Oral toxicity of essential oils and organic acids fed to honey bees (Apis mellifera)

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DOI: 10.1080/00218839.2007.11101398
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Abstract
Natural plant products have been studied for potential use as in-hive fumigants for suppression of parasitic mites and other pests. A more direct application through direct feeding of bees would avoid problems with fumigant volatility in cold climates and provide a more systemic route of exposure for the target pest. However, there must be a balance between toxicity to hive pests and toxicity (safety) to the bees. We focused on adult bee toxicity when testing ten products: cineole, clove oil, formic acid, marjoram oil, menthol, oregano oil, oxalic acid, sage oil, thymol, and wintergreen. Each product was tested at several concentrations in a sugar syrup fed to bees over several days, and dead bees were counted daily. Oxalic acid was the most toxic of the products tested. Menthol and cineole had mortality levels no different from controls fed plain syrup after 8 days of treatment. At 14 days of treatment, wintergreen was the least toxic, but neither menthol nor cineole were a part of the testing that went to 14 days. Our results indicate that the tested products could all be used safely for treating bees orally if dose is carefully managed in the hive.
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ORIGINAL RESEARCH ARTICLE
Oral toxicity of essential oils and organic
acids fed to honey bees (Apis mellifera).
Timothy A Ebert1*, Peter G Kevan2, Bert L Bishop3, Sherrene D Kevan2, and Roger A Downer1
1Laboratory for Pest Control Application Technology, Ohio Agricultural Research and Development Center, The Ohio State University,
1680 Madison Ave. Wooster, OH 44691, USA.
2Enviroquest Ltd., 352 River Road, Cambridge, ON N3C 2B7, Canada.
3Computing and Statistical Services, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison
Ave. Wooster, OH 44691, USA.
Received 27 March 2007, accepted subject to revision 8 June 2007, accepted for publication 17 June 2007.
*Corresponding author. Email: tebert@ufl.edu
Summary
Natural plant products have been studied for potential use as in-hive fumigants for suppression of parasitic mites and other pests.
A more direct application through direct feeding of bees would avoid problems with fumigant volatility in cold climates and provide
a more systemic route of exposure for the target pest.However, there must be a balance between toxicity to hive pests and toxicity
(safety) to the bees. We focused on adult bee toxicity when testing ten products: cineole, clove oil, formic acid,marjoram oil,
menthol, oregano oil, oxalic acid, sage oil, thymol, and wintergreen. Each product was tested at several concentrations in a sugar
syrup fed to bees over several days, and dead bees were counted daily. Oxalic acid was the most toxic of the products tested.
Menthol and cineole had mortality levels no different from controls fed plain syrup after 8 days of treatment. At 14 days of
treatment, wintergreen was the least toxic, but neither menthol nor cineole were a part of the testing that went to 14 days. Our
results indicate that the tested products could all be used safely for treating bees orally if dose is carefully managed in the hive.
Toxicidad oral de aceites esenciales y ácidos orgánicos en la
alimentación de la abeja de la miel (Apis mellifera)
Los productos naturales de plantas han sido estudiados para su uso potencial como agentes fumigantes de represión de ácaros
parásitos y otras plagas. Una aplicación más directa a través de la alimentación de las abejas evitaría problemas como la volatilidad de
los fumigantes en climas fríos y proporcionaría una vía más sistémica de exposición para las plagas. Sin embargo, debe haber un
equilibrio entre la toxicidad para las plagas y la toxicidad (seguridad) para las abejas. Nosotros nos hemos centrado en la toxicidad
sobre abejas adultas de diez productos: eucaliptol, aceite de clavo, ácido fórmico, aceite de mejorana, mentol, aceite de orégano,
ácido oxálico, aceite de salvia, timol y aceite esencial de wintergreen (salicilato de metilo).Cada producto fue probado con
diferentes concentraciones en un jarabe de glucosa que alimentó a las abejas durante varios días, las abejas muertas fueron contadas
diariamente. El ácido oxálico fue el producto más tóxico de todos los analizados. El mentol y el eucaliptol presentaron niveles de
mortalidad similares a los controles, que fueron alimentados únicamente con jarabe después de 8 días de tratamiento. Tras 14 días
de tratamiento, el aceite esencial wintergreen fue el menos tóxico, pero ni el mentol ni el eucaliptol se incluyeron en el análisis a los
14 días. Nuestros resultados indican que todos los productos testados pueden ser utilizados con seguridad por vía oral para el
tratamiento de las abejas si la dosis es administrada cuidadosamente en la colmena.
