2023 Box Trial of Matrices and Formulations for Prolonged-Unencumber Oxalic Acid

The extended-release manner for oxalic acid (OAE) is turning into extensively used all over the arena, particularly the place mites have advanced resistance to the overused artificial miticides. This yr we ran a box trial to check the efficacy of quite a lot of utility strategies, matrices, and ratios of OA to glycerin.

See my related announcement on this factor about EPA permitting us to make use of generic oxalic acid for varroa management. Whilst we have been looking ahead to a solution, I persevered my analysis on extended-release utility (OAE).

A Massive Box Analysis Undertaking

I obtain a large number of questions from beekeepers about the usage of OAE of their hives. I’m frequently not able to offer definitive solutions, since that is nonetheless experimental analysis in growth, with updates on this magazine, and at my web site [[1]]. 5 questions proceed to pop up:

What’s the most cost-effective and/or maximum efficacious matrix to make use of?
What’s the perfect components (ratio) of oxalic acid to glycerin?
Is it higher to make use of pads laid flat at the best bars, or strips hung between the frames?
Does it assist to switch the pads or strips after a month?
When is the most efficient time of season to use the strips?

This summer season we ran a massive box trial to try to assist solution the primary 4 questions.

Fabrics and Strategies

Experimental Design

Because of the inherent variability between colonies in any box trial, as a way to tease out “the sign from the noise” one should use a massive “n” of check hives for each and every check team, and reflect the trial in several yards. That is particularly the case with varroa therapies, since there are frequently really extensive hive-to-hive variations within the efficacy of precisely the similar remedy.

So I made up our minds to run a large-scale trial in 5 other apiaries (for replication), and to check each and every of 5 other matrices with 3 other ratios of oxalic acid to glycerin, stage of saturation of the matrix, and whether or not the remedy was once implemented best as soon as or changed at 30 days. My plan was once to then use research of variance (ANOVA) to tease out the variations between each and every variable. As an example, via checking out each and every components ratio on 5 other matrices, I may just search for consistencies for the efficacy of the ratio impartial of the matrix (and vice versa). This design would confidently permit me to get probably the most informative effects from a unmarried trial.

I’ve written prior to now about my checking out of quite a lot of absorbent utility matrices [[2]], on the lookout for matrices that have been biodegradable and non-contaminating, simple to organize, practice, and take away, efficacious in supply, and reasonably priced (possibly crucial attention for business beekeepers). My favourite so far have been the Swedish sponges bought as “If You Care” emblem [[3]], which I used as a “recognized” sure management (since on the 1:1 ratio it was once a remedy mixture that had a historical past of operating smartly for us).

In some earlier trials, I had examined chipboard hung strips from New Zealand, the place the advice has been to make use of a 1:1.5 OA:gly ratio, and to switch them after a month (because the hung strips generally tend to “dry out” and be chewed away via the bees; Determine 1). I used to be curious as to (1) whether or not a special ratio may well be extra efficacious, and (2) whether or not the volume of get pleasure from substitute was once value the price of the exertions concerned.

2023 Box Trial of Matrices and Formulations for Prolonged-Unencumber Oxalic Acid

Fig. 1 Freshly-prepared strips to the left; to the suitable strips got rid of 65 days after utility. Those strips tended to “dry out” extra briefly than did the opposite matrices (possibly having one thing to do with gravity), however can have persevered to dispense oxalic acid onto the bees all over the method of chewing and elimination. I thank the producer of the strips (Beequip, New Zealand [[4]]) for generously donating the uncooked strips for this undertaking.

Primarily based upon initial experimentation, I additionally examined the usage of citric acid slightly than oxalic. Citric acid supplies more or less the same quantity of overall acidity gram-for-gram as does oxalic, however it isn’t as sturdy an acid. Citric has the large benefit of being on EPA’s Minimum Possibility Pesticide checklist.

Desk 1 displays the 18 other matrix, acid, and components ratios examined.

Desk 1 The variables have been the two acids, the 5 matrices, and the 3 formulations, leading to 18 other therapies to check.

