 were found in both pollen and wax, nine (39%) were found only in pollen, and three (13%) were found only in wax.
Focusing on the herbicides detected in the pollen samples, 12 herbicides (60% of 20 herbicides detected in pollen) were detected only in a single year while four (20%) were detected in all 4 years. Atrazine was detected most frequently: it was found in 84 (50%) of the 168 pooled pollen samples. In pollen, the highest concentration of a single herbicide was of diuron (275 ppb) in 2011 (Table 3
In comb wax, six herbicides (50% of 12 herbicides detect in wax comb) were detected only in a single year, while two, atrazine and diuron, were detected in all years and these two herbicides were the most frequently detected in the wax samples: atrazine was found in 49 (34%) of the 142 samples, and diuron in 41 (29%). In wax, the highest concentration of a single herbicide was of halosulfuron-mythyl (386 ppb) in 2010 (Table 4
In foundation wax, only four herbicides were detected (Table 5
). The herbicides at highest concentrations were pendimethalin and prodiamine (17 ppb).
3.5. Insecticides in Pollen, Wax Comb, and Foundation Wax
In the 168 pollen samples, 142 wax comb samples, and 5 foundation wax samples there were a total of 304, 481, and 38 detections of 38 different insecticides, respectively. Of the 38 detected insecticides, 35 (92%) were detected in pollen samples and 24 (63%) in wax (comb and foundation) samples. Also, of the 38 detected insecticides, 21 (55%) were detected in both pollen and wax, 14 (37%) were found only in pollen, two (5%) only in wax comb and one (3%) only in wax foundation (Table 2
, Table 4
and Table 5
). The synergist piperonyl butoxide was detected only in wax samples: 56 times in wax comb and 3 times in foundation wax.
Pollen sample assays detected 35 different insecticides. Of these, 12 (34% of insecticides detected in pollen) were detected only in a single year while six (17%) were detected in all 4 years. Carbaryl was most frequently detected insecticide: it was found in 26% of the 168 pooled pollen samples. Imidacloprid or its metabolites were detected only 11 times (6% of the 168 samples) and it was not detected at all in 2010. In pollen, the highest concentration of a single insecticide was of spinetoram (645 ppb) in 2011 (Table 3
In comb wax, 22 different insecticides were detected (Table 4
). Of these, 10 insecticides (45% of insecticides detected in wax) were detected only in a single year while four (18%) were detected in all years. Even though no miticides were used in the colonies in this study, coumaphos was the most frequently detected insecticide/miticide: it was found in 122 (86%) of the 142 wax comb samples and 100% of the five foundation wax samples. Fluvalinate, another insecticide/miticide approved for use in honey bee colonies but never used in this study, was the second most detected insecticide: it was found in 111 (78%) of the 142 wax comb samples (Table 4
) and 100% of the five foundation wax samples (Table 5
). A metabolite of amitraz, another miticide used to control Varroa mites, also was found in 100% of the wax foundation samples.
3.6. Annual Pattern of Residues
In all years, more types of insecticides were observed in pollen and wax than were types of fungicides or herbicides: 38 (42%), 29 (32%), and 23 (25%), respectively (see Table 2
). In pollen, the greatest number of detections (223) and the greatest number of compounds (56) occurred in 2011. Also, in pollen, the smallest number of detections (138) and compounds (33) were found in 2009 (Table 3
). In wax comb, the greatest number of detections (458 detections in 65 samples (7 detections/sample)) occurred in fall 2012 and the greatest number of compounds (37) were found in fall 2010. However, the greatest number of detections per wax comb sample occurred in fall 2012: 64 detections in 7 samples (9.1 detections/sample) (Table 4
3.6.1. Annual Pattern of Residues within Pollen Samples
The largest number of residues detected in a single pooled pollen sample was 10, 9, 13, and 12 compounds in 2009, 2010, 2011, and 2012, respectively. No detectable residues were found in eight of the 168 pooled samples: one from Maine in 2009, two each from ME and FL in 2011, and three from PA in 2012. The maximum number of compounds found in a single pollen sample was 13 (WA, 2011). The overall mean (SD) number of detections per sample was 4.1 (2.7). Table 6
gives the mean (SD) number of detections per pollen sample broken down by year. The mean (SD) number of detections per pooled pollen sample varied by location from a low of 1.1 (1.1) in ME in 2011 to a high of 8.7 (2.1) in TX in 2012.