Keywords: Medicaments, oral toxicity, natural plant products, mortality, miticides, protectants
Journal of Apicultural Research and Bee World 46(4):220–224 (2007) © IBRA 2007
Oral toxicity of essential oils and organic acids fed to honey bees 221
Introduction
Various essential oils and organic acids have been evaluated as
materials to manage mite populations afflicting honey bees. In
general, these products are proposed to be used as in-hive
fumigants (Imdorf et al. 1999) or as contact treatments (Amrine
et al. 1996). In-hive fumigants have the disadvantage of needing
warmth for sublimation or evaporation, and therefore they are
less effective in colder weather (Scott-Dupree & Otis 1992).
Nevertheless, organic acids like formic acid, and essential oils like
thymol, have been found to be effective in management against
mite pests in honey bee hives (Imdorf et al. 1999). Oral
application circumvents the problems of fumigation in cold
weather, but this application strategy has been mostly neglected.
Menthol can be administered orally to honey bees, in
microencapsulated formulation, with beneficial effects in
suppressing population growth of tracheal mites (Acarapis woodi)
(Kevan et al. 1997;2003). If essential oils and organic acids are to
be considered as potential medicaments, rather than fumigants,
one must be sure that the target animals (e.g. honey bees) are
not poisoned. As with the research on menthol, we investigated
oral toxicity of various essential oils and organic acids with the
aim of assessing the potential problem of poisoning the patients
with the active ingredient of the medicine (Kevan et al. 1999). As
a control, we also used a plant compound, amygdalin, known to
be poisonous to honey bees (Kevan & Ebert 2005), and
reassessed menthol as a compound known to be innocuous to
honey bees (Kevan et al. 1999). Menthol provides a positive
control that is useful in evaluating the toxicity of other
medicaments.
This research was on the toxicity of various natural plant
compounds to honey bees with the intent to use these
compounds to treat hives for various hive pest problems. It is
likely that such treatments will involve exposing the colony to the
compound for weeks or months. It is therefore necessary to
assess how both the dose and the length of exposure influence
mortality.
Materials and Methods
Honey bees (Apis mellifera ligustica) were obtained from hives at
the Ohio Agricultural Research and Development Center
(OARDC) Honey bee Lab, and placed in cages similar to those
used by Kulencevic & Rothenbuhler (1973), and previously
described in Kevan & Ebert (2005). Each cage had an average of
48 bees (+/-15 S. D.; range 20–134). Fed bees were given sugar
syrup (69% sucrose), or a 69% sucrose syrup spiked with one of
10 possible natural plant products: cineole (CAS 470-82-6), clove
oil, formic acid (CAS 64-18-6), marjoram, L-menthol (CAS 2216-
51-5), DL-menthol (CAS 1490-04-6), oregano oil, oxalic acid
(CAS 144-62-7), sage oil, thymol (CAS 89-83-8), or natural
wintergreen oil (CAS 119-36-8). The clove oil was a commercial
extract from Eugenia caryophyllata Thunb. (Myrtaceae). The main
chemical components of clove oil are eugenol, eugenol acetate,
iso-eugenol and caryophyllene
(http://www.essentialoils.co.za/essential-oils/clove.htm). The
oregano oil was a commercial extract from Origanum vulgare L.
(Lamiaceae). The main chemical components are carvacrol,
p-cymene, y-terpinene, and b-caryophyllene (Chorianopoulos et
al. 2004). The marjoram oil was a commercial extract from
Origanum marjorana L. (Lamiaceae). The main chemical
constituents are sabinene, a-terpinene, y-terpinene, p-cymene,
terpinolene, linalool, cis-sabinene hydrate, linalyl acetate, terpinen-
4-ol and y-terpineol (http://www.essentialoils.co.za/essential-
oils/marjoram.htm). The sage oil was a commercial extract from
Salvia sclarea L. (Lamiaceae). The main chemical components of
sage oil are linalool, a-terpenol, linalyl acetate, neryl acetate, and
sclareol (Pitarokill et al. 2002). All solutions, including the control,
had 5ml ethanol added to bring the total syrup volume to 100ml.
Insoluble potential medicaments were dissolved in the ethanol
first, then mixed with the syrup to bring the volume up to 100ml.
In addition to these treatments, we included an unfed control,
with no food or water. The starvation treatment was necessary
because it was rumored that some of these medicaments would
reduce feeding. We needed a starvation treatment to
differentiate between toxicity and death due to starvation or
dehydration.
Bees were fed ad libitum for the duration of the experiments.
No additional water was provided, except what they could get by
feeding on the syrup solution. We will call this days of treatment
(DOT), since the bees are continually exposed for the entire
duration. Each treatment was replicated four times. Mor tality was
checked daily, and dead bees were removed. Bees were
considered dead when they would no longer move in response
to poking with forceps. At the end of the experiment, the live
bees were frozen and then counted. Cages were kept in open
laboratory conditions that ranged from 17 to 25°C with a R.H.
(Relative Humidity) from 18 to 37%.