Floor Space of the Therapies

I’ve prior to now made up our minds that the collection of sq. inches of matrix floor space is important for efficacy [[5]]. However it’s actually the volume of acid on that floor — versus any hidden deep inside the matrix — that permits for distribution via the bees and switch to the mites.

Vital word: All flat-laid therapies for this trial have been minimize into two pads having a complete floor space equivalent to that of the Swedish sponge: 60 sq. inches. Unusually, the advisable utility fee of 6 hung New Zealand strips supplies just about two times as a lot uncovered floor space: 112.5 sq. inches. And that doesn’t needless to say lots of the floor space of the flat-laid pads is blocked via the highest bars, while the bees are uncovered to either side of the hung strips. I do know — this remark is totally opposite to “commonplace sense,” so move determine!

Once more unusually, I discovered in earlier checking out that if that enormous quantity of saturated chipboard is as an alternative laid flat, that it may well overdose the bees, and purpose severe agitation, bearding, and brood kill.

Justification for the Ratios Examined

Our “sure management” components was once the 1:1 (weight:weight) ratio that we’ve used for years. Maggi [[6]] used a 1:2.5 ratio, so I cut up the variation between it and the most well liked New Zealand 1:1.5 system, and examined a 1:2 ratio. As well as, Kanelis [[7]] recommended the usage of a 1:2.7:1.7 (OA:gly:H₂0) ratio. This solvent-heavy ratio would have put little or no oxalic acid into the matrices, so I incorporated a 1:2:1 ratio, which ends up in a “drier” feeling matrix, very similar to the primary ratio that I used with store towels [[8]].

The quite a lot of formulations led to a spread of the volume of overall oxalic acid implemented in keeping with hive (Desk 2), despite the fact that with the extra absorbent matrices, a lot of OA remained within the matrices on the finish of the trial 65 days later.

The Take a look at Colonies

An added issue for me is that (as I described in September [[9]]) one of the crucial colonies that I had at my disposal became out to be mite resistant (what a bummer), and thus would now not be appropriate for checking out the efficacy of a remedy in opposition to varroa. So I may just best use colonies which exhibited a buildup of their mite ranges.

Experimental limitation: It’s just about not possible to equalize the mite infestation charges of a beginning team of colonies, since over part the mites are generally within the brood, and the mite inhabitants and replica dynamics can range a great deal from hive to hive. I handled this factor via the usage of a randomized block design to assign therapies (stratified project via their mite counts after 3½ months of mite buildup), thus now not best minimizing the impact of this variable, but additionally permitting us to check the impact of remedy over a spread of infestation charges.

I may just best scrounge up sufficient colonies with excessive sufficient mite counts to permit me an “n” (collection of replicates) of 12 hives in keeping with remedy (the minimal that I’ll use), however since each and every matrix was once implemented to 36 hives, and each and every components ratio to 72 hives, I was hoping that this repetition would permit me to tease out helpful findings. So we implemented therapies to 18 x 12 = 216 hives in overall (Determine 2).

Fig. 2 We numbered all of the check hives within the 5 yards, taken care of them via beginning mite rely, and randomly assigned therapies to each and every tier of 18 (a randomized block design). At each and every backyard, we unfold out the 18 tubs, and really moderately implemented the assigned remedy to each and every hive. Right here Brooke is appearing because the manager “Hawk,” monitoring and recording affirmation that Rose took each and every remedy from the suitable bathtub and positioned it on best of the proper hive for insertion.

For the reason that 3 formulations for each and every matrix appeared equivalent, we needed to be very cautious, and if truth be told (because of a conceivable error in correct utility) in two yards changed all of 2 therapies day after today (I’m a stickler for buying the whole lot precisely proper). The colonies numerous in energy, however all had bees in each the higher and decrease packing containers.

The women did a super activity at distributing and making use of the therapies, however they weren’t moderately as much as lifting 216 heavy higher brood chambers (Determine 3).

