Pesticides: Prevalence and HQ risk

A total of 93 different pesticide residues were identified in the analyzed samples (Table S1). A total of 13, 61, and 70 different pesticides and/or residuals were found in bees, beebread, and wax respectively (Tables S2, S3 and S4).

Bees: The maximum number of pesticides found in a single bee sample was 4. Five samples were pesticide-free, while all other bee samples (n = 33) had at least one product detected. Few pesticide residues were detected per bee sample (1.39 ± 0.15), with the main contributors being beekeeper-applied varroacides, coumaphos and fluvalinate, which were detected in 23.7% and 81.6% of samples, respectively (Table S2). Across all samples, only 1 of 38 live bee samples had an elevated HQ bee (HQ bee = 1,256), primarily a result of contamination with fipronil detected at 9.9 ppb. With its low LD 50 , fipronil contributed 1,222 points to the HQ bee . All other samples had an HQ bee < 50.

Beebread: Samples were retrospectively pooled from 3–4 colonies that perished at similar times, so that pesticide exposure at multiple time points during the season could be evaluated. The maximum number of different pesticide products found in a pooled beebread sample was 20. All samples (n = 147) had at least one product detected. On average, each pooled sample had 7.22 ± 0.30 different pesticides or metabolites. Across all pooled beebread samples, 1,061 pesticides and their metabolites were detected (see Table S3, Fig. S1); however, of these only 14.7% (n = 156) contributed at least 50 points to the HQ bbread score and were considered “relevant” (Table S3, Fig. S1). Seventeen individual pesticide detections contributed more than 1,000 points to the HQ bbread (chlorpyrifos = 9; fenpropathrin = 5; fipronil = 1; pyridaben = 2). Insecticides were most commonly detected (n = 363; 34.3%), followed by varroacides (n = 343, 32.3%), fungicides (n = 204, 19.2%), and lastly herbicides (n = 151, 14.2%). The average HQ bbread for all samples was 445 ± 62.8. The total HQ bbread score was greater than 1,000 in 15% of samples (n = 22). Insecticides were the largest contributor to the total HQ bbread score (85.9%) across all samples, followed by varroacides (10.6%), fungicides (3.5%), and herbicides that contributed only minimally (<0.01%). Fungicides were very common, appearing in 70.1% of all beebread samples (n = 103). In the current study, fungicide residues were detected 77 times at over 100 ppb (Table S5), over 2.5x more frequently than insecticides. All fungicides that contribute 5 points to the HQ bbread score were found at more than 100 ppb. The majority (91.2%) contributed less than 50 points to the total HQ bbread , generally because fungicides have relatively high LD 50 s that ranged from 25-2,430 ppb. Multiple fungicides in a single sample occurred frequently, with 44.9% (n = 66) of all samples tested having two or more fungicide residues (Fig. S2).

When grouped by their MOA, we found that HQ bbread scores were significantly elevated when more than two insecticides from group 1 (1.AChE, acetylcholinesterase inhibiting) occurred in the beebread sample (χ2 = 53.56, df = 5, n = 147, p < 0.0001). Samples with zero or one group 1 insecticide had a mean HQ bbread = 139.7 ± 28.7, while samples with 2 + products had a mean HQ bbread = 810.1 ± 119.5, ranging from 579.1 ± 140.7 to 895.1 ± 6167.4 depending on the number of products detected. One commonly detected varroacide, the organophosphate coumaphos, has a group 1 insecticide MOA; this product contributed 11.6% to the average HQ bbread score of 228.9 contributed by this MOA group. Group 2 insecticides (2.GABA, GABA-gated chloride channel blockers) had a similar impact (χ2 = 16.29, df = 4, n = 147, p = 0.0026), with HQ bbread scores significantly lower when no group 2 insecticides were detected in beebread (mean HQ bbread = 280.9 ± 71.3) compared to when group 2 products were present (mean HQ bbread = 369.4 ± 99.88 to 3,825.9 ± 0). No varroacides had a group 2 MOA.

