Ozone is a form of oxygen (O3) that is a powerful oxidant. It has the ability to
disinfect, eliminate odors, taste, and color, and remove pesticides, and is commonly used
as a water treatment for both drinking and swimming1. Recently, it has been developed as
an agricultural fumigant, being used, for example, on stored potatoes to prevent rot, and it
kills insect and fungal pests in stored grains 2-4. Ozone breaks down quickly into oxygen
(O2), so it will not persist in wax comb, wood, or plastic. The objective of the research
reported here was to determine how much ozone is needed to kill the greater wax moth
and to eliminate pesticide residues, without damaging the hive equipment or honeycomb.
This is the first round of tests designed to determine whether it is feasible to use ozone to
control pests of stored comb and to clean up comb that is contaminated with pesticides.
Tau-fluvalinate (a pyrethroid) and coumophos (an organophosphate) are the active
ingredients of Apistan Strips®i and Checkmite®, respectively, which are products
developed for varroa control. Both these chemicals can persist in the hive5-10. Amitraz is
occasionally used by beekeepers to treat for varroa mites, but amitraz breaks down
quickly in the environment on its own11, and for this reason, it is not included in this
study.
Ozone and Wax Moth Control.
To truly control wax moths, one needs to be able to kill all the life stages. Newly
hatched larvae (“small larvae”) and adults were the easiest to kill with ozone (Fig. 3C and
F). Small larvae were killed after only 1-2 hrs of exposure to even the lowest
concentration of ozone, and adults were killed, for the most part, after 6 hrs. However,
the eggs and pupae required much longer exposure times (Fig.3A, B, and E). Eggs were
the most resistant life stage, requiring 48 hours at 33.7°C (95°F) for complete kill. For the
most part, 1000 ppm was always more effective than 500 ppm; however, at the high
temperature (95°F), the difference may not always great enough to justify the additional
cost and effort that might be require to achieve 1000 ppm. One method to avoid the long
exposure times required to kill all the eggs may be to treat twice. Once to kill adults and
any larvae or pupae that may be present, and then a second short treatment 5-6 days later
to kill the newly emerged larvae that hatched from any eggs that survived the first
treatment.
We did not test the effects of ozone on small hive beetles, but it is likely that the
exposure times for this insect will be similar to that of the greater wax moth. Again, to
kill the adults and eggs, it may be most effective to apply two shorter treatments rather
than one treatment that lasts for several days.
One limitation to using ozone for controlling storage pests is that once the hive
supers and comb are treated, the chemical is gone, and the comb is susceptible to re
invasion of the pest. Thus, the supers will either need to be stored in such a manner that
the pests cannot re-infest, or repeated applications of ozone will be required (as is done
for potato storage). In the future, I plan to determine whether ozone will work to disinfect
comb contaminated with bee diseases, particularly spore-forming pathogens such as
chalkbrood and American foul brood
Ozone and Pesticide Degradation.
We evaluated the time required for complete decay of two acaricides: coumophos
and tau-fluvalinate. These tests were conducted at two different temperatures (25˚ and
35˚C [77˚ and 95˚F]), and with three different starting concentrations of the pesticides
(10, 25, and 50 ppm). We expected a greater amount of pesticide to take longer to decay
than a smaller amount, but we did not know how much longer, because the decay
response may not be linear (i.e. not additive).
To start, we obtained chemical grade coumophos and tau-fluvalinate, and these
were combined with acetylnitrile to make the standards of 10, 25 and 50 ppm. The
standards were created at the beginning of the experiment and stored at 4°C. For each
initial concentration of pesticide, 400 ul of pesticide-acetylnitrile solution was aliquoted
into a 2.0 ml glass vial. This vial was uncapped and heated to 46.5°C evaporate the
acetylnitrile, which took approximately 6 hrs. The vial with the pesticide residue was
then taken to either a control incubator or to the ozone incubator, and treated for a
prescribed amount of time (0, 6, 8,10,13, 20 or 24 hours). The incubator was kept at 50%
RH. The control treatments were exposed to the air in an incubator set at the same
conditions of temperature and RH as the ozone incubator. Thus, our degradation rates are
relative to the rate of degradation that would have occurred in the absence of ozone.
After treatment, 400 ul of acetylnitrile was added to the glass vials to dissolve any
remaining pesticide. The samples were analyzed using high-performance liquid
chromatography (HPLC)9. It has previously been shown that the effects of ozone on
pathogens are slower when they are placed on a non-porous surface, such as glass, than
on porous surfaces like wood or wax4. We assumed, then, that the tests of pesticide
6
degradation should also be conservative estimates for what might occur if we use hive
materials, and the use of glass allowed us to gain full recovery of the pesticide remaining
after ozone treatment.
During the first experiments with ozone, we also exposed pieces of wax
foundation because we were not sure what effect the ozone might have on wax quality.
Even exposures to 500-1000 ppm for two weeks had no discernable effect on the color or
flexibility of the foundation. Thus, even repeated exposure should be safe for the wax in
the comb.