Fig. 3 It was once dang scorching as we arrange the trial (Determine 4), and I equipped the muscle to crack open each and every hive for inspection and insertion of the remedy. The surfaces of our darkish hive covers reached over 150˚F, and simply resting them in opposition to my naked arm once I tipped them up would ship a flash of warmth thru my frame. I needed to pause every so often from lifting the heavy packing containers to keep away from heatstroke! Thank goodness that we stay delicate bees that don’t require us to put on a lot coverage.

Fig.4 The elements began out extremely popular and dry as we have been acting mite washes and making use of therapies, then cooled off slightly. Daylight humidity was once low early on, however in brief were given excessive in early September (there was once no rain all over the trial). Knowledge from KCAGRASS50 (2523 toes elevation).

The Matrices

In an effort to introduce you to the matrices that we examined, I took footage of Rose making use of them to a few hives (Figures 5 thru 10).

Fig. 5 Our “sure management” was once a Swedish sponge (If You Care emblem) minimize in part. All flat-laid therapies have been laid at the best bars of the decrease brood chamber, to the entrance and the rear of the hive, focused at the cluster. This enabled us to feed pollen sub to inspire brood rearing all over the lack (to permit for the mites to proceed to breed).

Fig. 6 I had disregarded corrugated cardboard all over earlier exploratory checking out, because the adhesive of the items that I attempted fell aside within the scorching oxalic resolution. However I lately spotted a delivery field comprised of double-layer, eco-friendly corrugated cardboard [[10]] and gave it a handy guide a rough check, which recommended that its glue may paintings for us. I showed that the double-corrugation board may just soak up a large number of oxalic resolution, so I incorporated it within the trial, since as a matrix it could be readily to be had to penny-pinching beekeepers.

Fig. 7 Sadly, we discovered that when a few days within the bathtub, the corrugated pads tended to delaminate, making them slightly tricky to use and to later take away. However the fee is true!

Fig. 8 I ordered chipboard sheets (very similar to that used for the New Zealand hung strips) from Uline [[11]]. They took a while to take in the answer, however have been simple to put in and take away. You’ll have spotted that we’ve shifted from the usage of nitrile gloves to meals dealing with gloves, which now not best supply ok coverage, however are a lot more straightforward to get off and on sweaty palms. And since they’re so reasonably priced we don’t hesitate to take away (and correctly cast off) them any time that we would like our palms to be freed from gloves (or acid).

Fig. 9 On the advice from a beekeeper whose title I’ve unfortunately out of place, I attempted King Zak biodegradable towels [[12]]. Those towels, despite the fact that skinny, have been unusually absorbent, and remarkably sturdy (they held in combination for elimination after 65 days). They appear promising for additional experimentation.

Fig. 10 For hung matrices, we implemented Beequip’s 1¼” x 15” chipboard strips on the fee of three strips in keeping with brood chamber [[13]]. As identified prior to now, this ends up in a some distance higher quantity of floor space publicity to the bees than with the flat-laid pads. The hung strips, despite the fact that slightly extra tedious to use, paintings smartly for treating nucs or unmarried brood chamber hives.

Following suggestions from New Zealand, I’d deliberate on changing the hung strips in part the check hives with contemporary ones at 30 days. On the other hand, when the time got here to take action, I made up our minds to additionally substitute all the pads within the different remedy teams in the similar yards as smartly (as a way to equalize that variable for all check teams). That then doubled the collection of remedy teams to check to a rely of 36!

Dose implemented to the bees

This brings us to the oblique dispersion of (via switch via the bees) of the lively aspect — oxalic acid — and its publicity to the mites (the supposed goal) (Determine 11).

A teaser: I’ve been the usage of chemical titration to trace the real quantity of acid residues at the bees’ our bodies due to quite a lot of utility strategies of oxalic acid (and feature introduced my initial findings at conventions and on Zoom). I’m hoping to quickly post my findings.

Fig. 11 Even though we’re satisfied to peer lifeless mites at the pads, in reality it’s not likely that they died from direct touch — it’s the volume of acid that will get disbursed from the pads onto the our bodies of the bees that it seems that does the trick. The wet floor of this sponge is because of the absorption of atmospheric moisture inside the cluster via the glycerin humectant.

