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


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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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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:
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
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
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
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
( 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 (
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
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.
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
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.
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.
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
... Boncristiani et al. [43] reported that thymol had increased the susceptibility of bees to N. ceranae infection through the reduced expression of the Dscam and Basket genes, which are significant cellular and humoral immune factors, respectively, in defending bees from parasites [44,45]. In several studies, treatment with thymol (orally or topically) did not induce toxic effects on bees [42,46,47], and bees even lived longer compared with the control [47]. These data together with earlier observations on the low toxicity of thymol [48], as well as its importance for beekeeping, led to thymol's approval by the European Union [49] for the control of the honey bee mite V. destructor in conventional and organic beekeeping [28,37]. ...
... The number of dead bees between the control group NI and each group treated with thymol was not statistically significantly different (p ≥ 0.095). This result is in accordance with those of Ebert et al. [46], Costa et al. [47], and Bergougnoux et al. [42], who found that thymol was not toxic to bees. Nevertheless, according to EU Regulation 834/2007 on organic production [49], thymol is authorized for use in Varroa control in organic beekeeping. ...
... Initial studies of the effect of thymol on bees infected with Varroa mites [31,46,68] did not report negative effects of thymol on bees. However, further research reported some negative effects of thymol on bees [43,[69][70][71], which was one of the reasons for our research. ...
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Nosema ceranae is the most widespread microsporidian species which infects the honey bees of Apis mellifera by causing the weakening of their colonies and a decline in their productive and reproductive capacities. The only registered product for its control is the antibiotic fumagillin; however, in the European Union, there is no formulation registered for use in beekeeping. Thymol (3-hydroxy-p-cymene) is a natural essential-oil ingredient derived from Thymus vulgaris, which has been used in Varroa control for decades. The aim of this study was to investigate the effect of thymol supplementation on the expression of immune-related genes and the parameters of oxidative stress and bee survival, as well as spore loads in bees infected with the microsporidian parasite N. ceranae. The results reveal mostly positive effects of thymol on health (increasing levels of immune-related genes and values of oxidative stress parameters, and decreasing Nosema spore loads) when applied to Nosema-infected bees. Moreover, supplementation with thymol did not induce negative effects in Nosema-infected bees. However, our results indicate that in Nosema-free bees, thymol itself could cause certain disorders (affecting bee survival, decreasing oxidative capacity, and downregulation of some immune-related gene expressions), showing that one should be careful with preventive, uncontrolled, and excessive use of thymol. Thus, further research is needed to reveal the effect of this phytogenic supplement on the immunity of uninfected bees.
... Regarding therapeutic hive treatments, comparisons with previous studies suggest that honey bees can tolerate both thymol and eugenol-rich clove oil at concentrations well above those needed to inhibit growth of gut parasites. For thymol, the 8 d LD50 [>1000 μg ml À1 (Ebert et al., 2007)] is well above the 28-54 μg ml À1 IC50 range for the parasites L. passim and C. mellificae, implying a >20-fold margin of safety for medication of bees with this compound. For clove oil, the 8 d LD50 [7800 μg ml À1 (Ebert et al., 2007)] is similarly nearly 30-fold higher than the 181-280 μg ml À1 IC50 range for the honey bee trypanosomatids (Table 3). ...
... For thymol, the 8 d LD50 [>1000 μg ml À1 (Ebert et al., 2007)] is well above the 28-54 μg ml À1 IC50 range for the parasites L. passim and C. mellificae, implying a >20-fold margin of safety for medication of bees with this compound. For clove oil, the 8 d LD50 [7800 μg ml À1 (Ebert et al., 2007)] is similarly nearly 30-fold higher than the 181-280 μg ml À1 IC50 range for the honey bee trypanosomatids (Table 3). However, these lipophilic compounds are rapidly absorbed from the intestine, with a half-life of $2 h and > 90% absorbance with 8 h in pigs (Michiels et al., 2008). ...
