Effect of OAV on the Rearing of Brood
Effect of OAV on the Rearing of Brood
Ernie Daley and Randy Oliver
First published in ABJ June 2024
I have used OAV (oxalic acid vaporization) for varroa control for over ten years and have wondered whether (1) the treatment could be applied proactively prior to the introduction of package bees, and (2) whether the treatment would adversely affect the development and survival of larvae. So, with guidance by Randy Oliver, I ran a couple of experiments.
SET UP OF EXPERIMENT #1
I wondered whether I could apply oxalic acid vaporization to drawn combs (as a proactive “residual insecticide surface treatment”) to control varroa, applied prior to installing the bees. My first question was whether such a treatment would affect the first round of brood laid by the queen (perhaps due to oxalic crystals in the cells). To answer that question, Randy suggested that I run a test by covering half of a frame of empty brood cells with plastic wrap (to provide a Control group unexposed to OA) and then expose the comb to oxalic acid vaporization. Then afterwards remove the plastic wrap and return the treated comb to the hive for the queen to lay eggs in.
So, in late May I ran two replicates of the test, preparing the combs (Figure 1), then placing each of them in the center of a bee-free hive consisting of a medium super with empty drawn combs on either side, and then applying 2 grams of OA by vaporization (Figure 2).
Fig. 1 Two frames of empty drawn comb, half wrapped with plastic wrap to provide both a treated Test and an untreated Control group on each comb.
Fig. 2 I used a 110 Volt OA vaporizer to heat two grams of oxalic acid to 315°F to vaporize and disperse it into a medium super containing 10 drawn frames (without bees) with the two Test frames in the center of the box, via a hole in the floor rim.
Ten minutes after applying the OAV, I peeled the plastic wraps off the Test combs, and placed them into individual excluder cages, which I then inserted back into the centers of their hives. I then introduced a 3-lb package of bees (containing their mother queens, caged for transport) into each hive, after first releasing and confining their queens onto their respective Test combs (Figures 3 &4).
Fig. 3Releasing the caged queen from the package into the excluder cage in which she will be confined.
Fig. 4 The excluder cages (with queens) returned to their colonies.
RESULTS OF EXPERIMENT #1
Three days after confining the queens, I removed the Test frames from the excluder cages, and observing that plenty of eggs had been laid, returned them (uncaged) to their respective colonies. Six days later I observed larvae and capped brood on both Test frames (Figure 5 and 6). There was no noticeable difference in the brood patterns of the OAV and Control halves of the Test frames in either colony.
Fig. 5 Test frame from colony “A” 9 days after pretreatment with OAV showing brood development on both the Control and OAV halves.
Fig. 6 Test frame from colony “B” 9 days after pretreatment with OAV showing brood development on both the Control and OAV halves.
EXPERIMENT #2
Now that I had determined that OA pretreatment did not adversely affect the survival of eggs or newly emerged larvae, I was ready to test whether treatment of combs with eggs and growing larvae already present would affect their survival or development.
To answer this question, Randy suggested that I repeat the first test, but this time with eggs and larvae already present when I applied OAV to the combs.
Working in early July I again used an excluder cage to confine a queen on a frame of drawn comb for six days. When I returned six days after confining the queen, I observed eggs and larvae on both sides of the frame. I then wrapped half of the Test frame with plastic wrap (Figure 7) and returned it to the hive without the excluder cage.
Fig. 7 The Test frame with eggs and larvae 6 days after confining the queen onto the comb, prior to applying OAV. One half is wrapped with plastic wrap to provide a Control group unexposed to OA.
I then applied two grams of oxalic acid by vaporization to the double-medium colony (the usual dosage I would apply when treating a double medium for varroa). Ten minutes after ending the OAV, I removed the Test frame from the hive, peeled off the plastic wrap, and returned it to the colony for 11 days. The ages of the eggs and larvae prior to OA vaporization were 1-6 days.
RESULTS OF REPLICATE #1
Eleven days after the OAV application, I removed the Test frame and inspected both sides for brood survival. There was no noticeable difference in brood survival or development between the OA-exposed and Control halves of the Test frame (Figures 8 and 9).
Fig. 8 Side one of the Test frame 11 days after OAV, showing brood development on both the Control and OAV halves.
Fig. 9 Side two of the Test frame 11 days after OAV, showing brood development on both the Control and OAV halves.
Replicate #2
At Randy’s suggestion, I replicated the experiment with younger eggs and larvae, to see whether I would get comparable results. I ran this replicate at the beginning of August, using the same double-medium hive. By now the colony had expanded and bees covered fourteen of the twenty frames. I confined the queen as before for 4 days. When I returned 4 days after confining the queen, I observed the queen laying (Figure 10) and eggs (Figure 11) and larvae on both sides of the frame.
Fig. 10 The queen laying an egg on the Test frame on the fourth day of confinement after removal from the excluder cage.