Wax: The maximum number of residues detected in a single wax sample was 39, with a mean of 10.17 ± 0.47 per sample. Of the 108 samples analyzed, all had at least three products detected. Altogether, 1,108 pesticide residues were detected, of which 32.3% (n = 358) contributed at least 50 points to the HQ wax and were considered relevant (Table S6). Total HQ wax was significantly lower in samples taken at the start of the beekeeping season compared to samples taken at the last inspection period (Fig. S3, χ2 = 5.50, df = 1, n = 108, p = 0.019), regardless of colony survival (χ2 = 0.024, df = 1, n = 108, p = 0.8751). Total HQ wax was above 1,000 for 77.98% (n = 85) and above 5,000 for 7.34% (n = 8) of wax samples. Residues that contributed more than 1,000 points to the HQ wax score included the varroacides coumaphos, fluvalinate, and the amitraz breakdown product DMPF, as well as the insecticides deltamethrin, fenpropathrin, fipronil, and permethrin (see Table S6). While never exceeding the 1,000 HQ wax threshold (largely on account of its high LD 50 ), the fungicide chlorothalonil was present in 68.8% of wax samples (n = 75) with a mean concentration of 1,635.0 ± 756.9 ppb, making it the most abundant wax contaminate after varroacides (see Table S6). In one wax sample, chlorothalonil levels were detected at 53,700 ppb, a higher concentration than any of the beekeeper applied varroacides, and contributed 483.8 points to the HQ wax of that particular sample. Low amounts of neonicotinoid insecticides (group 4, 4.nAChR, nicotinic acetylcholine receptor competitive modulators) were found in six wax samples; two were contaminated with imidacloprid at 2.4 and 13.6 ppb, contributing 60.3 and 341.7 points to the HQ wax ; four were contaminated with thiacloprid at 1.9 to 7.8 ppb, contributing 0.08 to 0.31 points to the HQ wax . The mean HQ wax score across all samples was 2,155 ± 192.4. The majority of this score came from the presence of varroacides (71.1%), followed by insecticides (28.3%) and fungicides (0.5%), while herbicides contributed minimally (<0.01%). Several MOA groups significantly increased the HQ when multiple products of that same MOA were detected. All wax samples had at least two group 1 acetylcholinesterase inhibiting products with a maximum of seven detected in a single sample. The HQ wax increased significantly when more than two products were detected (χ2 = 4.97, df = 1, n = 108, p = 0.026), raising the mean HQ wax from 1,539.6 ± 168.9 to 2,416.4 ± 258.5. All wax samples had at least one product of MOA insecticide group 3 (3.NaCh, sodium channel modulators) with up to 12 different products with this MOA detected in a single sample. In wax, 14.8% samples (n = 16) were free of MOA insecticide group 19 (19.Octo, octopamine receptor agonists) residues and had a mean HQ wax = 1,335.8 ± 174.8, while 33.3% (n = 36) had one group 19 residue HQ wax = 1,940.8 ± 272.3 and the remaining 51.9% had (n = 56) two detected with a HQ wax = 2,545.6 ± 318.8. Seven wax samples were positive for an insecticide residue with an unknown MOA; these few samples had significantly elevated HQ wax scores (χ2 = 28.37, df = 1, n = 108, p < 0.0001; MOA group unknown present HQ wax = 5,801.7 ± 1,933.0 vs absent HQ wax = 1,912 ± 133.0;). Fungicide MOA did not influence the total HQ wax score except for MOA fungicide group M (M.Multi, multi-site contact activity) (χ2 = 8.39, df = 1, n = 108, p = 0.004). When present, samples with group M fungicides had significantly elevated HQ wax = 2,502.7 ± 267.7 compared to samples where they were absent HQ wax = 1,429.2 ± 135.6.

Pesticide Prevalence and HQ in Different Operations and Foraging Environments

The participating commercial beekeepers moved their colonies among three different foraging environments: crop pollination, honey production, and holding yards. Only beebread samples were regularly collected and analyzed across all sampling time points in this study. The average total HQ bbread was not significantly different among operations but the number of total pesticide residues in beebread samples varied significantly (Fig. S4, HQ bbread : χ2 = 4.00, df = 2, n = 147, p = 0.135; Fig. S4, number of residues: χ2 = 130.28, df = 2, n = 147, p < 0.0001) as did the total number of “relevant” pesticides χ2 = 44.89, df = 2, n = 147, p < 0.0001).