We present the results from two tests. The first test
evaluated the toxicity of all potential medicaments every day over
an 8 day period at concentrations of 100 and 1000 ppm. The
second test evaluated the toxicity of clove oil, formic acid, oxalic
acid, oregano oil, and sage oil along with a fed and a starved
control over a 14 day period. Potential medicament
concentrations in the second test were at 100, 500, 1000, 5000,
10000, and 100000 ppm. Note; the 100000 ppm solutions for all
potential medicaments tended to separate, or crystals developed
in the solution. It is likely that the bees never experienced a
potential medicament at 100000 ppm, even though we tried
mixing the potential medicament back into the sugar syrup by
shaking up the bottles once per day. Also note that we tested
both D and DL menthol because sometimes a particular isomer
is more toxic than others. However, we could not find any
evidence of such a difference in the toxicity of these products.
Therefore, our discussion of menthol will be the results from the
combined data of the L-menthol and DL-menthol treatments.
Data were analyzed in SAS using a time-dose-mortality
analyses and probit analyses. The time-dose-mortality model was
a complimentary log-log model using a SAS program that was
written as an implementation of the work by Priesler & Robinson
(1989). However, the Hosmer-Lemeshow goodness-of-fit test
(Nowierski et al. 1996) was highly significant for all models. We
suggest that this lack-of-fit was caused by long tails in the data.
When individual time intervals were of special interest, we used
222 Ebert, Kevan,Bishop, Kevan, Downer
Proc Probit to estimate the LD50 values for that day. Although the
Proc Probit corrected for control mortality, the results from the
time-dose-mortality analysis did not correct for control mortality.
For this reason, the time-dose-mortality analysis overestimates
the toxicity of these products.
Results
We have included the results from the amygdalin (known to be
toxic to honey bees) trials for comparison (Kevan & Ebert 2003).
Relative to amygdalin, all of the materials we tested were
innocuous. Although oxalic acid is quite toxic relative to the other
materials (Table 1), Table 2 shows it to be somewhat less than
half as toxic as amygdalin on a molecule per molecule basis.
Starved bees do not live long, with 40% dying in the first 24
H, and all the bees dead within four days (Table 1). No other
treatment had higher first day mortality rates. High
concentrations of oxalic acid were the most toxic treatment, and
differed from the starvation treatment by having relatively low
first day mortality. Mortality was 100% at the highest oxalic acid
concentration, but bees at the lowest concentration only had
about 60% mortality after 14 days (there was 40% mortality in
the controls by this time). The second most toxic was formic acid,
for which the last survivors died eight days post treatment at the
highest concentration tested. However, mor tality was only 30%
after 14 days at the lowest concentration. A few other potential
medicaments also showed high mortality levels at the maximum
concentration tested. In contrast to these products, we conclude
that cineole, menthol, marjoram, and thymol are non-toxic to
bees because their mortality levels never exceeded background
mortality at any concentration tested (Table 2).
All products tested were much less toxic than amygdalin on a
molecular basis. Although the LT50 for oxalic acid and amygdalin
are similar, it takes over twice the number of molecules of oxalic
acid to achieve an equivalent level of toxicity (Table 2). However,
when comparing LD50s at 8 days of treatment (DOT), oxalic acid
appears more toxic (Table 3). The reason the model is non-
significant (α<_0.01) in this case is because mor tality in all
treatments was too high and too variable (average mortality in
lowest dose was 50% +/- 40% S. D.). The reason the model for
sage is also non-significant (α<_0.01) is because, even at the
highest dose, mortality was only 60% +/- 40% S. D.
The critical feature in the potential utility of all these products
is their long term effects on bee health. This was assessed by
keeping bees in the cages as long as possible. Treatment with sage
oil resulted in highly variable mortality, for which we have no
explanation. Oxalic acid, with the lowest LD50 value, was most
toxic and wintergreen was the least (Table 4).
All LD50 levels decline over time. However, most show a rapid
decline in LD50 values within the first few days, followed by a
leveling off. In part this trend reflects ever increasing levels of
background mortality. However, most cages of bees had a few
individuals that lived many days longer than their sisters. This
made the tails of the mortality distribution long, and probably
accounted for most of the significance in the lack-of-fit tests.
Day 1 Number
Potential Average Average % mortality of Bees
8 DOT 14DOT
Medicament % mortality 1000 ppm 100,000 ppm*Tested
Amygdalin 0 79** 221
Cineole 2 11 214
Clove oil 3 28 96 927
Control – fed 1 10 40 1189
Control – unfed 46 100*** 100*** 495
Formic acid 1 33 100 1438
Marjoram oil 2 34 308
Menthol DL 1 25 256
Menthol L 1 21 239
Oregano oil 1 41 92 953
Oxalic acid 6 96 100 1217
Sage oil 3 21 87 1289
Thymol 8 43 201
Wintergreen 1 24 99 1308
Table 1. Twenty four hour mortality and average mortality at 8 days and 14 days exposure (DOT) for various essential oils and
organic acids fed to honey bees, together with the total number of bees tested (sum of all replicates and dosages).