... In addition, comparisons between parasite species that differ in phytochemical tolerance could illuminate the mechanisms that confer resistance to antiparasitic compounds, with potential to improve the efficacy of phytochemical-based treatments for neglected tropical diseases. Honey bee LD50 estimates are for 8 days' exposure time, as measured in Ebert et al. (2007). Concentrations are in μg ml À1 . ...
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Gut parasites of plant-eating insects are exposed to antimicrobial phytochemicals that can reduce infection. Trypanosomatid gut parasites infect insects of diverse nutritional ecologies as well as mammals and plants, raising the question of how host diet-associated phytochemicals shape parasite evolution and host specificity. To test the hypothesis that phytochemical tolerance of trypanosomatids reflects the chemical ecology of their hosts, we compared related parasites from honey bees and mosquitoes-hosts that differ in phytochemical consumption-and contrasted our results with previous studies on phylogenetically related, human-parasitic Leishmania. We identified one bacterial and ten plant-derived substances with known antileishmanial activity that also inhibited honey bee parasites associated with colony collapse. Bee parasites exhibited greater tolerance of chrysin-a flavonoid found in nectar, pollen, and plant resin-derived propolis. In contrast, mosquito parasites were more tolerant of cinnamic acid-a product of lignin decomposition present in woody debris-rich larval habitats. Parasites from both hosts tolerated many compounds that inhibit Leishmania, hinting at possible trade-offs between phytochemical tolerance and mammalian infection. Our results implicate the phytochemistry of host diets as a potential driver of insect-trypanosomatid associations, and identify compounds that could be incorporated into colony diets or floral landscapes to ameliorate infection in bees. This article is protected by copyright. All rights reserved.
... However, none of them applied alone may ensure long-lasting Varroa control, mostly because of their insufficient or variable efficacy (Goswami et al., 2014;Jack et al., 2020;Pietropaoli & Formato, 2018, 2019Underwood & Currie, 2005;Vandervalk et al., 2014). In addition, both organic acids and plant products may negatively affect adult honey bees and their brood (Brasesco et al., 2017;Damiani et al., 2009;Ebert et al., 2007;Floris et al., 2004;Gashout & Guzm an-Novoa, 2009;Mo skri c et al., 2018;Pietropaoli & Formato, 2019;Satta et al., 2005;Underwood & Currie, 2005). Even weak water solution (0.5%) of oxalic acid, alone or mixed with thymol (0.12%), after double trickling in autumn, caused remarkable toxic effects to bees and considerable weakening of colonies during the following winter (Toomemaa, 2019). ...
... In some reports the efficacy of natural-based products (plant essential oils, hop beta acids and oxalic acid) against Varroa in hive conditions (Gregorc et al., 2017;Loucif-Ayad et al., 2010;Maggi et al., 2016;Tlak-Gajger and Su sec, 2019) was rather high (91-98%) and comparable to that achieved with Licit here and LiCl in the study by Ziegelmann, Abele, et al. (2018). However, there were also evidences of undesirable (often toxic) effects caused by natural compounds on adult bees, brood and whole colonies, although different approaches and various methodologies were applied in different studies (Brasesco et al., 2017;Damiani et al., 2009;Ebert et al., 2007;Floris et al., 2004;Gashout & Guzm an-Novoa, 2009;Gregorc et al., 2018;Mo skri c et al., 2018;Pietropaoli & Formato, 2019;Satta et al., 2005;Toomemaa, 2019;Underwood & Currie, 2005). ...