Each participating commercial beekeeper had a different migration route (Fig. 1), and so the differences in pesticide prevalence and abundance among operations is not surprising. Clear peaks in the HQ bbread and number of residues detected occurred when colonies were in or had just been moved out of certain specialty crops, especially citrus, apple, cranberry, and cucumber (Table 1). Notably, in OP1, HQ bbread was significantly higher and the number of pesticides detected greatest when colonies were sampled in May 2007 immediately after apple pollination. In OP2, the HQ bbread was elevated during citrus bloom and late season cucumber pollination. Late cucumber pollination was associated with significantly more pesticide residues than any other crop. In OP3, HQ bbread and pesticide residues were highest when bees were foraging in citrus groves at the start of the study and were also elevated during cranberry pollination (see S1, Fig. S5).

Table 1 Mean Bee Bread Hazard Quotients ± SE and Mean Number of Pesticide Residues ± SE by Operation and Crop Exposure. Full size table

Overall, the total HQ bbread is significantly elevated in pollination environments compared to honey production or holding yards (Fig. S6, top row, χ2 = 39.13, df = 2, n = 147, p < 0.0001). Of the beebread samples that had total HQ bbread score greater than 1,000 (15%; n = 22), the majority were collected in March (n = 9) and May (n = 9) during pollination of apple or lowbush blueberry.

When the HQ bbread was subdivided into the four categories of insecticides, fungicides, herbicides, and varroacides, the trends of elevated risks during pollination were consistent across all pesticide groups except for varroacides (Fig. S6). Varroacide levels increased significantly in beebread samples collected while colonies were held in holding yards, presumably because these were the times when beekeepers applied treatments for Varroa control.

Fungicide prevalence was low outside of pollination events and was completely absent in 54% and 44% of samples taken from colonies in holding yards and honey production yards, respectively. Only 9% of beebread samples taken from colonies during pollination events were absent of fungicides. These few fungicide-free beebread samples occurred early in the season during citrus and apple pollination. Of the 8.8% of analyzed samples that had a fungicide with an HQ bbread > 50 (HQ bbread fungicide range = 88.3–239.6), all were collected during blueberry (n = 9) and cranberry (n = 4) pollination. Total fungicide residues measured in ppb per sample for in-hive beebread and wax are frequently high (bee bread: mean = 1,706.4 ppb, max = 26,600 ppb; wax: mean = 1,137 ppb, max = 53,704.8 ppb), but were rarely detected in live in-house bees at the start of the season (mean = 1.32 ppb, max = 35.8 ppb).

Environment strongly influenced the number of products of a specific MOA found in a beebread sample (Table 2). For insecticides, MOA group 18 (18.EcRs, ecdysone receptor agonists) were more prevalent in pollination environments and reduced when colonies were placed for honey production (Fig. 2), while insecticide MOA group 2 GABA-gated chloride channel blockers were highest in holding yards (predominantly because of endosulfan residues). MOA group 19 octopamine receptor agonists were highest during honey production, which is attributed entirely to DMPF residues, a breakdown product of Amitraz. Several fungicide MOA groups were also elevated during pollination. These included fungicides from the groups C (C.Resp, respiration), G (G.Sterol, sterol biosynthesis in membranes, and M (M.Multi, multi-site contact activity) MOA groups (Fig. 2).

Table 2 The number of residues detected in bee bread collected from colonies in different beekeeping environments, grouped by MOA. Full size table

Figure 2 The mean number (+− SE) of products found in bee bread, grouped by their MOA, which differed between environments (as indicated by different letters in each group). Full size image

HQ wax at the start of the study did not differ among operations (χ2 = 0.16, df = 1, n = 54, p = 0.69), although the total number of relevant pesticides varied between the wax of the two operations sampled (χ2 = 11.88, df = 1, n = 54, p = 0.0006), with significantly higher relevant residues in OP3 (mean = 4.22 ± 0.54) compared to OP1, both when the packages in OP1 were included (mean = 2.44 ± 0.17) or excluded (mean = 2.82 ± 0.27).