Sensible utility: Glycerin absorbs just about part its weight in water at cluster humidity (which runs at about 50-60% impartial of ambient humidity). My sons and I’ve discovered to test for a pad’s attainable for acid switch from its floor via touching them flippantly with a fingertip, after which tasting that finger for acidity [[14]]. As long as the pad’s floor stays wet and bitter tasting, it may well proceed to disburse acid onto the bees.

Effects

So how a lot overall acid would the implemented therapies include? I weighed them as we keen them to peer (Desk 2).

Desk 2 The quantities proven in crimson are the full implemented dose in keeping with hive. Notice that at upper solvent (glycerin and water) ratios, much less overall acid is contained within the matrix. And the addition of water ends up in there being much less glycerin as a humectant (and a drier floor).

So simply how essential is the full quantity of acid in a pad or strip? This could be regarded as because the “dose in keeping with hive,” however doesn’t account for the way a lot acid degradation happens [[15]] or by no means will get dispersed prior to the pad is got rid of via the beekeeper or the bees (I’ve showed that oxalic acid degrades relatively hastily when involved with natural fabrics, however some matrices maintained really extensive acidity (via the style check) on their floor for over two months in my setting).

Even though the full quantity of OA contained within the matrix does now not essentially mirror the volume that makes it onto the bees, it did correlate with efficiency (Determine 12). The 1:1 sponges and NZ strips held probably the most acid (66 & 62 g respectively; each carried out smartly), and the 1:2:1 King Zak fabric and chipboard the least (12 & 18 g respectively; each carried out poorly).

Fig. 12 There gave the look to be a susceptible correlation between the gross quantity of oxalic acid within the pads or strips, and the ensuing % mite relief. This may merely be as a result of there was once extra acid at the floor upon utility, or extra of a reservoir of acid to diffuse to the outside over the years. Notice that the best possible doses have been not more efficacious than part the dose, particularly with the sponges, KZ fabric, and corrugated cardboard — the matrix gave the look to be extra essential than the dose! Unusually, the sponges with the 1:2:1 ratio carried out really well in all check hives however two.

Replace: It’s transparent that it doesn’t require 50 grams of oxalic acid to acquire just right efficacy. The brand new product Varroxan comprises 7 grams of OA dihydrate in keeping with strip, with 4 strips advisable in keeping with hive, which equals best 28 grams – a determine supported via the information above.

In my analyses, I used median values slightly than method. Way may also be a great deal skewed via a unmarried hive with an excessively excessive mite rely. Medians extra mirror what a beekeeper is concerned with — the midpoint, with part the hives showing decrease, and part upper values. Statistically, one contains the usual deviation or a field plot. However with this huge information set, I discovered that merely appearing the uncooked information in blue and crimson tells us all we wish to know [[16]](Determine 13).

Fig. 13 Effects 65 days after utility of the therapies. The extra crimson in keeping with remedy team, the poorer the efficiency; the extra blue the easier. I highlighted the most efficient performers in yellow. However take note of the median mite relief figures within the small packing containers beneath each and every remedy.

Abstract of my interpretation of the chart

Vital word: The chart does now not point out “efficacy” of remedy, however best relief (typically) from the beginning rely. In any hive by which its crimson column is not any taller than its blue column, its mite infestation fee didn’t building up (typically it diminished). I stacked the columns on this chart for higher visibility — in case you see any blue in any respect, that colony’s mite rely went down. Any column appearing most commonly blue would point out excessive efficacy, however I may just now not calculate the price, since we didn’t have detrimental controls.

Formulations: So far as components, the 1:1 ratio carried out the most efficient total (probably the most blue), with a couple of different wonder showings. Its constant efficiency throughout quite a lot of matrices is telling.