presents the three most common pesticides found in pollen samples for each year of the study (summarized from Table 3
). Over the four years of the study, the herbicide atrazine was the most commonly detected pesticide; it was also the most frequently detected pesticide in each individual study year. Carbaryl, an insecticide, was among the three most frequently detected pesticides detected in years 2010 and 2012. It was also the third most commonly detected pesticide over the four study years. The fungicide carbenzadim made the “top three” most frequently detected pesticides in only one study year (2011); however, it was the second most commonly detected pesticide over the four study years.
Most pollen residues tended to be episodic—occurring in some but not all locations and in some but not all months or years. Thus, predicting the presence of a residue based upon prior detection was not possible. For example, imidacloprid was detected in PA in July and August of 2009, MN in August and September of 2011, TX in July and MN in August of 2012 but in no other locations or months/years. Tebuconazole was not detected in any of the six locations in 2009 and 2010. In 2011, it was detected once in TX in August, twice in WA in June and August and in 2012 it was found only in TX in March. Propiconazole in 2009 was observed in WA in two non-sequential months—June and August—but then was found in 2010 through 2012 from May–September. In ME, FL and MN propiconazole was only episodic. It was detected in ME in three months—May 2010 and June and July 2011 while in FL and MN the only detection was in November 2010 and August 2011, respectively. In MN and FL dimethoate was detected only in June 2009 and it was never detected in PA or ME.
While most pollen residues were episodic, in some locations a few residues occurred more continuously. Dimethoate was found in WA from May/June through September in all four years. Azoxystrobin was detected in WA in June and July 2009 and from May/June through September 2010, 2011, and 2012, and in MN from July through September 2011 and 2012. Atrazine was found in all locations but somewhat continuous only in MN, PA, and WA. It was detected in MN from May/June through September 2010, 2011 and 2012, in PA from June through August 2012, and in TX from May through December 2009, February through July 2010 and in March through July 2012.
3.6.2. Annual Pattern of Wax Comb and Wax Foundation Residues
summarizes detections in wax comb taken from active colonies, while Table 5
summarizes detections in foundation wax. A total of 58 different compounds were detected in these samples. More compounds were detected in drawn wax comb (49) than in wax-coated foundation (27). Not all compound detections in wax comb and wax foundation were the same. Thirty-three compounds were found only in wax comb and eight were found only in the foundation wax. Fifteen compounds (five fungicides, two herbicides, and eight insecticides) were found in both comb and foundation wax.
In drawn wax comb, there was a mean (SD) of 7.1 (1.7) compounds per sample, and a total of 49 compounds detected. Of the 49 detected compounds, 14 were fungicides, 22 were insecticides, 12 were herbicides, and one was a synergist. The greatest number of fungicides (11 of 14) and insecticides (19 of 22) detected in any single year were detected in the fall 2010 wax comb samples, while the greatest number of herbicides (8 of 12) detected in any one year was in the fall 2012 wax comb samples. The maximum number of residues per wax comb sample varied by location from three in FL to ten in TX.
When colonies were established in 2011, the wax used to coat the plastic foundation was from two different batches (lot numbers) of wax (see Section 2.1
). Therefore, foundation wax samples from PA 2009 (purchased and sampled in 2009), PA 2011 (purchased in 2009 but sampled in 2011) and ME 2011 (purchased and sampled in 2011) were compared. There was a weak but positive association in contaminants between the samples from the same manufacturer but different batches; the PA 2009 and PA 2011 (same batch) and ME (different batch, same manufacturer); r
= +0.433, p
= 0.0273 and r
= +0.326, p
= 0.105; PA 2009 vs. ME 2011 and PA 2011 vs. ME 2011, respectively. There was a high correlation between the PA 2009 and 2011 foundation wax samples (r
= +0.948, p
< 0.001); they were from the same manufacturer and batch.
