Ozone Sterilization

[Countryboy]

I have read of other sterilizing combs by spraying a 10% bleach solution on old combs and boxes. Ozone is supposedly a better sterilizer than bleach. It's my understanding that many parts of Europe sterilize drinking water with ozone instead of chlorine.

Has anyone tried sterilizing old combs and boxes with ozone? You can pick up an ozone purifier for hot tubs or aquariums on eBay for $100. If you pumped the ozone into an old chest freezer, you could gas 8 or 10 boxes at once.

Has anyone tried this? How much ozone production do you need to be effective and how long would you need to gas boxes? Or would it be better (more practical) to ozonate the water you mix the 10% bleach solution with and mist combs and boxes?

If you could sterilize combs and boxes in a reasonable amount of time, this might be practical for a sideliner beekeeper. (A commercial beekeeper would likely need one of the larger ozone generator models so they could gas a whole storage room at once.)

[Allen Dick]

Interesting idea. Gasses are easier to handle in many ways, especially common and disposable ones. Maybe not in California, though.

ETO (Ethylene Oxide gas) was used here in Alberta and Manitoba for a while. Alberta had a huge mobile unit which did 500 boxes at a time. Its use was discontinued due to concerns about the reactivity of ETO and the unknown and potential toxic or carcinogenic by-products possible when placing painted wooden boxes nailed with galvanized nails with beeswax and honey and pollen together with ETO under pressure. Moreover, the process was not completely reliable, since any honey or pollen in frames impeded penetration by the gas. There was always, also, concern for operator safety. ETO is explosive and also it dissipates slowly and the operator must remove the boxes from the chamber at some point. ETO was superseded by electron beam radiation, which is now the sanitizing method of choice in Alberta and BC. It also has problems with penetration if there is much pollen or honey in the combs.

Ozone on a small scale is different from ETO, but can still be dangerous to the operator. Wikipedia -- http://en.wikipedia.org/wiki/Ozone -- says:

Due to the strongly oxidizing properties of ozone, ozone is a primary irritant, affecting especially the eyes and respiratory systems and can be hazardous at even low concentrations. The Canadian Center for Occupation Safety and Health reports that: "Even very low concentrations of ozone can be harmful to the upper respiratory tract and the lungs. The severity of injury depends on both by the concentration of ozone and the duration of exposure. Severe and permanent lung injury or death could result from even a very short-term exposure to relatively low concentrations."]

Using O3 on a small scale could be practical, though, especially since the generator may be cheap to buy and there are no consumables. If the chamber devised is small -- as suggested -- and there is no need to cycle many boxes through as quickly as practicable as there was with large facilities like the ETO chambers we had in Manitoba (stationary) and Alberta (mobile). O3 is heavier than air and clearing the device and the area of ozone before entering or handling boxes in the device would be the biggest concern I can see. A good fan should be able to accomplish this, I would think.

[Allen Dick]

Jerry bromenskenk reports on BEE-L:
"Rosalind James at the Ogden Bee Lab has been testing ozone fumigations."

[Allen Dick]

Jerry Bromenshenk posted this to BEE-L:
>Rosalind James at the Ogden Bee Lab has been testing ozone fumigations.

Then Dave Black posted this:
Ozone is frequently used in cool stores dealing with fruit, in my case
kiwifruit. Purefresh is one US proprietary system, Smartfresh I think
another. It is used to deplete ethylene, and as a 'cleanser' killing exposed
fungi and bacteria. Kiwifruit in store is particularly susceptible to
ethylene and botrytis rots.

A search for 'cool storage ozone' will start you off and give some idea of
the concentrations used etc. I have not had the chance to throw some bee
boxes in, mostly because in our case (but not elsewhere) we use it in
controlled atmosphere rooms sealed for several months. It is quite routine
and I haven't the slightest doubt that it would work. Fruit being a seasonal
business you may be able to arrange an out of season 'trial' with a local
store when the stores are not being used. Clearly, it will only work on
exposed surfaces, spores and so on buried in wax will be unaffected.

Very low concentrations are used and venting the rooms is not a problem,
safer actually than venting a CA room in some respects. Yes if you want to
clear it quickly a big fan is used. I haven't really thought about it but
the rooms are something like 12,000cubic metres, and safe in less than eight
hours.

[Countryboy]

After doing some further research, I was able to find this study:
http://www.reeis.usda.gov/web/crisproje ... 15067.html

This year, laboratory experiments in a small fumigation chamber (10 cubic ft) to determine the concentration of ozone needed to kill honey bee pests and pathogens. Tests were conducted using 1000 ppm for up to 3 days. Significant mortality was achieved for chalkbrood, but not for foulbrood. Wax moths infesting comb (all stages) were also found to be readily killed using 500 ppm of ozone.
The pesticides coumophos and fluvalinate were significantly reduced by the ozone treatments, especially at high concentrations. It was difficult to reduce the pesticides to below 100 ppb. Elimination of pathogens from the comb was also evaluated. Chalkbrood in non-sporulating bee cadavers placed in the comb was completely eliminated, and the viability of the spores from cadavers was greatly decreased by the treatments. Methods to better quantify the actual spore mortality levels were developed as result, but experiments using these methods are still underway. Foulbrood scales were also placed in the comb, however, ozone had no measurable effect on the viability of this pathogen at the test concentrations used.