Matrices: Sadly, chipboard laid flat was once unimpressive, even on the repeated 1:1 ratio. On the other hand, when hung within the New Zealand strips, it carried out smartly. Swedish sponges have been probably the most constant performers total, adopted intently via the New Zealand strips, double-corrugated cardboard (particularly on the 1:1 ratio), and the wonder appearing via the skinny King Zak towels (as opposed to with the 1:2:1 ratio). I didn’t check Maximizer pads [[17]], since they’ve carried out very in a similar way to Swedish sponges in earlier trials.

Repeating the appliance: Unusually, changing the therapies at 30 days didn’t fortify their efficiency for any check team as opposed to the King Zak towels. I’m now not transparent on why the Kiwis suppose that it’s of receive advantages with the hung strips.

The citric acid therapies: I didn’t display the result of the citric acid therapies, since at our spot tracking on the trial midpoint, many in their mite counts have been exploding, so we got rid of them from the trial and handled them with oxalic. Bummer, since I had excessive hopes for citric.

Dialogue

Aid vs. efficacy: This was once a comparative trial of the matrices and formulations, by which shall we evaluate the % mite discounts via the quite a lot of therapies, however now not decide efficacy, which might have additionally required an untreated detrimental management team.

As an example, if in a detrimental Keep an eye on team the mite rely quadruples over the 2 months of a tribulation (a sensible building up), a colony that maintained its authentic beginning infestation fee would show off a % mite relief of 0, however an efficacy of 75% (relative to the Keep an eye on) via the Henderson-Hilton calculation.

Sensible utility: Primarily based upon the foremost will increase within the infestation charges of the citric therapies and the opposite deficient performers, we will conclude that the efficacies of lots of the 1:1 remedy teams have been if truth be told moderately excessive.

Some other factor to note within the chart is that you just generally tend to peer proportionally extra crimson in colonies that began with low mite counts, in comparison to those who began with excessive mite counts. That is one thing that I’ve spotted prior to with different miticide therapies [[18]] – that you just get your biggest quantity of “remedy disasters“ in colonies beginning with decrease counts. I’ve transformed the information in Determine 14 to turn the trend.

Fig. 14 Notice that there are proportionally extra will increase in mite counts in the ones hives beginning with decrease counts (to the left) than in the ones with upper beginning counts (to the suitable). I’m now not transparent as to why that is, however it was once once more obvious on this trial.

Sensible utility: Mite therapies usually generally tend to show off a better % relief (and thus calculated efficacy) when implemented to high-mite hives than to low-mite hives. I want that I may just provide an explanation for why!

Musing on different research

There are a variety of actually just right research on OAE, however I should take the result of plenty of others with a grain of salt. Some don’t take into account that despite the fact that the remedy reasons increased mite drop all over the primary week, that it might also purpose an building up within the infestation fee for the primary month, taking two complete months to score complete efficacy (possibly because of further modes of motion as opposed to acute toxicity). And as proven above, it makes a distinction whether or not the check colonies get started with low or excessive mite infestation charges (so beginning with low-count hives would possibly lead to extra confounding outliers).

As well as, as evidenced via this trial:

It’s now not best in regards to the overall dose implemented.
The ratio of oxalic acid to glycerin makes a large distinction.
As does the supply matrix used.
The stage of saturation of the matrix (“sloppy” matrices would possibly paintings higher than “dry” ones).
The volume of floor space of the of the pads or strips is important.
In addition to is their placement — bees it seems that should make touch as a way to distribute the acid to the mites.
The sudden remark that hung strips require a better quantity of floor space than do pads laid flat around the best bars (no less than when implemented to double brood chamber hives).
Whether or not the outside of the supply matrix stays wet and acidic.
The truth that we’re now not but transparent at the modes of motion that oxalic remedy has upon the mites (it’s now not simply acute toxicity — watch this area!).
And upload the impact of the beginning infestation fee, in addition to
The huge variations in hive-to-hive efficiency in the similar backyard.

Complicated? Sure. Does OAE paintings? Sure. Will we nonetheless have so much to be told? Sure!

Ultimate Notes

The most productive use of this remedy seems to be utility firstly of the honey float. We want extra analysis on its efficacy at different instances of the season.