The foundation used to initiate the colonies in 2009, 2010, and 2011, was found to have 26 pesticides or metabolites with a mean (SD) of 14.2 (4.2) detections per sample. Of the 26 compounds detected in the foundation wax, 11 were fungicides, four were herbicides, 10 were insecticides, and one was a synergist. The number of detections per sample ranged from nine to 19. Two metabolites (amitraz DMPF and coumaphos oxon), two insecticides (fluvalinate and coumaphos), and one fungicide (boscalid) were found in all of the wax foundation samples. One herbicide (pendimethalin), one fungicide (azoxystrobin), and three insecticides (chlorpyrifos, fenproximate, and propargite) were found in 80% of wax foundation samples.
There were 16 contaminants found in common to both the wax comb and foundation wax (five fungicides, two herbicides, and eight insecticides/miticides, and one synergist).
In contrast to the pollen sampling, wax sampling was less frequent and not at consistent intervals (see Section 2.2.2
). Whether the data were log transformed or not, there was no evidence of a correlation between the frequency of detection of insecticides in wax comb and foundation wax for compounds that were found in both in either 2009 or 2011 (p
> 0.10). When data were pooled over years there was a weak correlation when the data were transformed (log ppb) between concentrations of pesticides in foundation wax and wax comb (r
= +0.448, p
The contaminants in the wax foundation used to initiate the hives in 2009 was not correlated with the mean monthly, maximum, or total seasonal contaminants found in pollen brought into hives in 2009 for any of the six apiaries in the 2009 trial. Only three of the apiaries in 2009 shared some of the top five most abundant contaminants in pollen with those most abundant compounds in the foundation wax (fluvalinate in two apiaries and coumaphos in three apiaries).
3.7. Comparison of Wax Comb Residues from Living vs. Dead Colonies
Comparison of wax comb sampled from both living and dead colonies in the same apiary on the same date revealed that pesticide detection number did not differ (p > 0.10); however, the total pesticide concentration (sqrt ppb) showed a possible trend of lower total concentration in wax combs from living colonies than from wax comb from dead colonies (F(1,6) = 5.3566, p = 0.06).
3.8. Lethal Dose Levels of Residues in Pollen and Wax Comb
Of the parent pesticides and metabolites for which both honey bee oral and contact LD50 values were available (51 compounds), 12 (24% of the 91 detected compounds) had the same LD50 values for the contact and oral mode of entry, 18 (35%) had lower oral than contact LD50 values (oral exposure was more toxic than contact exposure) and 21 (41%) had lower contact than oral LD50 values (contact exposure was more toxic than oral exposure). There is no evidence, based upon the oral versus contact LD50 values for the detected compounds, to suggest that, independent of quantity of pesticide used, oral exposure to a pesticide of a honey bee is more lethal than contact exposure (χ2(2) = 2.0799; p = 0.35).
While some pesticides were frequently detected, most never were detected at a level that was lethal to honey bees. For example, the herbicide atrazine, was never detected at a level higher than 10−6 of its contact LD50. Likewise, the most frequently detected fungicide in pooled pollen was carbendazim, which was never detected higher than 0.1% of its contact LD50. Among the compounds detected in pollen, the greatest acute risks were posed by insecticides: while imidacloprid and its metabolites were infrequently detected in the pooled pollen samples (11 of 168 samples), in one sample imidacloprid was measured at 137% of its oral LD50. Another insecticide, dimethoate, was detected at near 35% of its oral LD50. The largest measured residue of two other insecticides, phosmet, and pyridaben, were 13% of their respective LD50s.
The herbicide, fungicide and insecticide residues in comb wax posed a lower acute risk than pollen. In wax comb, the highest concentration of a single fungicide was diphenylamine at 227 ppb, for which no oral or contact LD50 has been determined. No fungicide was detected in wax comb at a level greater than 0.2% of its LD50; no herbicide was found in wax comb at a level greater than 0.01% of its LD50. In wax comb the greatest risk was, again, for insecticide exposure: in 2010, 2011, and 2012, the maximum fluvalinate concentrations were 862 ppb, 512 ppb, and 537 ppb, respectively, which correspond to 43%, 25%, and 26% of fluvalinate’s 2000 ppb contact LD50.
