Two Separate Toxicity Studies Conducted on Apis Melifera (Honey Bees)

Introduction

This report outlines two separate toxicity studies conducted on Apis melifera. The first study follows the parameters of the EPA Litmus test and the second study delves deeper into possible BAPC toxicity surpassing the standards of the EPA definitive test. Each study was performed under EPA guidelines including harvesting location, race, containment conditions, test duration, age, acclimation, diet and feeding solutions, and care/handling.

For both studies, Apis melifera (honey bees) were collected from one of eight locally maintained hives, from the same geographical location (GPS 39° 17′ 8.03713″ N 99° 32′ 54.76309″ W) and were subjected to Branched Amphiphilic Peptide Capsules (BAPCs) to establish toxicity values. BAPCs were obtained from Phoreus Biotech lyophilized and were reconstituted with MilliQ water at a concentration of 50 µM. Bees were stored in bee buses utilized for transport and feeding studies. All bee studies were completed at Fort Hays State University in an insect incubator with hive lighting conditions and environmental parameters set at 32oC and 50-60% relative humidity for the duration of the studies mimicking normal hive conditions. Prior to beginning the study, bees were allowed to acclimate to the conditions for 24 hours although this parameter was not required per EPA guidelines.

Study 1: EPA Litmus Test

According to EPA guidelines for a Limit test, for test substances expected to have relatively low toxicity, as in the case for BAPC’s in place of a definitive test a limit test may be conducted with a single contact dose level, the limit dose, plus a control group. In these situations, it is only necessary to ascertain that the 48-hr LD50 is above the limit dose (i.e., 48-h LD50 > limit dose). In a honey bee acute contact limit tests at least 25 bees are exposed to the “limit dose” with the same number of bees in appropriate controls. For most pesticides the limit dose is 25 μg of active ingredient per bee (25 μg a.i./bee). BAPCs were utilized at these concentrations for four trials including a control. These bees were subjected to a diet with BAPCs in the standard honey bee diet consisting of a 50% weight/volume (w/v) solution of sugar/water (500 grams/liter) provided ad libitum throughout the holding and test periods. Distilled MilliQ water was used for the sugar diet solutions. Following test guidelines, 25 bees were used, and the test was conducted for 48hrs.

At test termination (48 hours), since one or fewer bees were dead at the limit dose, the acute contact LD50 is considered to be greater than the limit dose (i.e., LD50 > limit dose). This is because the Binomial Theorem predicts that when 25 bees are tested, the probability of seeing <1 dead bee if the true 48-h LD50 is at or below the limit dose is <0.001. Conversely the probability of seeing 2 or more dead bees if the true 48-h LD50 is at or below the limit dose is >0.999. Therefore, if <1 mortality occurs among the 25 bees tested, the 48-h LD50 is reported as greater than the limit dose (i.e., 48-h LD50 >25 μg/bee for pesticides). Data (table 1.) shows that following the Litmus Test, BAPCs pose no significant mortality to honey bees.

Study 2: Enhanced Long Duration Tests

For this test, I wanted to evaluate long term exposure to BAPCs as it pertains to my research goals and is vital information for future applications of BAPC by Phoreus. It is important to note that although bees in the wild can and will persist and live for 6 to 9 months, in a laboratory setting the lifespan is near the two week mark typically around 16-18 days.

Housed within the incubator, containment vessels fed bees consisting of a sucrose solution mixed with BAPCs at concentrations of 50µM, 5µM, 500nM, 50nM, 50pM, 5pM and a control with no BAPCs. Each trial consisted of 400 bees and was simultaneously conducted at all BAPC concentrations. Daily counts of bee deaths were utilized to establish death curves to allow for statistical comparison (see figure 1.)

Although increased BAPC concentration initially appears at first glance to cause an increased death of bees, the statistical results indicated by the hazard rates in the log-rank comparison tests show that they actually are no different than that of the controls. The log- rank test, utilized at a confidence interval of 99% showed that the highest concentration tested, 50µM is statistically insignificant and therefore all others are also insignificant. Parameters of each Log-rank test is listed in Table 2.

Based on previous literature of aphid feeding studies, a commercial dosage of BAPCs in application would be in the nano to pico molar ranges. Environmentally, I can state with that information in mind, it is obvious that BAPCs pose no threat to honey bees at the exposure ranges that would be seen in the environment if utilized in a similar commercial application. Moreover, as a typical commercial application would be a one-time application spanning a few days, versus a continuous application as seen in these studies, I would be unsurprised if those few deaths associated at the 50µ and 5µM concentrations would fall off and mirror the control studies. Unequivocally these results show that BAPCs used at commercial concentrations pose no threat to one of the most significant pollinator species seen in the world.

Conclusion

These two studies show statistically that for both the short term and long term exposure to BAPCs, there is no significant death to honey bees. BAPC could be considered as an inert delivery vector for the treatment of bees, and they do not pose a significant threat to bee populations in the commercial and noncommercial levels utilized in the aforementioned trials.

Tables and Figures

BAPC Litmus Test: Live Bees

Table 1. Litmus Test toxicity studies.

Log-rank test Results @99% Confidence Interval

Table 2. Bee cytotoxicity Log-rank test statistical data.

Control

Table 3. Bee cytotoxicity control. No BAPC present in diet.

50uM

Table 4. Bee cytotoxicity test. 50µM BAPC present in diet.

5uM

Table 5. Bee cytotoxicity test. 5µM BAPC present in diet.

500nM

Table 6. Bee cytotoxicity test. 500nM BAPC present in diet.

50nM

Table 7. Bee cytotoxicity test. 50nM BAPC present in diet.

50pM

Table 8. Bee cytotoxicity test. 50pM BAPC present in diet.

5pM

Table 9. Bee cytotoxicity test. 5pM BAPC present in diet.

Figure 1. Bee cytotoxicity death curves by various BAPC treatment present in diet.