In a cage experiment, lithium chloride (LiCl) and lithium citrate hydrate (Li-cit) were tested for varroacidal efficacy and impact on bees. Treatment with Li-cit (4, 7.5, 10, and 25 mM) resulted in 100% varroacidal efficacy and 100% bee survival. Due to better results in the cage experiment, Li-cit was further tested in field experiments on full-sized free-flying colonies treated three times in 6-day intervals. All the concentrations of Li-cit in the field experiment (5, 10, 15, 20, and 25 mM) expressed high varroacidal efficacy: 93.2–95.5%, significantly (p < 0.01) greater than in the negative and positive (amitraz-treated) controls. Lithium residues in honey from brood chambers were much higher nine months after the last treatment (169.3–1756.0 μg/kg) than seven days post-treatment (19.2–27.8 μg/kg). In honey from honey chambers (eligible for human consumption), the average lithium residues were 26.9 μg/kg and 33.7 μg/kg seven days after the last treatment. In wax combs taken from the brood chamber nine months post-treatment, lithium residues ranged from 410 μg/kg to 2314 µg/kg, without significant differences from the negative control. Lithium residues in wax matrices seven days after the last treatment were in a narrow range of 234.3–300 µg/kg, in wax combs and cappings being significantly lower than in commercial wax foundations. For the first time, Li-cit proved to be effective against Varroa destructor under field conditions.
... For all seasonal treatments, despite initial reductions of weed coverage observed 4 weeks post treatment, 12 weeks post application the contact-based products did have a significant effect on reducing or suppressing weeds at the Aspendale site. Interestingly, in spite of organic acid based products (acetic acid and hydrochloric acid) and plant oil (essential oil) based products (clove oil and pine oil) being established as disinfectants, pesticides or herbicides (by either chemical burning or blocking oxygen access) no impacts to arthropods, bacteria or fungi relative abundance was observed in our study [74][75][76][77][78]. This is likely due to the large dilution of the products across the surface area of the treatment sites, rainfall events and/or within the soil profile. ...
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Glyphosate-based foliar spray herbicides are the most common method for urban weed control due to their broad-spectrum and efficacy for burndown applications. As interest in glyphosate alternatives has increased in recent years, this project assessed the efficacy of the following non-glyphosate-based alternative weed management strategies: glufosinate, imazapyr, MCPA + dicamba, prodiamine, pine oil, clove oil, nonanoic acid, acetic acid + hydrochloric acid and steam against untreated (negative) controls and glyphosate-treated sites. Across all four seasonal treatments (winter, spring, summer and autumn), glyphosate and glufosinate reduced weed coverage (>65% after 4 and 12 weeks); imazapyr reduced weed coverage by >80% after 12 weeks; and steam reduced weed coverage by >80% after 4 weeks, and after 12 weeks showed to reduce weed coverage by >20% after the second application. The MCPA + dicamba, prodiamine, pine oil, clove oil, nonanoic acid and acetic acid + hydrochloric acid treatments had mixed impacts on weed coverage. Minimal alterations to soil physicochemical properties were observed across the two sites for all treatments. Assessment of impacts the different weed management strategies had on arthropod and microbial relative abundance showed minimal alterations; with only steam observed to reduce relative microbial abundance. Glufosinate, imazapyr and steam may be considered alternatives to glyphosate for reducing weed coverage but may not be as effective or have undesirable off-target effects. Overall, glyphosate provided the most consistent weed reduction at both sites over 12 weeks, without any recorded negative off-target or soil biota impacts.
... For instance, savoury or spearmint oils were investigated and showed acaricidal properties with low rate of honeybee mortality while dillsun induced higher death rates [157]. Menthol in sugar syrup displayed encouraging short-term results [150,158] whereas neem oil killed mites [159], but also increased brood mortality and reduced the worker's walking activity [160]. Finally, another plant extract, relying on hop leaves, was shown to contain polyphenols with high miticide effect and low acute toxicity for bees [161,162]. ...