*Cells with missing data are left blank. ** Dosage for amygdalin was 2250 ppm. *** All bees were dead in 4 days.
Oral toxicity of essential oils and organic acids fed to honey bees 223
Table 2. Estimated LT50 for potential medicaments at 1000 ppm. In this analysis, data from tests 1 and 2 were combined to estimate
the LT50. Except as noted, all terms in the models were significant (α<_0.01), and all lack-of-fit tests were also significant (α<_0.01).
Material Molarity*LT 50 Lower 95% Upper 95%
in days Fudicial Fudicial
Limit Limit
Amygdalin** 0.0049 4.6 3.0 6.3
Cineole 0.0065 NS
Clove Oil 11.2 10.2 12.8
Controls – Fed 17.0 15.9 18.7
Controls – unfed 1.9 1.7 2.1
Formic Acid 0.0217 11.8 8.1 30.3
Marjoram Oil 27.0 15.0 3935.0
Menthol 0.0064 NS
Oregano Oil 10.8 9.9 12.1
Oxalic Acid 0.0111 4.8 2.6 5.9
Sage Oil 11.5 10.6 12.7
Thymol 0.0067 NS
Wintergreen 0.0029 14.4 12.6 17.7
NS = no significant model. Mortality in these tests did not reach 50%, so accurate estimates cannot be made.
* These plant extracts are blends of several compounds so it is not possible to calculate the molarity of each component.
** Amygdalin concentration was 2,250 ppm, the lowest concentration tested by Kevan and Ebert (2005).
Table 3. Estimated LD50 for the tested essential oils and organic acids at 8 Days exposure (DOT).
Material LD50 Lower 95% Upper 95%
in ppm Fudicial Fudicial
Limit Limit
Amygdalin 1600 1300 1800
Clove Oil 7800 2300 18100
Formic Acid 5600 3500 8100
Oregano Oil 14900 0 43400
Oxalic Acid NS
Sage Oil NS
Wintergreen 13500 8500 21700
NS = model non-significant (α<_0.01). Oxalic acid was too toxic and mortality by Sage Oil was too variable.
Table 4. LD50 values 14 days exposure (DOT).
Material LD50 Standard Lower 95% Upper95%
in ppm Error of Fudicial Fudicial
Log10 (dose) Limit Limit
Clove Oil 240 0.1483 80 710
Formic Acid 450 0.0653 280 720
Oregano Oil 600 0.1327 230 1620
Oxalic Acid 80 0.0635 50 130
Sage Oil*NS
Wintergreen 800 0.0632 500 1270
*dose was not a significant variable in the time-dose-mortality model.
Discussion
Looking at mortality in Table 1, we conclude that wintergreen,
menthol, sage oil, and cineole all are relatively innocuous, and that
marjoram oil is quite benign. At the other extreme, oxalic acid
and, as expected, amygdalin were the most toxic, and has the
potential to cause high levels of mortality if the dosage is high.
These results also demonstrate that development of hive
treatment protocols require balancing the exposure time and the
dose given to the hive. Our results are sufficient for dosing bees
for up to 14 days, but we expect that dosage would have to be
more limited if the exposure period is lengthened. If continuous
dosing is the best treatment option, then additional research
would be needed to assess toxicity during the over-wintering
period and the effects these products may have on egg laying
viability of the queen, and development of larvae. However, all of
these products have a low enough toxicity that they could be
used as ingested medicines (rather than fumigants) with minimal
effects on the hive. Clearly, the next phase is to determine if safe
doses of these products can be fed to bees to effectively manage
hive pests, and whether it is better to shock the hive with a short
term massive dose, or to try long term exposures at low dosages.
We would like to caution readers that these results are most
relevant to adult worker health. It is possible that the adult
workers could feed the medicaments to larvae, and that the
larvae may be more sensitive. It is also possible that the
medicaments fed to the queen or drones could affect their
reproductive capacity. However, exposure of these individuals is
buffered through the workers. Unless the only source of food for
the entire hive is the treated sugar water, there will be a dilution
effect where the treated sugar water is mixed in the hive with
nectar from outside sources. A queen that gets one drop of
treated syrup from one worker followed by a droplet of nectar
from outside the hive is effectively consuming a nectar at half the
dosage of the treated syrup. Consequently, workers will be
exposed to greater dosages of these medicaments than will other
members of the hive. Therefore, testing the toxicity of the
medicaments to the workers is a natural first step, and it may be
the only necessary step unless other problems occur as these
products are developed.
Acknowledgements
We thank SBIR (Small Business Innovation Research) of the
United States Department of Agriculture for support to Robert
A. Stevens and Betterbee Inc. (Greenwich, NY). Alison Skinner
and team of the Ontario Beekeepers’ Association assisted with
bee wrangling and technical instruction. Jim Tew and the Ohio
State University Honey bee Lab supplied the bees for the
research in Wooster. Rebecca Eber t provided additional
laboratory support in Wooster.