Pesticides and colony survivorship during the entire beekeeping season

As previously reported in vanEngelsdorp et al.26, colony survival varied among operations (Fig. S4) with OP2 experiencing the fewest colony losses. OP2 managed their colonies at the apiary level, equalizing colony strength and replacing dead colonies throughout an apiary and the season, while OP1 and OP3 managed for survival at the individual colony level. For all operations, we considered a colony to be dead when it had no adult bees in the hive at the time of inspection.

The mean number of residues detected in brood nest wax samples at the start of the study did not differ between colonies that survived versus those that died over the course of the study (χ2 = 0.11, df = 1, n = 108, p = 0.736). This analysis included both established colonies and colonies that were installed as packages on drawn comb that had previously had only honey stored in it (as such, colony type strongly influenced the statistical model; χ2 = 12.31, df = 1, n = 108, p = 0.0005). Colonies established from packages had significantly fewer total and relevant residues in their wax (6.26 ± 0.55 and 2.11 ± 0.17, respectively) when compared to established colonies (12.0 ± 1.03 and 3.54 ± 0.33, respectively) at the first sampling period (t 52 = 3.93, p = 0.0002; t 52 = 3.91, p = 0.0003, respectively). Because of the differences in pesticide residues in packages and established colonies, we excluded packages from the analysis for colony survival. At the start of the study, HQ wax was not significantly different between colonies that lived or died (Fig. 3a, χ2 = 1.88, df = 1, n = 70, p = 0.17). However, established colonies that died during the season had significantly more total pesticide residues in their wax over all sampling periods than did colonies that lived (Fig. 3b, χ2 = 7.29, df = 1, n = 70, p = 0.0069). Increased exposure followed a similar pattern for relevant pesticides (Died: mean = 3.70 ± 0.27 vs. Survived: 3.0 ± 0.20), but was not significant statistically (χ2 = 2.25, df = 1, n = 70, p = 0.13).

Figure 3 (a) Mean HQ (±S.E) and (b) mean number of total residues (±S.E) found in wax comb in established colonies that survived the entire beekeeping season (green) compared to those that died (red). Significant differences (α = 0.05) indicated by different letters. Full size image

Additionally, we analyzed the total pesticide residues by grouping them according to their MOA. The number of products that are MOA group 19 insecticides (octopamine receptor antagonists) increased from the first sampling period to the last sampling period, irrespective of whether colonies lived or died (Fig. 4a, χ2 = 24.72, df = 1, n = 108, p < 0.0001). This group is exclusively comprised of the breakdown products of the varroacide amitraz, which likely reflects the use of this product for the control of Varroa over the course of the season. In colonies that died, the total number of group G fungicides (sterol biosynthesis in membranes) in wax increased between first and last sampling periods (Fig. 4b, Died: χ2 = 5.86, df = 1, n = 72, p = 0.0154; Survived: χ2 = 1.41, df = 1, n = 36, p = 0.23). A similar increase was seen in fungicides with group M (multi-site contact activity) MOA (Fig. 4c, Died: χ2 = 16.36, df = 1, n = 72, p < 0.0001; Survived: χ2 = .09, df = 1, n = 36, p = 0.30).

Figure 4 Mean number of total residues detected in wax ± S.E. for different MOA that changed significantly over time during the course of study; (a) Insecticide MOA group 19 (octopamine receptor agonists); (b) Fungicide MOA group G (sterol biosynthesis in membranes); (c) Fungicide MOA group M (multi-site contact activity). Wax samples taken at first inspection light colored, and at last inspection dark colored. Significant differences indicated: *p < 0.05; **p < 0.01; ***p < 0.001. Full size image

