We observed no significant difference in mortality between honeybees exposed to chemical-free PM in the circulator compared to control honeybees (F1,14 = 2.398, P = 0.144; see Table 1, Fig. 2).
Table 1
Source of variation table for one-way analysis of variance on honeybee mortality data.
Source of variation
|
Df
|
SS
|
MS
|
F
|
P
|
Treatment
|
1
|
0.016
|
0.016
|
2.398
|
0.114
|
Residuals
|
14
|
0.093
|
0.007
|
|
|
Similarly, mortality did not differ between male and female mason bees exposed to PM and respective controls (male: F1,14 = 2.461, P = 0.139; female: F1,14 = 0.021, P = 0.887; see Tables 2, 3, Fig. 3).
Table 2
Source of variation table for one-way analysis of variance on male mason bee mortality data.
Source of variation
|
Df
|
SS
|
MS
|
F
|
P
|
Treatment
|
1
|
0.164
|
0.016
|
2.461
|
0.139
|
Residuals
|
14
|
0.093
|
0.007
|
|
|
Table 3
Source of variation table for one-way analysis of variance on female mason bee mortality data.
Source of variation
|
Df
|
SS
|
MS
|
F
|
P
|
Treatment
|
1
|
<0.001
|
<0.001
|
0.021
|
0.887
|
Residuals
|
14
|
0.172
|
0.012
|
|
|
Further, mortality of male and female mason bees exposed to PM in the circulator was not different (F1,3 = 0.028, P = 0.867). Limited mortality observed among control bees (mean of 7.72% in honeybees, 9.93% in male mason bees, and 12.15% in female mason bees) was possibly due to stress from wind in the circulator. While mortality of bees exposed to PM in the circulator (mean of 11.97% in honeybees, 16.32% in male mason bees, and 12.95% in female mason bees) was not significantly different than that of controls, it was consistently higher. This suggests a slight increase in mortality due to PM introduction into the circulator system, but it was not a major driver of mortality. Mortality observed in both control and PM-exposed treatments can serve as a measure of background mortality for future in-situ exposure experiments with chemical-laden PM. Of the many potential contributors to the toxicity of PM (e.g., oxidizing species, PAHs ;Kelly and Fussell, 2012), the relatively inert substrate of dried manure in PM used in these tests did not appear to be a significant contributor to pollinator mortality.
Bees exposed to PM in the circulator became coated with PM on all surfaces of their body. Some bee species engage in grooming, a behavior which can vary between species (Hamiduzzaman et al., 2017). Specifically, A. mellifera individuals groom themselves and other individuals in the hive in response to accumulation of dust and pollen (Božič and Valentinčič, 1995), infestation of Varroa mites (Hamiduzzaman et al., 2017), and to pack pollen collected by the individual (Parker et al., 2015). Grooming behavior may serve to reduce or exacerbate toxic effects of agrochemical-laden PM. It is worth noting that studies have documented altered grooming behavior in bees as a result of pesticide exposure (de Mattos et al., 2017; Morfin et al., 2022). Investigation into chronic exposure to contaminated PM and potential mitigating effects of grooming behavior is thus warranted.
Little data exist regarding the toxic effects of chemicals bound to fugitive PM. Anecdotal evidence collected at field locations suggest increased mortality of honeybees and mason bees when exposed to fugitive feedyard PM in situ. However, this study indicates that exposure to uncontaminated PM is not a major driver of pollinator mortality. Traditionally, efforts to investigate pesticide toxicity among pollinators involves acute contact toxicity tests (e.g., OECD Test No. 214), wherein a pesticide is dissolved in a solvent (e.g., acetone) and directly applied to the test subject; or oral toxicity tests which involve dosing an animal’s food (e.g., sugar water; OECD Test No. 213). These direct application methods are not reflective of environmental pesticide exposure occurring among pollinators. Toxicity of pesticides used in agriculture (e.g., pyrethroids, neonicotinoids, and macrocyclic lactones) to pollinators (e.g., bees (Peterson et al., 2021a; Zhao et al., 2020); butterflies (Bargar et al., 2020); and other pollinating insects (Peterson et al., 2021c)) is well documented. For example, Osmia lignaria (blue orchard mason bee) experience toxic effects from a variety of pesticides11 that have been detected in fugitive beef cattle feedyard PM (Peterson et al., 2020, 2021a). Further, Bombus terrestris (bumble bees) and Osmia bicornis (red mason bee) are twice as susceptible to clothianidin compared to honeybees (Heard et al., 2017). Pollinators can be exposed via drift of aerial applications, overspray or accidental application, dust from seed coatings, residues in nesting materials, and other methods not readily simulated via established laboratory protocols (Sanchez-Bayo and Goka, 2014). The present study provides a method to evaluate toxicity from exposure to contaminated PM. Future studies will involve manufactured PM spiked with one or more of the many pesticides found in contaminated PM (Peterson et al., 2020) and then introduced into the circulator. In addition to single chemical toxicity tests, mixtures of pesticides can be added to manufactured PM in order to evaluate potential interactive toxicity. Further, other pollinator species (e.g., non-apis bee species, butterflies, etc.) could be subjected to agrochemical-laden PM exposure in this system to gain a more complete understanding of pollinator risk associated with aerially transported pesticides. Particulate matter from a variety of other sources (e.g., mining and smelting operations, industrial processes) could also be examined for pollinator toxicity in the above-described system.