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224 Ebert, Kevan,Bishop, Kevan, Downer
  • ... However, there is still scarce information about the effect of prolonged systemic administration of EOs containing carvacrol on honeybees (Ebert et al., 2007;Porrini et al., 2017). Therefore, the aim of this study was to evaluate and compare the sublethal effects of long term consumption of carvacrol and thymol on Carnolian honeybee workers (Apis mellifera carnica). ...
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    Nosema ceranae is a microsporidian parasite that causes nosemosis in the honey bee (Apis mellifera). As alternatives to the antibiotic fumagillin, ten nutraceuticals (oregano oil, thymol, carvacrol, trans-cinnmaldehyde, tetrahydrocurcumin, sulforaphane, naringenin, embelin, allyl sulfide, hydroxytyrosol) and two immuno-stimulatory compounds (chitosan, poly I:C) were examined for controlling N. ceranae infections. Caged bees were inoculated with N. ceranae spores, and treatments were administered in sugar syrup. Only two compounds did not significantly reduce N. ceranae spore counts compared to the infected positive control, but the most effective were sulforaphane from cruciferous vegetables, carvacrol from oregano oil, and naringenin from citrus fruit. When tested at several concentrations, the highest sulforaphane concentration reduced spore counts by 100%, but also caused 100% bee mortality. For carvacrol, the maximum reduction in spore counts was 57% with an intermediate concentration and the maximum bee mortality was 23% with the highest concentration. For naringenin, the maximum reduction in spore counts was 64% with the highest concentration, and the maximum bee mortality was only 15% with an intermediate concentration. In the longevity experiment, naringenin-fed bees lived as long as Nosema-free control bees, both of which lived significantly longer than infected positive control bees. While its antimicrobial properties may be promising, reducing sulforaphane toxicity to bees is necessary before it can be considered as a candidate for controlling N. ceranae. Although further work on formulation is needed with naringenin, its effect on extending longevity in infected bees may give it an additional value as a potential additive for bee feed in honey bee colonies.
  • ... During the search of substances for the control of honeybee pathogens, the toxicity against the host organism must be evaluated (Eguaras and Ruffinengo 2006). In several studies, the oral toxicity of natural substances such as propolis, organic acids, essential oils, and their main components has been tested against bees (Ebert 2007;Maistrello et al. 2008;Maggi et al. 2010;Damiani et al. 2017;Porrini et al. 2017). Here, the oral toxicity of GA, NP1, NP2, and NPs against workers and toxicity of NP2 against larvae of A. mellifera were proved for the first time. ...
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    The aim of this work was to evaluate antimicrobial activity against Paenibacillus larvae and oral toxicity against workers and larvae of Apis mellifera of gallic acid (GA) and two nanohybrids of GA and silica. Also, the physicochemical, structural, and energetic properties of GA and the nanohybrids were determined through structure–activity relationship (SAR). The minimum inhibitory concentration (MIC) against P. larvae was determined. GA showed MIC values between 62.5 and 125 μg/ml, whereas the nanoparticle functionalized through the GA carboxylic moiety (NP2) showed the best antimicrobial activity with a MIC value of 23 μg GA/ml for four of the five isolates used. SAR analysis showed that electronegativity, chemical hardness, and dipolar moment are reliable estimators of the antimicrobial activity. NP2 showed the lowest toxicity against workers and was innocuous for bee larvae. Therefore, the nanohybrid NP2 was the best antibacterial and resulted in non-toxic against workers and larvae of honeybees, becoming a potentially effective and safe agent for the treatment of American Foulbrood disease.
  • ... However, OA and EO have significant disadvantages: their efficacy is insufficient and depends on climatic and in-hive conditions, and means of application [49,75,93]. Some natural preparations exert side effects on bees and/or their brood [95,97]. Luckily, some plant-derived formulations, rather effective, but without unwanted effects on colony development and productivity have been used recently [73,96]. ...