Total HQ bbread was elevated in established colonies during the first half of the beekeeping season from March through June that subsequently died during the beekeeping year (Fig. 5a, HQ bbread : χ2 = 10.79, df = 1, n = 34, p = 0.0010). The HQ bbread varied significantly by collection date, with the highest scores detected in March (Fig. 5a, Collection Date: χ2 = 11.32.32, df = 3, n = 34, p = 0.0101). Insecticides were the greatest contributor to the HQ bbread for these colonies, contributing 940.9 ± 157.6 points to colonies that died during the season compared to 448.5 ± 151.0 points to colonies that survived the entire beekeeping season. We separately analyzed the new colonies (packages) installed on drawn honey comb and determined that an elevated HQ bbread was not associated with colony losses in these newly established packages early in the beekeeping season from March-June (χ2 = 0.05, df = 1, n = 15, p = 0.821). However, later in the season (from June through September), packages showed a similar pattern of elevated HQ bbread in colonies that perished (Fig. 5b, χ2 = 5.48, df = 1, n = 19, p = 0.0193), suggesting that any advantage to providing colonies with “clean” comb only slightly delayed possible links with pesticide buildup and colony mortality. The HQ bbread scores in packages did not vary by collection date (χ2 = 1.93, p = 0.5861). We also examined if colonies that survived differed in their HQ pesticide category compared to colonies that perished, and only fungicides were significant. Since fungicides are only detected during the active beekeeping season, we focused on samples collected between March-September. During this time period, HQ bbread fung was lower in colonies that lived compared to levels in those that perished (Fig. 6, χ2 = 5.54, df = 1, n = 82, p = 0.0186). Collection date had no significant influence on the model (χ2 = 4.75, df = 6, n = 82, p = 0.5766).

Figure 5 Mean HQ bbread ± SE and colony survival. Mean HQ bbread , segregated by colony survival during the beekeeping year. HQ bbread in the first half of the beekeeping season is significantly elevated in established colonies that perish during the beekeeping year. Red = all colonies in the pooled sample die before Jan 2008, Green = all colonies in the pooled sample live. (a) established colonies from March–June; (b) new colonies established from packages from June-September. Full size image

Figure 6 Mean fungicide HQ bbread (±S.E.) during the active beekeeping season from March through September, segregated by colony survival. Red = pooled samples in which all the colonies die before the end of the year; green = pooled samples where all colonies survived the season. Full size image

Pesticides and imminent colony death

An elevated HQ bbread was not predictive of imminent (within ~30 days) colony loss (χ2 = 2.15, df = 1, n = 107, p = 0.14). In contrast, the total number of products found in beebread trended toward being elevated in colonies that perished (χ2 = 3.59, df = 1, n = 107, p = 0.0582), while the number of relevant products contributing at least 50 points to the total HQ bbread was significantly elevated in colonies that died before the next sampling period during the beekeeping season from April through September (Fig. 7, χ2 = 14.06, df = 1, n = 107, p = 0.0002). Imminent colony death also varied by collection date (Fig. 7, χ2 = 18.34, df = 5, n = 107, p = 0.0025), but there was no significant interaction of collection date and HQ bbread (χ2 = 6.62, df = 5, n = 107, p = 0.25).

Figure 7 Imminent colony loss and number of residues contributing 50 + to HQ bbread . The mean number of residues (±S.E.) excluding miticides used for Varroa treatment contributing at least 50 points to the total HQ bbread , segregated by imminent death. Red = pooled samples in which at least one colony dies before the next sampling period; green = pooled samples where all colonies are still alive the next inspection period. Full size image

Despite their low overall contribution to the HQ bbread , fungicides were significantly elevated during the summer season from May-August in colonies that died within ~30 days of sampling (Fig. 8a, χ2 = 5.72, df = 1, n = 78, p = 0.0168). The fungicide HQ bbread varied significantly by collection date during that same period (χ2 = 46.50, df = 3, n = 78, p < 0.0001). No fungicides were detected during citrus pollination. Samples from citrus were thus excluded when determining how HQ bbread Fung varied by crop. HQ bbread Fung was elevated during blueberry and cranberry pollination compared to other crops (Fig. 8b, Crop: χ2 = 138.90, df = 4, n = 55, p < 0.0001), but only elevated fungicide scores associated with blueberry pollination were linked with imminent colony loss (t 20 = 2.29, p = 0.033).