In foundation wax the most acute risk was posed by the insecticide fluvalinate, which was measured at 7.7 times its LD50
. Amitraz, because of its ability to interact with fluvalinate and coumaphos [8
], likely also posed a significant risk (a metabolite of amitraz was detected at 582.3 ppb); however, its LD50
, acting alone or in combination with fluvalinate and coumaphos, is unknown. Of the remaining pesticides, the insecticide phosmet, posed a low—though not negligible—risk with its largest measured residue level being 5% of its LD50
. The variation among replicate samples of foundation, estimated by the coefficient of variation when including all 26 contaminants, was 105.6%. This level indicates the vast majority of the 21 contaminants have low mean concentrations.
3.9. Modes of Action Distributions in Pollen, Wax Comb, and Wax Foundation
Different pesticides may act on target organisms in similar or in different ways. Pesticides that act in the same way are said to be of the same class; pesticides that act in different ways are of different classes. The different classes of pesticides detected in pollen, wax comb, or foundation wax, are summarized in Table 8
. Whether in pollen, wax comb, or foundation wax, a greater number of classes of insecticides (14) were observed than of fungicides (11) or herbicides (8). The fungicides that were detected ranged from benalaxyl, an inhibitor of nucleic acid synthesis to chlorothalonil, a multi-site activity fungicide. Herbicides detected varied from atrazine, an inhibitor of photosynthesis, to sethoxydim, an inhibitor of acetyl CoA carboxylase. Insecticides detected varied from spiriclofen, an inhibitor of acetyl CoA carboxylase (as is the herbicide sethoxydim) to imidacloprid, a nicotinic acetylcholine receptor agonist. Among the fungicides, the largest number of compounds (9) was found in class G (MOA: sterol biosynthesis in membranes). The herbicide group with the greatest number of compounds (10) was class C (MOA: inhibition of photosynthesis at photosystem II). Insecticide class 1B contained the greatest number (11) of compounds (MOA: acetylcholinesterase inhibition).
In pollen, the most frequently detected pesticide was the herbicide atrazine, a photosynthesis inhibitor. Of the insecticides detected in pollen, acetylcholinesterase inhibitors were detected in more samples than were any other class of insecticides. Among the fungicides, three compounds with different modes of action (B1, C2 and G1: see Table 5
) occurred most frequently. If aggregated, the acetylcholinesterase inhibitors (239 detections) were detected more frequently in pollen than were the photosynthesis inhibitors such as atrazine (127 detections). If we suppose that each acetylcholinesterase inhibitor insecticide acts independently, then we might reasonably estimate the cumulative risk to this class of insecticides, in the “LD50
” sense, as the sum of the ratios of each compound’s measured residue to its own LD50
. If all acetylcholinesterase inhibitor insecticides exposure occurred simultaneously at the maximum detected level, then the total exposure for this class of insecticide would be 130% of the “net” acetylcholinesterase inhibitor LD50
In wax comb and foundation wax, a greater number of classes of insecticides (10) were observed than classes of fungicides (9) and herbicides (7). Some modes of action were more commonly observed than others: the most frequently detected modes of action were the irreversible and reversible acetylcholinesterase inhibitors (358 of 559 insecticide detections, or 64%), followed by photosynthesis inhibitors such as atrazine (119 of 173 herbicide detections, or 69%), followed by respiration inhibitors such as azoxystrobin (131 of 275, or 48%, of fungicide detections) [40
3.10. Potential Residue Interactions—Multiple Compounds
Linear correlations (co-occurrence) of pesticides in pollen were evaluated for pesticides of the same class, and between different pesticide classes. All correlations with individual p
< 0.05 are reported in Table 9
. Likely co-occurrences in pollen were observed between class B1 fungicides and C2 herbicides (r
= 0.9660; p
< 0.0001), between class C3 fungicides and class A herbicides (r
= 0.9561; p
< 0.0001), between class D1 fungicides and class C1 herbicides (r
= 0.9955; p
< 0.0001), between class C2 fungicides and class 28 insecticides (r
= 0.9910; p
< 0.0001), and between class A herbicides and class 28 insecticides (r
= 0.9962; p
This is the only honey bee study to date to use a prospective study design sampling the same colony from the time of its establishment until its death. The colonies, once established, were not moved; thus the samples can be used to describe the temporal changes in pesticide residues in bee forage.