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    Colony losses, including those induced by the colony collapse disorder, are an urgent problem of contemporary apiculture which has been capturing the attention of both apiculturists and the research community. CCD is characterized by the absence of adult dead bees in the hive in which few workers and a queen remain, the ratio between the brood quantity and the number of workers is heavily disturbed in favor of the former, and more than enough food is present. Robbing behavior and pests usually attacking the weakened colony do not occur. In the present paper, the causes of the emergence of this problem are discussed, as well as the measures of its prevention. The following factors, which lead to colony losses, are analyzed: shortage of high-quality food (pollen and honey); infestation with parasites, primarily with Varroa destructor, and mixed virus infections; bacterial infections (American and European foulbrood), fungal infections (nosemosis and ascosphaerosis) and trypanosomal infections (lotmariosis); and, finally, general management of the apiary. Certain preventive measures are proposed: (1) providing ample high-quality forage and clean water, (2) avoiding sugarisation, i.e. superfluous use of sugar syrup, (3) meeting the nutritional needs of the colony, (4) when feeding bees, taking care of the timing and the composition of diet, avoiding pure sugar syrup which in excessive quantities may induce energetic and oxidative stress, (5) when there is a shortage of natural feed – honey in the brood chamber – use sugar syrup with natural/artificial supplements to avoid protein starvation, (6) organized control of V. destructor in the colonies is obligatory due to its vector role, and (7) compliance with hygienic and sanitary measures and principles of good apiculture practice and management in apiaries. To conclude, all preventive measures are feasible in compliance with rules and regulations concerning regular spring and autumn bee health monitoring by licensed veterinarians, who can propose adequate treatments if necessary.
  • ... Ariana et al. (2002) showed that spraying thyme, savory, or spearmint oil on adult bees caused varroa mortality without apparent harmful effects to the bees. Also, menthol fed to bees in sugar syrup resulted in bee mortality levels that did not significantly differ from controls fed only sugar syrup (Ebert et al. 2007). In other studies, the LD 50 found for thymol was 210.3 μg per bee (Gashout and Guzman-Novoa 2009) and 55.1 μg per bee (Johnson et al. 2013), whereas that for formic acid was 450 μg per bee (Ebert et al. 2007). ...
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    In this study, honey bees (Apis mellifera L.) were exposed to LD05 and LD50 doses of five commonly used acaricides for controlling the parasitic mite, Varroa destructor. LD50 values at 48 h post-treatment showed that tau-fluvalinate was the most toxic, followed by amitraz, coumaphos, thymol, and formic acid. However, the hazard ratios, which estimate the hive risk level based on a ratio of a standard dose of acaricide per hive to the LD50 of the acaricide, revealed that tau-fluvalinate was the most hazardous followed by formic acid, coumaphos, amitraz, and thymol. The expression of the honey bee acetylcholinesterase gene increased after treatment with the LD05 and LD50 acaricide doses and could distinguish three patterns in the timing and level of increased expression between acaricides: one for amitraz, one for tau-fluvalinate and formic acid, and one for coumaphos and thymol. Conversely, changes in cytochrome P450 gene expression could also be detected in response to all five acaricides, but there were no significant differences between them. Changes in vitellogenin gene expression could only detect the effects of tau-fluvalinate, amitraz, or coumaphos treatment, which were not significantly different from each other. Among the acaricides tested, coumaphos, amitraz, and thymol appear to be the safest acaricides based on their hazard ratios, and a good marker to detect differences between the effects of sub-lethal doses of acaricides is monitoring changes in acetylcholinesterase gene expression.
  • ... For individual compounds, toxicity data are available only for thymol. It was shown to be not toxic at the tested concentrations towards adult bees in cage experiments 61,62 , whereas larval weight and pupal survival were reduced after six days of feeding with 500 ppm 63 . Short term thymol exposure (48 h, 2 nd instar larvae, acute toxicity) led to a LD 50 value of 2010 ppm, while chronic toxicity was estimated with 703 ppm (LD 50 , 5 th -instar larvae) after six days of permanent feeding 63 . ...
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    Honey bees are highly prone to infectious diseases, causing colony losses in the worst case. However, they combat diseases through a combination of their innate immune system and social defence behaviours like foraging for health-enhancing plant products (e.g. nectar, pollen and resin). Plant secondary metabolites are not only highly active against bacteria and fungi, they might even enhance selective foraging and feeding decisions in the colony. Here, we tested six major plant terpenes and their corresponding acetates, characterizing six natural Thymus vulgaris chemotypes, for their antimicrobial activity on bacteria associated with European foulbrood. Comparison of the inhibitory activity revealed the highest activity for carvacrol and thymol whereas the acetates mostly did not inhibit bacterial growth. All terpenes and acetates are present in the nectar and pollen of thyme, with pollen containing concentrations higher by several orders of magnitude. The physiological response was tested on forager and freshly emerged bees by means of antennal electroantennography. Both responded much stronger to geraniol and trans-sabinene hydrate compared to carvacrol and thymol. In conclusion, bee-forageable thyme product terpenes (mainly from pollen) yield effective antibiotic activity by reducing the growth of bee disease-associated bacteria and can be detected with different response levels by the honey bees’ antennae. This is a further step forward in understanding the complex pathogen-pollinator-plant network.
  • ... Both OA and TH are relatively toxic to bees. A study to determine the oral toxicity of 10 essential oils and organic acids that were fed to bees in a sugar syrup showed that OA was the most toxic (Ebert, Kevan, Bishop, Kevan, and Downer, 2007). TH is a neurotoxic acaricide (Charpentier, Vidau, Ferdy, Tabart, and Vetillard, 2014) and was found to be the most toxic of four selected essential oil products tested on adult bees and larvae (Gashout and Guzm an-Novoa, 2009). ...