Figure 8 Fungicide contributions to HQ bbread by sampling period and crop pollinated. During the summer season elevated mean fungicide HQ bbread (±S.E.) were linked with colony death 1 month later in June and July. Fungicide HQ varied significantly by crop. (a) Fungicide HQ by sampling period; (b) Fungicide HQ by crop. Different letters indicate significant differences. Red = pooled samples in which at least one colony dies before the next sampling period; green = pooled samples where all colonies are still alive during the next inspection. Full size image

To determine if the MOA of particular pesticides was implicated in imminent colony loss, we examined if the total number of products with the same MOA was elevated in beebread in colonies that perished before the next sampling period. No particular mode of action was significantly associated with imminent colony loss, though there was a trend toward elevated residues of insecticide MOA group 18 insecticides (ecdysone receptor agonists) in colonies that died (mean = 0.25 ± 0.08) compared to colonies that lived (mean = 0.10 ± 0.03) (χ2 = 3.44, df = 1, n = 139, p = 0.063). A similar pattern was seen with insecticides categorized as an unknown MOA (χ2 = 2.94, df = 1, n = 139, p = 0.087), which were somewhat elevated in colonies that died (mean = 0.14 ± 0.07) compared to those that lived (mean = 0.05 ± 0.02) to the next sampling period.

Specific pesticides and colony mortality

The insecticide fipronil was found in one adult bee sample (see above), in one beebread sample, and in one wax sample. These samples came from different colonies, and in all cases the colony from which the sample was collected died before the next sampling event. The presence of the fungicide chlorothalonil in pooled beebread samples was common and often exorbitant. Of the 147 beebread samples analyzed, 87 had detectable levels of chlorothalonil, with 19.5% (n = 17) contaminated at more than 1,000 ppb and 13.8% (n = 12) at more than 10,000 ppb. In beebread, the maximum chlorothalonil residue was 26,600 ppb, eight times higher than the maximum varroacide residue detected (3,260 ppb for coumaphos). Colonies that perished during the beekeeping year had significantly higher HQ bbread chlorothalonil during the summer season from May-August than colonies that survived (Fig. 9, χ2 = 5.62, df = 1, n = 54, p = 0.0177).

Figure 9 Mean HQ bbread (±S.E.) contributed by chlorothalonil in colonies that perished during the beekeeping season in bee bread samples collected May-August, months when this fungicide was detected. Full size image

Pesticide prevalence, load and queen replacement events

As described in vanEngelsdorp et al.26, one of the leading predictors of imminent colony mortality was a queen event—that is, evidence that the queen was recently replaced (e.g., presence of a virgin queen), was being replaced (e.g., supersedure cells), or the colony was queenless. Colonies that were diagnosed with this condition were more than three times as likely to die over the next ~50 days. All three models (HQ wax , total pesticide residues, and relevant residues) were higher in the wax of colonies that experienced queen events (Fig. 10: HQ wax : χ2 = 22.38, df = 1, n = 108, p < 0.0001; Total products: χ2 = 5.04, df = 1, n = 108, p = 0.025; relevant pesticides: χ2 = 8.08, df = 1, n = 108, p = 0.005). All colonies with HQ wax scores above 6,500 (n = 5) experienced queen events during the season. When analyzed by MOA, the number of group 3 insecticides (sodium channel modulators) was significantly higher in the wax of colonies that experienced a queen event than colonies that remained queenright (Table 3, Fig. 11). Though their presence was much lower, a similar pattern was seen with group G fungicides (sterol biosynthesis in membranes) and group M fungicides (multi-site contact activity) (Table 3, Fig. 11).

Table 3 Queen Events and total number of pesticides products found in comb wax at the start of the study, grouped by MOA. Full size table

Figure 10 Queen events and pesticide contamination in wax. (a) Mean HQ wax (±S.E.) for colonies that experienced a queen event compared to colonies that did not lose or replace their queen (queenright), (b) mean total number of pesticide residues detected, (c) mean number of relevant pesticide residues (50+) detected. Significant differences (α = 0.05) indicated by different letters. Full size image