insects

Article
Effects of Oxalic Acid on Apis mellifera
(Hymenoptera: Apidae)
Eva Rademacher *, Marika Harz and Saskia Schneider
Institute of Biology/Neurobiology, Freie Universität Berlin, Königin-Luise-Str. 28-30, 14195 Berlin, Germany;
marika.harz@lwk.nrw.de (M.H.); saskia.schneider@kabelmail.de (S.S.)
* Correspondence: e.rademacher@fu-berlin.de; Tel.: +49-30-8385-6537

Academic Editor: Brian T. Forschler

Received: 12 June 2017; Accepted: 2 August 2017; Published: 7 August 2017

Abstract: Oxalic acid dihydrate is used to treat varroosis of Apis mellifera. This study investigates
lethal and sublethal effects of oxalic acid dihydrate on individually treated honeybees kept in cages
under laboratory conditions as well as the distribution in the colony. After oral application, bee
mortality occurred at relatively low concentrations (No Observed Adverse Effect Level (NOAEL)
50 µg/bee; Lowest Observed Adverse Effect Level (LOAEL) 75 µg/bee) compared to the dermal
treatment (NOAEL 212.5 µg/bee; LOAEL 250 µg/bee). The dosage used in regular treatment
via dermal application (circa 175 µg/bee) is below the LOAEL, referring to mortality derived
in the laboratory. However, the treatment with oxalic acid dihydrate caused sublethal effects:
This could be demonstrated in an increased responsiveness to water, decreased longevity and a
reduction in pH-values in the digestive system and the hemolymph. The shift towards stronger
acidity after treatment confirms that damage to the epithelial tissue and organs is likely to be
caused by hyperacidity. The distribution of oxalic acid dihydrate within a colony was shown by
macro-computed tomography; it was rapid and consistent. The increased density of the individual
bee was continuous for at least 14 days after the treatment indicating the presence of oxalic acid
dihydrate in the hive even long after a treatment.

Keywords: Apis mellifera; Varroa destructor; oxalic acid dihydrate; toxicity; tolerance; sublethal effects

1. Introduction
Oxalic acid dihydrate (OAD) is one of the most important organic acids used for the control
of Varroa destructor. It has been known to be effective against the parasite since the end of the 20th
century [1]. The European Group for Integrated Varroa Control developed OAD for the final application
stage in beekeeping [2,3]. Three different application methods of OAD exist: trickling, spraying and
evaporation. There are principal points to be considered concerning the medical treatment of honey bee
colonies: the tolerability of the ingredient to bees, and the toxicity to mites, as well as its distribution in
the colony, which again is directly affects the toxicity and efficacy of a substance. The trickling method
of OAD combines high efficacy against V. destructor and low bee mortality. The tolerability in the bee
colony has been documented in a concentration of 3.5% (w/v) OAD and a dose of 30–50 mL per colony
for Central Europe [4]. Approval as a veterinary drug has been given in many countries worldwide
over recent years, for Germany in 2006 [5].
So far, toxicological data on individual bees in the laboratory without combinatory effects has not
been available. The mode of action in the colony was only partially clarified. In order to get a better
understanding we tested the toxicity of OAD after dermal or oral application in the laboratory. Our
aim was to define the no observed adverse effect level (NOAEL) and the lowest observed adverse
effect levels (LOAEL) for OAD, including a safety margin for the dosage used in practical beekeeping.
With this focus in mind, it is most important to look primarily at the dosage range of up to 10% bee

Insects 2017, 8, 84; doi:10.3390/insects8030084 www.mdpi.com/journal/insects

Insects 2017, 8, 84 2 of 17

mortality, as higher mortality rates are not acceptable for practical beekeeping. Therefore, we decided
not to test very high dosages as it is unreasonable to kill large numbers of bees for non-relevant
information. Furthermore, we wanted to understand which sublethal effects can be found and how
OAD is distributed in the colony.