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Varroadestructor is a real challenger for beekeepers and scientists: fragile out of the hive, tenacious inside a bee colony. From all the research done on the topic, we have learned that a better understanding of this organism in its relationship with the bee but also for itself is necessary. Its biology relies mostly on semiochemicals for reproduction, nutrition, or orientation. Many treatments have been developed over the years based on hard or soft acaricides or even on biocontrol techniques. To date, no real sustainable solution exists to reduce the pressure of the mite without creating resistances or harming honeybees. Consequently, the development of alternative disruptive tools against the parasitic life cycle remains open. It requires the combination of both laboratory and field results through a holistic approach based on health biomarkers. Here, we advocate for a more integrative vision of V. destructor research, where in vitro and field studies are more systematically compared and compiled. Therefore, after a brief state-of-the-art about the mite’s life cycle, we discuss what has been done and what can be done from the laboratory to the field against V. destructor through an integrative approach.
Trypanosomatid gut parasites are common in pollinators and costly for social bees. The recently described honey bee trypanosomatid Lotmaria passim is widespread, abundant, and correlated with colony losses in some studies. The potential for amelioration of infection by antimicrobial plant compounds has been thoroughly studied for closely related trypanosomatids of humans and is an area of active research in bumble bees, but remains relatively unexplored in honey bees. We recently identified several floral volatiles that inhibited growth of L. passim in vitro. Here, we tested the dose-dependent effects of four such compounds on infection, mortality, and food consumption in parasite-inoculated honey bees. We found that diets containing the monoterpenoid carvacrol and the phenylpropanoids cinnamaldehyde and eugenol at >10-fold the inhibitory concentrations for cell cultures reduced infection, with parasite numbers decreased by >90% for carvacrol and cinnamaldehyde and >99% for eugenol; effects of the carvacrol isomer thymol were non-significant. However, both carvacrol and eugenol also reduced bee survival, whereas parasite inoculation did not, indicating costs of phytochemical exposure that could exceed those of infection itself. To our knowledge, this is the first controlled screening of phytochemicals for effects on honey bee trypanosomatid infection, identifying potential treatments for managed bees afflicted with a newly characterized, cosmopolitan intestinal parasite.
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With an almost global distribution, Varroa destuctor is the leading cause of weakening and loss of honey bee colonies. New substances are constantly being tested in order to find those that will exhibit high anti- Varroa efficacy at low doses/concentrations, without unwanted effects on bees. Lithium (Li) salts stood out as candidates based on previous research. The aims of this study were to evaluate Li citrate hydrate (Li-cit) for its contact efficacy against Varroa , but also the effect of Li-cit on honey bees by estimating loads of honey bee viruses, expression levels of immune-related genes and genes for antioxidative enzymes and oxidative stress parameters on two sampling occasions, before the treatment and after the treatment. Our experiment was performed on four groups, each consisting of seven colonies. Two groups were treated with the test compound, one receiving 5 mM and the other 10 mM of Li-cit; the third received oxalic acid treatment (OA group) and served as positive control, and the fourth was negative control (C group), treated with 50% w/v pure sucrose-water syrup. Single trickling treatment was applied in all groups. Both tested concentrations of Li-cit, 5 and 10 mM, expressed high varroacidal efficacy, 96.85% and 96.80%, respectively. Load of Chronic Bee Paralysis Virus significantly decreased ( p < 0.01) after the treatment in group treated with 5 mM of Li-cit. In OA group, loads of Acute Bee Paralysis Virus and Deformed Wing Virus significantly ( p < 0.05) increased, and in C group, loads of all viruses significantly ( p < 0.01 or p < 0.001) increased. Transcript levels of genes for abaecin, apidaecin, defensin and vitellogenin were significantly higher ( p < 0.05— p < 0.001), while all oxidative stress parameters were significantly lower ( p < 0.05— p < 0.001) after the treatment in both groups treated with Li-cit. All presented results along with easy application indicate benefits of topical Li-cit treatment and complete the mosaic of evidence on the advantages of this salt in the control of Varroa .