    Article
    In this study, the effects of a weak oxalic acid (OA) water solution (0.5%) and a mixture of oxalic acid (0.5%) and thymol (0.12%) (OA/TH) on Varroa mites and honey bees were investigated by trickling method. Prior to the experiments, queens in colonies were caged for 21 days to prevent egg laying and hives were provided with screened bottom boards. In experiments with caged bees in laboratory and brood-free colonies in field conditions, mite mortality and bee tolerance were assessed. Trickling caged bees once with OA resulted in 46.8 ± 4.4% effectiveness, while OA/TH had an effectiveness of 91.6–98.7%. Aqueous thymol (TH) solution had no acaricidal effect, but if mixed with OA doubled the effectiveness. Sugar solutions with low concentrations of OA and OA/TH (1:1) were considerably less effective than aqueous solutions. OA/TH was more harmful to bees than OA alone: respectively 3.2 and 1.2% of bees perished in trials. In field conditions, trickling with OA/TH (25 mL/comb space) was highly effective in autumn experiments: 97.6–99.0% for single and 99.2–99.8% for double trickling. Trickling once in spring was considerably less effective (77.0 ± 3.0%). The acaricidal effect of OA double trickling in autumn appeared more slowly, but had similarly high effectiveness: 99.6–99.8%. OA/TH double trickling in autumn was shown to be toxic to bees: colonies weakened considerably the following winter. Water provision reduced weakening of colonies in winter. Trickling of colonies with OA/TH or OA at a lower dosage (15 and 20 mL/comb space, respectively) earlier in autumn ensured high effectiveness and good bee tolerance.
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    Background: Honey bees are associated with gut microorganisms essential for their nutrition and health. The microbial community composition can be used as a biological health indicator and characterized employing biomarker fatty acids. Commonly gut microorganisms are exposed to pathogens and to an array of chemical and biological pest control methods. Results: We found a strong negative effect on the microbial gut community composition when exposed to the bee pest control chemicals oxytetracycline, oxalic acid, imidacloprid as well as when inoculated with the bee pest Nosema spp. and the potential bee pest biocontrol agent L. plantarum. Results from the in vitro test with bee pest chemicals showed a differential response of Lactobacillus spp. At the community level some taxonomic groups were more affected depending on treatment, but sharp shifts in the microbial structure were caused by compounds generally considered as bee safe. Conclusion: Our results show that pest as Nosema spp. and pest control methods alter the composition of bee gut microorganisms, which may have severe consequences for pathogen defence, physiology and general honey bee health. In addition L. plantarum show potential as a biocontrol agent against Nosema spp. This article is protected by copyright. All rights reserved.
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    Hoarding behaviour was measured in the laboratory by putting 50 newly emerged bees into a small box-like cage and noting the time the bees required to remove 20 ml of sugar syrup from their feeder, some of which was consumed and some stored in their comb. Bees from brood of the same colony usually required similar times; bees from brood of different colonies varied widely. The time required by bees from 21 colonies in these laboratory tests was negatively correlated with the weight gained by the colonies in the field (r = −0.33). It is suggested that the laboratory test may therefore provide an indication of colony hoarding behaviour, which directly affects the weight gained and consequently the honey production by the colony.
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    A number of plants produce nectar or pollen that contains toxins which can adversely affect honey bees. Almond (Amygdalus communis L. (Rosaceae)) produces amygdalin, a toxic cyanoglycoside, in its nectar and pollen. Although beekeepers regard almond as a valuable nectar and pollen plant, and honey bees are deployed extensively for almond pollination, prolonged reliance on almond may be detrimental to honey bees' health. Our results suggest that almond nectar is not sufficiently rich in amygdalin to pose a hazard, but that almond pollen could be toxic if exclusively consumed by honey bees for much more than a week. Further tests are needed to determine if honey bees in almond orchards are at risk, or if they somehow cope with the toxin.
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    Tracheal mites, Acarapis woodi (Rennie) (Acari: Tarsonemidae), are important parasites of honeybees, Apis mellifera L. (Morse and Flottum 1997). They enter the prothoracic tracheae and feed through the walls on haemolymph. Currently, these mites are controlled by applying 50 g of menthol crystals in a perforated bag per hive when ambient temperatures are 15°C or more. Even then, it takes 2 weeks for the menthol to perfuse the hive (Herbert et al. 1988; Wilson et al. 1990). In cold climates, sublimation of menthol is too slow to be effective in controlling mites, especially in spring and fall when control is most needed (Scott-Dupree and Otis 1992). Because menthol fumes enter the bees' trachea and kill the mites therein, it may be possible to kill the mites with menthol that is delivered to them via the haemolymph. Therefore, finding if menthol could become systemic when ingested by bees and it could kill tracheal mites by that route, an ingestible menthol medicament for the bees would hold potential for mite control (Kevan and Kevan 1997).