Repeated utility of oxalic acid with out rotation of miticides with different modes of motion would possibly smartly make a selection for the evolution of resistant mites (don’t wager in opposition to evolution). Thymol is a brilliant observe up after you’ve pulled your honey. Formic acid works smartly within the springtime. You’ll be able to additionally rotate in Hopguard or a man-made miticide. And naturally our objective is to make use of resistant bee inventory that can best require a unmarried remedy a yr!

A request: Please don’t write me for main points on utility till you’ve learn this directions web page (which I will be able to attempt to stay up to date):

https://scientificbeekeeping.com/instructions-for-extended-release-oxalic-acid/

Acknowledgements

Due to my helpers Rose Pasetes, Brooke Molina, and Corrine Jones.

Citations and Notes

[1] https://scientificbeekeeping.com/instructions-for-extended-release-oxalic-acid/

[2] https://scientificbeekeeping.com/extended-release-oxalic-acid-progress-report-2/ (First printed in American Bee Magazine, October 2017)

https://scientificbeekeeping.com/extended-release-oxalic-acid-progress-report-3/ (First printed in American Bee Magazine, January 2018)https://scientificbeekeeping.com/extended-release-oxalic-acid-progress-report-4/ (First printed in American Bee Magazine, November 2018

https://scientificbeekeeping.com/extended-release-oxalic-acid-progress-report-2019/ (First printed in American Bee Magazine, December 2019)

https://scientificbeekeeping.com/2022-extended-release-oxalic-update-part-3/ (First printed in American Bee Magazine, Might 2022)

https://scientificbeekeeping.com/testing-cotton-matrices-for-oae/ (First printed in American Bee Magazine, January 2023)

[3] To be had on Amazon, or a lot inexpensive if bought in bulk a variety of 500 or 1000 (touch Oliver Weiss [email protected]).

[4] https://beequip.nz/

[5] https://scientificbeekeeping.com/7701-2/ (First printed in American Bee Magazine, March 2022)

[6] Maggi, M, et al (2016). A brand new components of oxalic acid for Varroa destructor management implemented in Apis mellifera colonies within the presence of brood. Apidologie 47: 596-605.

[7] Kanelis, D, et al (2023): Analysis of oxalic acid with glycerin efficacy in opposition to Varroa destructor (Varroidae): a 4 yr assay. Magazine of Apicultural Analysis, DOI: 10.1080/00218839.2023.2169368

[8] https://scientificbeekeeping.com/extended-release-oxalic-acid-progress-report/ (First printed in American Bee Magazine, July 2017)

[9] Selective Breeding Development File 2023

[10] Pratt Eco Choices, in a field from House Depot. I’ve now attempted a couple of manufacturers, and none actually grasp in combination lengthy.

[11] https://www.uline.com/Product/Element/S-18997/Corrugated-Pads/8-1-2-x-11-Chipboard-Pads-050-thick

[12] From Amazon: “Eco Pleasant Reusable Cleansing Cloths, Reusable Paper Towel Material, 30 Sheets, All Goal Dress, Biodegradable.” Thanks to the beekeeper who recommended those, and whose title I’ve out of place.

[13] For the occasional weaker colony, we implemented strips proportionally, as a way to deal with constant publicity.

[14] I in fact am now not recommending that you just do that. However needless to say a unmarried serving of spinach would possibly include a complete gram of oxalic acid.

[15] Those figures are the acid content material right away after preparation. I’ve now not but carried out titrations to decide how hastily the acid degrades after preparation (it’s on my to do checklist!).

[16] I do know — with out a calculated p-value, trusting our eyes and mind to select a trend would by no means go peer evaluate. However I frequently believe my eyes greater than I believe convoluted statistics.

[17] 2022 Prolonged-Unencumber Oxalic Replace Section 1 https://scientificbeekeeping.com/7701-2/ (First printed in American Bee Magazine, March 2022)

[18] https://scientificbeekeeping.com/mite-control-while-honey-is-on-the-hive-part-2/ (First printed in American Bee Magazine, December 2020)