Nosema ceranae is a microsporidium parasite that silently affects honey bees, causing a disease called nosemosis. This parasite produces resistant spores and germinates in the midgut of honey bees, extrudes a polar tubule that injects an infective sporoplasm in the host cell epithelium, proliferates, and produces intestinal disorders that shorten honey bee lifespan. The rapid extension of this disease has been reported to be widespread among adult bees, and treatments are less effective and counterproductive weakening colonies. This work aimed to evaluate the antifungal activity of a prototype formulation based on a non-toxic plant extract (HO21-F) against N. ceranae. In laboratory, honey bees were infected artificially, kept in cages for 17 days and samples were taken at 7 and 14 days post infection (dpi). At the same time, in field conditions we evaluated the therapeutic effect of HO21-F for 28 days in naturally infected colonies. The effectiveness of the treatment has been demonstrated by a reduction of 83.6% of the infection levels observed in laboratory conditions at concentrations of 0.5 and 1 g/L without affecting the survival rate. Besides, in-field conditions we reported a reduction of 88% of the infection level at a concentration of 2.5 g/L, obtaining better antifungal effectiveness in comparison to other commercially available treatments. As a result, we observed that the use of HO21-F led to an increase in population size and honey production, both parameters associated with colony strength. The reported antifungal activity of HO21-F against N. ceranae, with a significant control of spore proliferation in worker bees, suggests the promising commercial application use of this product against nosemosis, and it will encourage new research studies to understand the mechanism of action, whether related to the spore-inhibition effect and/or a stimulating effect in natural response of colonies to counteract the disease.
Among pests of bees and beehives, arthropods make up a large and important group. Mites like Varroa destructor, Acarapis woodi, or Tropilaelaps spp., beetles (Aethina tumida, Oplostomus spp.), and lepidopterans (Galleria mellonella, Achroia grisella) decrease honey bee population and vitality, with subsequent significant colony production losses. Synthetic chemicals have been traditionally used to protect honey bee colonies from pests’ infestations but they have often been of poor selectivity, consequent high toxicity to bees and humans, and resistance development by the targeted apiary pests. The current European policy encourages the usage of eco-friendly methods to combat bee pests and the international research highlights plant secondary metabolites as candidate alternatives of significance. In this review, we argue the potential of plant-derived substances in the protection of the bee colonies against their arthropod pests. The before mentioned major apiary arthropods are briefly described followed by the recent reports on the botanical extracts and notable constituent compounds exhibiting activity against them. We discuss the different ways the essential oils are reported to be applied to the bee or the apiary, along with the importance of the application method to the exhibited efficacy. We designate synergism issues of blends, attractants, and repellency cases, as well as selectivity and mode of action as reported for bees or insect pests.
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Nutrition is vital for health and immune function in honey bees ( Apis mellifera ). The effect of diets enriched with bee-associated yeasts and essential oils of Mexican oregano ( Lippia graveolens ) was tested on survival, food intake, accumulated fat body tissue, and gene expression of vitellogenin ( Vg ), prophenoloxidase ( proPO ) and glucose oxidase ( GOx ) in newly emerged worker bees. The enriched diets were provided to bees under the premise that supplementation with yeasts or essential oils can enhance health variables and the expression of genes related to immune function in worker bees. Based on a standard pollen substitute, used as a control diet, enriched diets were formulated, five with added bee-associated yeasts ( Starmerella bombicola , Starmerella etchellsii , Starmerella bombicola 2 , Zygosaccharomyces mellis, and the brewers’ yeast Saccharomyces cerevisiae ) and three with added essential oils from L. graveolens (carvacrol, thymol, and sesquiterpenes). Groups of bees were fed one of the diets for 9 or 12 days. Survival probability was similar in the yeast and essential oils treatments in relation to the control, but median survival was lower in the carvacrol and sesquiterpenes treatments. Food intake was higher in all the yeast treatments than in the control. Fat body percentage in individual bees was slightly lower in all treatments than in the control, with significant decreases in the thymol and carvacrol treatments. Expression of the genes Vg , proPO , and GOx was minimally affected by the yeast treatments but was adversely affected by the carvacrol and thymol treatments.
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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.
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.