Differences in the number of compounds detected among Mullin et al. [22
] and Stoner and Eitzer [25
] and this study may be due to differences in the sampling protocols (same colonies sample throughout the foraging season and over multiple years versus single sample per colony), matrices sampled (pollen only versus pollen/bee bread and wax versus pollen and wax), time of year when sampling occurred, migratory versus stationary bee management, in-hive pest management methods, land use differences, length of time wax is in the hive, or the use of wax-coated plastic foundation or pre-drawn wax comb with pesticide residues. To fully characterize the exposure of honey bees to pesticides it is important to sample multiple colonies at multiple locations, with dissimilar management strategies, various colony longevities and different study methodologies.
A total of 91 pesticides, metabolites, and synergists from 335 pooled and individual pollen and comb and foundation wax samples, collected over four years, were detected in this study. Mullin et al. [22
] detected 121 compounds in wax and pollen/bee bread while Stoner and Eitzer [25
] detected 70 parent compounds and metabolites in pollen. A higher proportion (72% of 121) of pesticides in wax was reported by Mullin et al. [22
] than in this study (48% of 91). It is not surprising that fewer compounds (44) were detected in 1 to 2-year-old wax from this study while a greater number of compounds (85) were reported by Mullin et al. [22
] where some wax samples where older than 2 years. In this study atrazine, carbendazim, and carbaryl were the most frequently detected in pollen (48.6%, 27.7% and 25%, respectively). Mullin et al. [22
] reported fluvalinate, coumaphos and chlorpyrifos (88.3%, 75.1%, and 43.7%, respectively) were the most frequently detected residues in pollen/bee bread and Stoner and Eitzer [27
] reported coumaphos, carbaryl, and phosmet were most frequently detected residues in pollen (46.6%, 40.6%, and 32.9%, respectively).
The frequency of residue detection in pollen was similar between Stoner and Eitzer [25
] and our study; <50% of the pollen samples were positive for at least one pesticide residue. This differed substantially from Mullin et al. [22
], who reported 88.3% of the pollen samples positive for at least one pesticide residue. Differences in the types of compounds found in pollen and number of samples positive for residues may be due to an artifact of data reporting. Mullin et al. [22
] reported pollen and beebread residues as a single category while our data and Stoner and Eitzer [25
] are pollen samples collected from foragers prior to their entry into the hive. In-hive contamination of the beebread resulting from the use of amitraz, fluvalinate or coumaphos in the management of Varroa
may explain the difference in the frequency of detection. The source of coumaphos found in the pollen analyzed in our study or by Stoner and Eitzer [25
] is unknown. The pollen was never inside the colony, thus eliminating contamination by in-hive use of coumaphos, fluvalinate or amitraz. At this time there are no approved crop uses of coumaphos (the only approved use other than in honey bee management of V. destructor
is as a cattle dip) unlike all the other compounds detected. As none of the pollen collected in this study or by Stoner and Eitzer [25
] was ever in the colony, the source of coumaphos is of great interest. Possibly the source of coumaphos (and possibly fluvalinate and the amitraz metabolite) in the pollen in both our study and Stoner and Eitzer [25
] could be due to residue transfer from the surface of the foragers’ body to the pollen. The source of surface contamination of the forager’s body could be bees from a package source where coumaphos was used, contamination picked up from wax residues in the colony or contamination of bee forage from unknown sources. Another potential source is the wax-coated foundation but a number of pesticides residues present in the wax-coated foundations samples were never detected in the drawn comb wax. A third possibility is that some coumaphos- or fluvalinate-contaminated pollen could have been obtained by bees stealing from honey bee colonies not a part of this project but located within flying distance to our apiaries. The remaining pesticides detected in pollen likely originated from pollen contamination from crop application.
Generally, insecticides were present more frequently and at higher concentrations than fungicides or herbicides. It would be interesting to explore the possible reasons for this result. One possibility would be to investigate potential correlations between prevalence of different types of pesticides to high versus low humidity conditions, differences in nighttime temperatures or the length of the growing season. Other possible explanations for the differences insecticide versus herbicide and fungicide detection that could be explored include similarities and differences in the chemical characteristics among insecticides, herbicides and fungicides, the types crops grown, regional differences in recommendations by Extension or pesticide company representatives and differences in the research emphasis by land grant universities.