While it doesn't appear to have much efficacy against foulbrood, one has to wonder if ozone would be effective against other viral transmissions via combs and boxes also since it is effective against chalk.

It is interesting to note the reduction of chemical buildup in the combs of checkmite and apistan. For beekeepers who are having fertility problems with drones and queens due to high levels of these chemicals in the combs, this may be a way of cleaning up old combs.

[Allen Dick]

Excellent. Also, on BEE-L, randy Oliver came up with this:

--- begin quote ---

Allen, I have researched ozone sterilization extensively.
Rosalind James in Utah has done the most work.
Hard to kill AFB.
Takes a high concentration, far more than any cheap generator can produce.
Does not penetrate well, but will oxidize anything on the surface.
Sensitive to moisture.
Some commercial beeks in Utah have built rooms, but there is little data as to efficacy.
When venting room, can pass through a catalyst to "neutralize."
Have not yet run trials myself.
Sure wouldn't hurt, and may have effect on viruses and nosema not buried in pollen or honey.
Frank Eischen found that phostoxin was effective against nosema spores.

Randy Oliver

-- end quote ---

[Allen Dick]

Bump.

>Has anyone tried sterilizing old combs and boxes with ozone? You can pick up an ozone purifier for hot tubs or aquariums on eBay for $100. If you pumped the ozone into an old chest freezer, you could gas 8 or 10 boxes at once.
>
>Has anyone tried this? How much ozone production do you need to be effective and how long would you need to gas boxes? Or would it be better (more practical) to ozonate the water you mix the 10% bleach solution with and mist combs and boxes?

My phone rang this morning and a friend asked me about this idea again. He is considering setting up a sizeable room with a generator if they can get enough information as to dosage, potential hazards and pitfalls and efficacy. I've been compiling what I can find at http://www.honeybeeworld.com/diary/articles/ozone.htm So far that has been mostly a matter of compiling this thread, but I expect to add more as I gather responses and resources.

They have lined up a generator they can rent initially at http://www.absoluteozone.com/ and purchase later if desired. They have a large room that is reasonably well sealed, so now it is just a question of whether to proceed -- and how.

Has anyone anything to add to what we have covered here already? I'm writing several researchers in hopes of getting more specific ideas and will share what i learn. I understand that there are some commercial beekleepers in the US who have been using ozone, but have neither their contact info or word of their results.

Is ozone the best choice or should we consider phostoxin? I looked that chemical up and it is controlled up here. It can be had, but is a seriously dangerous product and there are some hopps to jump through. Of course ozone is dangerous, too, but it is not controlled and can be generated quite cheaply on demand.

Anything anybody can offer will be appreciated.

[Countryboy]

I had thought about starting a thread asking how you were going to sterilize your deadouts, but hadn't gotten around to it yet.

I decided not to get a ozone generator. I figured that for me doing only a few boxes, it would be simpler/quicker to use a hand pump sprayer to spray a bleach solution on combs. I figured it would be more practical to look at ozone once i had more hives and could do a room of boxes at once.

Are you planning on reusing a few hives without sterilizing, to see if they crash again? If those hives crash or do poorly, and the sterilized hives do fine, that would be a good indication that it was a virus/bacteria causing the problems. If the unsterilized hives do just as well, you will know the sterilizing may not have had an effect. If you sterilize all the deadouts, it's harder to isolate the problem.

Phostoxin is often used as a fumigant for grain bins to control bugs. It is also used for controlling burrowing rodents. A deceased farmer I used to help would get phostoxin tablets. We would run a garden hose down a groundhog hole and bury the entrance. Then we would drop about 10 tablets down the hose, and pour some water down, since moisture activates the phostoxin. It was a restricted pesticide here, and you had to have a special certification to buy it. The farmer was not certified for fumigation (he was certified for spray chemicals) but they would still sell it to him even though they weren't supposed to since they knew he wasn't going to misuse it. If you know any guys at the local feed mill, they may be able to get some phostoxin for you, even though you aren't certified to use it. (I didn't say that.) I only used phostoxin outdoors - I wore gloves when handling it. The fumes are extremely nasty - I would start to feel ill when treating groundhog holes within a couple hours, and outdoors is as well ventilated as you can get without oxygen tanks.

Does phostoxin work on anything besides wax moths and nosema? Does it affect viruses?

Have you considered spraying combs with bleach water?

Just out of curiousity - have you looked at your deadout combs to see if there is much entombed pollen?

[Allen Dick]

> Are you planning on reusing a few hives without sterilizing, to see if they crash again?