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    Mortality of last instar western spruce bud worm, Choristoneura occidentalis Freeman, exposed to a series of increasing doses (topical application) or concentrations (diet incorporation) of 10 insecticides was analyzed over 7 d by time-dose-mortality regression. Regression based on the complementary log-log model was useful for analyzing time trends for all dose levels simultaneously. Each of the 10 insecticides acted more quickly when applied topically. In both kinds of experiments, speed of lethal action of the insecticides depended on dose or concentration. The statistical methods developed to estimate lethal doses as functions of time and median times of death as functions of dose are more efficient than methods published previously.
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    Essential oils and essential oil components offer an attractive alternative to synthetic acaricides for the control of Varroa jacobsoni. They are generally inexpensive and most pose few health risks. Terpenes (mainly monoterpenes) are the main components of essential oils, comprising about 90 % of the total. More than 150 essential oils and components of essential oils have been evaluated in laboratory screening tests. Very few of them, however, have proven successful when tested in field trials. Thymol and thymol blended with essential oils or essential oil components offer a promising exception. Mite mortality obtained with these formulations typically exceeds 90 % and often approaches 100 %. In addition, residues in honey are low, even after long-term treatments. The exact conditions under which these formulations will yield reliable and effective control, however, have only been determined for certain European regions. Based on the available studies, relying solely on a single treatment with an essential oil or essential oil component is generally not sufficient to maintain mite populations below the economic injury level. Therefore, efforts are necessary to optimize the use of these substances and to incorporate them, along with other measures for limiting mite populations, into an integrated pest management strategy for control of Varroa jacobsoni. © Inra/DIB/AGIB/ Elsevier, Paris
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    Four miticides in various formulations and concentrations were applied in a fall treatment program to honey bee colonies infested with Acarapis woodi. The miticides tested were menthol, Amitraz, fluvalinate (Apistan®), and cymiazole (Apitol®). None of the miticides significantly reduced mite prevalence in treated colonies relative to the control treatment. Additional studies should be undertaken in temperate climates to determine whether the efficacy of the miticides tested in this study can be improved by administering them at other time during the year or by adjusting dosages rates to improve their efficacy when applied to infested colonies during a fall management program.
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    A complementary log-log (CLL) model was used to model time-dose-mortality relationships from bioassay tests of 26 fungal isolates mostly from Madagascar, Africa, against three acridid species, all referred to here as "grasshoppers." The fungal pathogens included 15 isolates of Beauveria bassiana, 9 isolates of Metarhizium flavoviridae, and 2 isolates of Paecilomyces spp. Grasshopper species tested included Melanoplus sanguinipes, Locusta migratoria migratorioides, and Schistocerca gregaria. The scaled deviance, mean deviance, Pearson X2 statistic, Hosmer-Lemeshow (H-L) C statistic, and three-dimensional time-dose-mortality graphs were used to assess extra-binomial variation, data points that were potential outliers, conformance of the scaled deviance statistic and Pearson's X2 statistic to a chi2 distribution, and the fit of the CLL model. The H-L C statistic also was found to be useful in showing the goodness of fit of the CLL model for the fungal isolates prior to modeling the extra-binomial variation. After the extra-binomial variation was modeled using Williams' method, the slope from maximum likelihood estimation, modified log(LD50) estimates (which were corrected for background mortality using the CLL model), a dynamic ranking of the log(LD50) values over time, and a three-dimensional plot of time, dose, and mortality of the three grasshopper species were used to evaluate the effectiveness of the fungal isolates. In general, the CLL model provided a rather poor fit of the fungal isolates which had a large number of replicate trials in the bioassay tests (i.e., a large sample size) due to extra-binomial variation. The CLL model provided an excellent fit of the time-dose-mortality relationships of such isolates after the extra-binomial variation was modeled and included in the CLL model. Metarhizium isolates MFV and SP5 were found to be the most virulent isolates tested against M. sanguinipes, followed by Metarhizium isolates: SP8, SP7, SP9, SP6, and SP1, and Beauveria isolate S33B. Metarhizium isolates SP3, SP5, SP6, and SP9, and Beauveria isolates SP11, SP12, SP13, and SP16 showed higher levels of virulence against L. migratoria migratorioides over more of the time periods tested than the other pathogen isolates examined. Metarhizium isolates SP9 and SP5 were the most effective isolates tested against S. gregaria. In general, the Metarhizium isolates were more virulent against the grasshoppers than the Beauveria isolates, which were more virulent than the Paecilomyces isolates. The CLL model was found to be very useful in describing grasshopper mortality as a function of time and dose. This approach combined with model and fungal isolate assessment statistics will be helpful for determining which pathogen isolates have the greatest potential for controlling grasshoppers and other pests in the future.