The pattern of pollen and wax residues differed substantially by month and year. The number of detections was highest in 2011 and lowest in 2009. A compound may have been detected only once across all locations and years, at one or all locations but only once in a foraging season, multiple times (months) within one foraging season but at only one location or, at multiple locations or multiple years at multiple locations and multiple times (months) per foraging season. The differences by month, year and location are likely due to differences in target organism and the types of pesticides applied by growers, homeowners, and others to honey bee forage. Different pesticides may be used by growers because different crops have been planted, to address the occurrence of different pests or to reduce the development of pesticide resistance by pests. Differences among the apiaries may be a result of the surrounding land use or differences in climate. Two of the three apiaries surrounded by the highest concentration of agricultural land, TX and WA, tended to have the greatest number of residues and the most continuous exposure to the same compounds. Surprisingly, FL with its high humidity and long growing season had consistently fewer and more episodic detections than TX and WA.
Substantial variation in the quantity of residue detected in wax was found. Without information on the history of a wax sample, it is difficult to determine the reasons for the large variation in the quantity and types of pesticides detected. All the comb wax samples in this study were from stationary hives less than two years old, managed without the use of miticides or other pesticides and with a variety of environments surrounding the apiaries. Mullin et al. [22
] obtained their samples from a variety of sources including colonies managed by small beekeepers, migratory beekeepers and commercial sources. Martel et al. [27
] used commercially available wax. It is likely that the wax from these two studies included some old (>2 years) wax samples, samples from hives where beekeeper management likely included both registered and unregistered miticides, and wide-ranging surrounding environments. More studies with detailed information concerning the conditions experienced by the wax samples are needed to arrive at a clearer understanding of the presence and absence of compounds when studies are compared.
Interestingly, the type and the quantity of pesticides found in the same batch of wax used for coating foundation wax were not consistent. These data show that a better understanding of the dispersion of pesticide contaminants in wax is needed to explain the variation in types and quantities of compounds observed from the same batch of wax. Two likely reasons for the variation are the uneven distribution of residues in wax and the degradation of residues over time. Not knowing the causes of the variation nor the degree to which each cause influences the variation limit our ability to predict honey bee exposure.
Fewer studies of honey bee oral exposure to pesticides have been conducted than contact exposure. Of the 91 compounds detected in this study, two (2.2%) had neither an oral nor a contact LD50
, two (2.2%) lacked a contact LD50
and 35 (38.5%) lacked an oral LD50
). If the oral and contact LD50
for the remaining 52 compounds are compared, excluding the eight compounds with identical oral and contact LD50
, the same number of compounds posed a greater oral exposure risk (lower oral LD50
) as compounds posing a greater contact risk (lower contact LD50
) (Table 2
). Thus, the lack of oral LD50
studies may be less of a problem than expected.
Increased attention is needed on the MOA of various pesticides. Currently, the data on pesticide modes of action focus on the pests for which the pesticide has been developed and on resistance management for the target organism. Thus, important the modes of action for honey bees and other beneficial or non-target insects may be missed. An example is the herbicide atrazine with plants as the target organism; its published MOA is photosynthesis inhibition [41
]. Atrazine has been studied in several non-target organisms including honey bees where the carotenoid-retinoid system is altered at exposure levels observed in agricultural systems. In rats, fish and quail the endocrine system is altered [43
]. Thus, additional research is needed on pesticide modes of action for honey bee, other pollinators and non-target organisms. An additional area requiring research is, at minimum, to determine methods for summing exposures within the same MOA and how multiple modes of action may interact with one another.
Though the number of potential pesticide interactions and the varied types of target and non-target organisms is daunting, studies have been conducted on the combined impact from simultaneous and sequential exposure to pesticides [8
]. Our study provides likely real-world examples of multi-residue exposure for future research. Because multiple residue exposure can result from the presence of more than one pesticide within a single pollen sample and from the all the residues in all pollen within a colony, it is important to gather data on pesticide residues in pollen and other hive products over multiple years and from many locations. One group of compounds found in our study to frequently co-occur was the reversible and irreversible acetylcholinesterase inhibitors and three of the miticides (amitraz, coumaphos, and fluvalinate) used for Varroa
control. Other multiple exposures of interest, based upon pesticides detected in this study, include the possibility interaction of fluvalinate with resmethrin, the only other insecticide detected with the same MOA as fluvalinate, and the interaction of coumaphos with 10 other insecticides with the same MOA—acephate, azinphos-methyl, chlorpyrifos, diazinon, dichlorvos, dimethoate, malathion, omethoate, phorate, and phosmet.