At this point, I have no intention of doing anything to sterilize comb, except allow time to work on them. I really doubt that bleach does much and it is a lot of work. I had considered laying combs out in the bright sun, though.

We'll see what happens with my friends' idea. I'm researching now and won't have time to present everything here yet, but I did write to Rosalind James and Frank Eischen and they both replied. I also started a notes summary page at http://www.honeybeeworld.com/diary/articles/ozone.htm

I have a copy of Rosalind's article, Potential of ozone as a fumigant to control pests in honey bee (Hymenoptera: Apidae) hives
http://www.ncbi.nlm.nih.gov/pubmed/21510179

The objective of the research reported here is to determine whether ozone can kill greater wax moths
> and spore forming pathogens. and uso, to develop the response curves relative to different ozone concen
trations. Honey bees can be infected b a variety of pathogens. but the spore-forming pathogens are the
pathogens most likely to be transmitted on comb due to their long persistence. Spores are also the most difficult
life stage to kill because they are in a dormant state with very low metabolic activity. Two spore-
forming pathogens were tested here, Ascosphaera apis (the cause of chalkbrood) and Paenibacillus larvae
(the cause of foulbrood). Bacterial spores can be extremely difficult to kill, being resilient to both heat and
chemical treatments. P. larvae is resistant to many antibiotics (Kochansky et al. 2001), and the spores can
withstand temperatures of >60°C (Forsgren et al. 2008,Generseh 2008). This pathogen can survive for
years on hive material (Lindström 2006). Spores of care less heat resistant (Anderson et al.
1997, James 2005b) bt also remain viable for many years (Toumanoff 1951). Nosema apis and Nosema
ceranae are also highly virulent spore-forming pathogens of honey bees, but they were not tested here.
These Nosema spp. are more temperature sensitive than the other two pathogens mentioned above, and
they have a much lower environmental persistence on the comb, <1 mo (Malone et al. 2001), suggestive that
ozone conditions adequate to kill Paenibacillus and Ascosphaera are probably adequate to kill Nosema as
well. Furthermore, methods for measuring Nosema viability were not vet available at the time of this study,
making it difficult to determine when ozone was lethal to this pathogen.

I'm assuming that the problem goes away with time. We'll see, but if my friends get fumigation going, I may use it. The whole process takes time I don't have, however. I plan to pick up my hives, split if necessary, super otherwise and head east until July.

> Does phostoxin work on anything besides wax moths and nosema? Does it affect viruses?

Frank Eischen says:

In the May issue of the American Bee Journal (pages 508-509), we published an abstract of some of our work. Phostoxin was effective in killing nosema spores on stored honeycomb. We are still trying to figure out if ozone will work.

Just out of curiousity - have you looked at your deadout combs to see if there is much entombed pollen?

Not yet.

Here are some links:

http://www.powershow.com/view/10ba00-MD ... esentation
http://mj3.biz/slideridgehoney/OzoneTes ... ipment.ppt (Same as above, but a real .ppt file)
http://www.absoluteozone.com/

[cam bishop]

What about acetic acid like Robert Hack uses? Might do the trick much cheaper. I think I'll try it this fall on my combs.

[Allen Dick]

Acetic acid is easier to use, but it seems to me that it does not do as much. It also is really hard on any metal.

My friends who are considering the ozone chamber used acetic in a used ocean container and they say it was very hard on it.

[armust]

I have read of other sterilizing combs by spraying a 10% bleach solution on old combs and boxes. Ozone is supposedly a better sterilizer than bleach. It's my understanding that many parts of Europe sterilize drinking water with ozone instead of chlorine.

Has anyone tried sterilizing old combs and boxes with ozone? You can pick up an ozone purifier for hot tubs or aquariums on eBay for $100. If you pumped the ozone into an old chest freezer, you could gas 8 or 10 boxes at once.

Has anyone tried this? How much ozone production do you need to be effective and how long would you need to gas boxes? Or would it be better (more practical) to ozonate the water you mix the 10% bleach solution with and mist combs and boxes?

If you could sterilize combs and boxes in a reasonable amount of time, this might be practical for a sideliner beekeeper. (A commercial beekeeper would likely need one of the larger ozone generator models so they could gas a whole storage room at once.)

I have never tried this but it sounds very interesting. Will definitely talk with a good friend who is also a beekeeper about this. Maybe we will give it a try. Additionally I will also try to rent such a generator which I think won't be a problem because I normally always use a generator rental. Already excited how this will work and maybe the good friend of mine already tried.

[joannej45]

Hi! Is there a ph level for acetic acid so that the comb will not be destroyed?

[Allen Dick]

Acetic acid will eat nails and excluders, given enough exposure, but is usually not problematic if used as recommended. As far as I know, it has little effect on the comb, but could have some slight effects on the pollen, honey and cocoons.

I do not use acetic acid or recommend it. IMO, acetic acid disinfection is just another of those things beekeepers do so they do not feel as helpless against nature. Strong bees in summer can clean up old combs as long as they don't have AFB scale.