Fig. 11 Eggs and larvae observed on the Test frame 4 days after confining the queen.
As before, I wrapped half of the Test frame with plastic wrap and returned it to the hive. I then applied two grams of OAV, and after ten minutes unwrapped the plastic from the Control side and returned the Test frame to the colony for 13 days. The age of eggs and larvae prior to OAV treatment was 1-4 days.
RESULTS OF REPLICATE #2
When I returned 13 days after OAV application, I removed the Test frame and inspected both sides. Both the OAV and Control halves had similar brood patterns with no observable difference in development or survival (Figures 12 and 13).
Fig. 12 Side one of the Test frame 13 days after OAV showing brood development on both the Control and OAV halves.
Fig. 13 Side two of the Test frame 13 days after OAV showing brood development on both the Control and OAV halves.
DISCUSSION
As far as I could tell there were no adverse effects on the development of eggs or larvae whether brood frames had been pretreated with OAV prior to egg laying, or if OAV was applied when eggs and larvae were already present. The amount of brood, the pattern, and the development on the OAV half of the frame appeared to be the same as that of the Control half in both experiments.
OXALIC RESIDUES FOLLOW UP IN CALIFORNIA (by Randy)
Following the field experiments by Ernie, I was curious as to how much oxalic acid actually remained in each cell after Ernie performed his vaporizations (without or containing larvae). I calculated how much total surface area there is in a medium super of drawn combs (including the woodenware (top, bottom, and sides) and ten frames (tops, bottoms, ends, and faces of combs): it worked out to 3736 square inches. Dividing 2 grams of oxalic acid by that area, I got a theoretical result of 535 micrograms (µg) per square inch of surface area (83 µg pr cm2), or 23 µg per cell face.
Note on titrations: I’ve done extensive work (as yet unpublished) on quantifying the amounts of oxalic acid on bees’ bodies after treatment with oxalic acid applied by different methods. Since titration doesn’t identify the actual type of acid, my microgram values are for “OA equivalents.”
The theoretical dose from the registered application methods is ~100 µg per bee, but that amount is seldom achieved in the field. An actual dose of ~30 µg per bee will kill mites. An adult bee can handle 200 µg of oxalic acid applied as a dribble, but the theoretical 23 µg per cell face may have the potential to affect a newly-emerged larva.
So along with my helpers Rose Pasetes and Corrine Jones, we ran six separate tests on different days, each time applying 2 grams of oxalic acid by vaporization (using a ProVap 110) to different deep supers (since I didn’t have any mediums) of empty drawn brood comb (for which there would be theoretical residues of 17 micrograms per cell face). We first confirmed that there were no preexisting acid residues in the cells by using a pipette and indicator solution.
Run 1, with plastic squares on the comb faces
For our first run, we also pressed 1 cm2 pieces of plastic (cut from a deli container) into the comb surfaces to quantify the amount of residues on the comb surface to compare to the residues inside the cells.
After allowing a half hour for the OA vapor to settle, we removed the treated combs and again used a pipette to test haphazardly-chosen cells for acid residues (measured in OA equivalents) (Figure 14).
Fig. 14 We dropped 5 drops of indicator solution into each cell to test, then sucked it back out with a pipette. Here, in a practice run, Rose has placed a Control drop in the center, and drops from tested cells around it. The indicator dye turns green and then orange relative to the amount of acid residues. To our surprise, there were only 1-2 micrograms of OA equivalents per cell, and only 0-10 µg per cm2 on the plastic test squares –– far less than the expected 83 µg!
Out of curiosity, I scooped some beebread out of three cells, knowing that bees use lactobacilli to preserve pollen with lactic acid. The scoops of beebread titrated at 450, 360, and 860 µg OA equivalents of acidity! Although lactic acid is not quite as reactive as is oxalic, it helps to explain how bees can tolerate treatment with OA.
Anyway, the low amounts in the cells and on the plastic surprised us, and when we later lifted the treated box, we noticed that a portion of the OA had crystallized at the entrance where the stream of vapor hit the bottom board (Figure 15).
Fig. 15 This pile of OA crystals amounted to only a fraction of a gram, but shows that it is important for anyone applying OAV to make sure that the stream of vapor does not directly hit anything inside the hive!
So we ran five more experiments:
Run 2, with dead bees pinned to the combs
In Run 2 we pinned dead bees on the combs (Figure 16) to compare the amount of OA that accumulated on each bee to the amount that we’d previously found when we titrated bees from clusters after OAV treatment (there is typically ~10 µg per bee shortly after application).
Fig. 16 We pinned dead bees to the combs to see how much OA stuck to them as opposed to the plastic squares used in the first run. See Fig. 17 for the results.