Most efforts to understand pesticide interactions have focused on developing predictive models, not studies of specific compounds [49
]. Due to the sheer number of potential combinations of pesticides, predictive models are a logical method to evaluate potential risk. Unfortunately, at least for our understanding of the risks to honey bees, the models have been built using common laboratory animal and plant model organisms, i.e., Daphnia magna
, Vibrio fischeri
, and Stellaria media
]. Thus, the models are of little use to those attempting to predict real-world impacts of multiple pesticide exposure on honey bees.
Most of the pesticides detected in our study are for plant protection rather than honey bee colony management. Fungicides and herbicides may be applied to crops or horticultural plantings but would not be applied within a honey bee colony. Even among the insecticides detected, most are never used within the hive. Potential interactions between pesticides used and never used for colony management need to be explored along with interactions between pesticides only used for crop or horticultural plants.
Some studies focused on honey bees exposed to multiple pesticides have been conducted usually including at least one pesticide that may be used in honey be management. Colin and Belzunces [52
], Pilling and Jepson [47
], and Pilling et al. [48
] explored the synergistic impact from paired pyrethroid and fungicide exposures. Sub-lethal exposures, either simultaneous or sequential, resulted in an increase in honey bee mortality; a reduction in the rate of pyrethroid biotransformation in the presence of the fungicide was proposed as the mechanism of action. More recently, Thompson and Wilkins [53
] obtained similar results when they paired eight different fungicides and two pyrethroid insecticides but the evidence provided suggested a different mechanism of action. The presence of a fungicide decreased the repellency of the pyrethroid resulting in a higher exposure to the pyrethroid. Johnson et al. [8
] reported that acaricides with different modes of action interact, usually resulting in decreased the LD50
compared to the single compound control in adult bees. Additionally, they showed that some fungicides interact with acaricides to lower the LD50
of the acaricide and that piperonyl butoxide lowers the LD50
of tau-fluvalinate. Data from our study show that fluvalinate and coumaphos could be found in the same sample of pollen, drawn comb, and wax-coated foundation and the piperonyl butoxide may co-exists with fluvalinate in wax.
Using honey bee larvae, Zhu et al. [54
] tested fluvalinate, coumaphos, chlorothalonil, and chlorpyrifos alone and in combination. At moderate levels of exposure, the toxicity of fluvalinate-chlorothalonil and coumaphos-chlorothalonil increased the toxicity of the insecticides but at very low doses (10-fold reduction) the fluvaninate-chlorothalonil exposure became antagonistic. Complicating the picture further, when coumaphos was added to the fluvalinate-chlorothalonil mixture, the toxicity was reduced. The risk potential to adult and immature bees from pollen consumption, and to nurse bees, who recycle hive wax, and to new adult bees, who chew through wax to emerge from their brood cells, should be explored.
If pesticide exposure is evaluated using only the LD50
from each individual pesticide, based upon the results from this study, the risk posed to honey bees is low. In our pollen samples, one sample out of 168 exceeded the pesticide residue’s oral LD50
. While much attention has been focused on the newer types of pesticides, ie. Neonicotinoids, and their impact on bee health, from this study it is clear than some of the older pesticides, specifically the carbamates and organophosphates, may continue to pose a risk. When the ratio of exposure (ppb) to its LD59
for the acetylcholinesterase inhibitors was summed, the ratio exceeded 1. While few samples had residues approaching the pesticide’s LD50
, the pesticides residues detected are likely to pose a high sublethal risk to honey bee survival [7
]. Currently it is difficult to evaluate the risks from sublethal exposures as there are no agreed upon outcome measures similar to the LD50
for the various sublethal outcomes. Additional work is needed, first, to define the most important sublethal outcomes and, second, to quantify the impact of sublethal exposures from various pesticides so a risk evaluation can be conducted.