Fig. 17 The reference tube it to the left. Most bees had less than 5 micrograms, but the yellow one tested at 60. There was surprising variation, despite them all having been placed between central combs well above the vapor stream. Again we titrated minuscule levels of OA in the cells.
Run 3, with a handful of live bees loose on the combs.
So I decided to put a handful of young live bees loose on the ten combs of a different super. I plucked off a batch of them ten minutes after OAV application (Figure 18), and then ten more after they had walked on the combs for an hour (Figure 19).
Fig. 18 At 10 minutes post application, most bees exhibited only slight amounts of acidity, although a few titrated at 40-50 microgram equivalents. Again, surprisingly wide variation for a treatment applied as a fog.
Fig. 19 But by an hour, all the bees had picked up much more oxalic acid, presumably from walking over the treated comb surfaces.
Run 4, with a tiny cluster of live bees
Still intrigued by our findings, we went out to a stack of recently brought-home deadout supers to look for a cluster of dead bees to test, and to my surprise found a hand-sized cluster of live bees with a queen between the upper portions of two combs. So we placed that box between a bottom board and cover, and applied 2 grams of OAV (Figures 20- 24).
Fig. 20 When we removed the cover after 10 minutes, we could immediately see that the bees had a “dusting” of white oxalic microcrystals on their setae (“hairs”).
Fig. 21 The bees were far more “frosty” looking than after a normal vaporization.
Fig. 22 A close up of a typical bee from the previous photo covered with oxalic acid crystals ‑‑ far more than I see when I OAV a full-sized cluster! I hesitated to titrate them…
Fig. 23 I only titrated the first three bees, since it took so many drops of titrant. Their OA equivalents were 630, 1330, and 150 micrograms! I have no idea as to why this tiny cluster of bees got hit so heavily by exactly the same vaporization that we’d been giving for the other test runs.
Fig. 24 The cells also contained far more acidity than in the other runs: ranging from 5 to 20 micrograms. Go figure!
Intrigued again, we performed yet another experiment.
Run 5, live bees in push-in cages
This time I placed a few live bees into each of several push-in cages (wide Mason jar lids with 1/8” screen), located between different combs, near the top, and again applied the same amount of OAV via the entrance. The results are shown in Figure 25 and Table 1.
Table 1 The acid residues on the bees were higher than those for Runs 2 and 3, but far less than those in Run 4.
Fig. 25 Despite the relatively-high amounts of acid on the caged bees, the amounts of residues in the cells were again barely detectable (reference drops below, test drops from the combs above).
Conclusions
To our great surprise, in the five experimental runs –– using the same vaporizer on the same stand, with nearly-identical boxes of combs –– there were large differences in the amounts of acid residues on bees on the combs, and within the cells. The amounts of acid on the bees was generally far more that I observe when I’ve titrated full hives (mostly 8-10 frame clusters). So I’m not surprised that Ernie did not observe any adverse effects from OAV upon eggs or larvae.
Experiment 6, Live bees introduced after vaporization
At this point of time, I realized that we hadn’t yet addressed Ernie’s first objective –– to determine whether he could preload boxes of drawn comb with oxalic acid by vaporization, prior to installing a package of bees, for the purpose of killing mites on the introduced package bees. This was based upon the assumption that the acid residues on the combs would transfer to the bees. The results of Run 3 certainly indicated that they would.
So we ran yet another experiment. We again set up a box of acid-free drawn brood combs and vaporized it with 2 grams of oxalic, then allowed it to sit for an hour for all the acid to settle. We then shook bees from an untreated colony, and confirmed by titration that they were free of acidity. We allowed any older bees to fly off to ensure that when we introduced them onto the treated combs, that they would remain in the new “hive.” We introduced a cupful of the presumably young bees, replaced the hive cover, and allowing them to remain in the “hive,” freely walking on the combs. We took samples of them after 10 minutes, 1 hour, and two hours (by which time their acid levels ceased increasing (Table 2).
Table 2. Similar as in Run 3, the acid levels of the bees increased during the first hour, and then stabilized. Our finding: Bees can readily “pick up” a substantial amount of OA microcrystals by walking over treated combs for an hour.
Experiment 7, to be done
The acid levels on the bees above would be plenty to kill mites –– provided that the acid crystals were distributed over the bees’ bodies. But in a prior preliminary experiment I had found that acid residues of this level, when only on the bees’ feet, did not cause any varroa mortality in an incubator trial. Unfortunately, by this time it was already November and my dang sons had already treated any hive with measurable mites, so we need to wait until next season to test Ernie’s assumption –– which we’ll do by comparing mite drops on stickyboards after introducing high-mite packages to boxes of treated or untreated combs. If anyone wants to join us in performing this experiment, we’d love to see your results!
ACKNOWLEDGEMENTS
I wish to thank Randy Oliver for his mentorship, assistance in experimental design, and the writing of this article. And we thank Rose and Corrine for their help with all the titration experiments.