2. Materials and Methods

2.1. Laboratory Tests: Treatment of Individual Bees with OAD

2.1.1. Investigation of Lethal Effects—Acute Oral and Dermal Toxicity

The toxicity tests were conducted during August and September using Apis mellifera carnica
bees from our apiary at the Institute of Biology/Neurobiology, Berlin (Germany). The colonies were
managed according to good beekeeping practice.
Worker bees were recruited from brood combs by brushing about 100 individuals into small cages.
The test bees were approximately five to ten days old. The cages are made of wood (105/65/120 mm
length/width/height) with bee wire and a glass plate on the sides. The caged bees were kept under
laboratory conditions in the dark at 22 ◦ C and 65% relative humidity, draft-free. The bees formed
small clusters and were starved for 24 h to ensure an even distribution of food before being treated
with OAD. After the application, they were held in small groups of 10 bees per cage and received food
(Apifonda, Südzucker AG, Mannheim, Germany) and water ad libitum.
OAD (Caelo, Hilden, Germany) dissolved in sucrose solution (50% w/w) was applied to bees
individually using two application forms: trickling 5 µL OAD solution onto the abdomen (dermal) or
feeding 10 µL OAD solution (oral). Topical treatment with 5 µL was the maximum volume of solution
applicable without considerable loss of agent. In feeding trials, 10 µL solution allows the feeding of
even high dosages by lower concentrations, so they were well accepted by the bees. The test design
for both treatments was identical. Each dosage was tested on 30 bees (three cages per dosage and
ten bees in every cage). Each trial was replicated at least once leading to a minimum number of 60
bees tested for each dosage and treatment method. The control groups (three cages with ten bees
per cage, one replicate) were treated in the same way but received only sucrose solution (50% w/w).
The acute dermal toxicity test was conducted with different OAD concentrations: 3.5, 4.25, 5, 7.5 and
10% (w/v) and dosages: 175, 212.5, 250, 375 and 500 µg OAD/bee, respectively. The acute oral toxicity
test consisted of concentrations respectively dosages: 0.1, 0.5, 0.75, 0.8 and 1% (w/v) corresponding to
10, 50, 75, 80 and 100 µg OAD/bee. Bee mortality data for test and control groups were collected 24, 48
and 72 h after the respective applications. Directly after the treatment, bees were observed for four
hours and in time intervals up to 72 h for signs of behavioral changes.

2.1.2. Investigation of Sublethal Effects

Responsiveness to Water and Ascending Concentrations of Sucrose Solution

The proboscis extension response (PER) was used to test bees’ responsiveness to water and
ascending concentrations of sucrose solution (ACSS). Bees were recruited out of the colony as described
for the previous experiments and individually marked. Bees were treated dermally with 5 µL of 3.5%
OAD in sucrose solution (50% w/w) trickled onto the abdomen, the controls received sucrose solution
only (n = 60). The proportion of animals releasing a PER was calculated for water and six concentrations
of sucrose solution (0.1%, 0.3%, 1%, 3%, 10% and 30%). Each bee was tested twice: prior to the treatment
and 24 h afterwards. Between tests the bees were kept in cages and starved for three hours prior to
each test to ensure equal motivation. Solutions were applied to the antennae with a three-minute
inter-trial interval. To ensure equal motivation, bees were starved for 3 h prior to the tests. Without
anesthetization, every bee was carefully moved into a little plastic tube allowing only the head with
antennae and proboscis to move freely. Bees were tested for water and then for sucrose responsiveness
Insects 2017, 8, 84 3 of 17

by applying first a drop of water followed by the ascending sucrose solutions to the antennae with a
3-min inter-trial interval. The proportion of animals releasing a PER was calculated.

Longevity under Laboratory Conditions

To obtain bees of the same age, a brood-frame was taken from a colony and put into an incubator
at 34.5 ◦ C. The hatched bees were removed daily. The young bees were kept in small cages and
provided with food (Apifonda, Südzucker AG, Mannheim, Germany) and water ad libitum. Pretests
showed a mortality rate of up to 60% when bees younger than five days were treated with OAD; for
this experiment bees at the age of five days were chosen and treated dermally with 5 µL of 3.5% OAD
in sucrose solution (50% w/w) trickled onto the abdomen or sucrose solution (controls), respectively (n
= 50). Dead bees were counted and removed from the cages daily until the last bee had died. The test
was repeated four times, a total of 200 bees per group were treated.

pH Values of the Digestive System and the Hemolymph

Worker bees were brushed from honey combs and individually treated with OAD. Dermal
treatment was conducted with an amount of 5 µL 3.5% OAD in sucrose solution (50% w/w), dosage:
175 µg/bee, n = 120); oral treatment was performed with 10 µL 0.35% OAD in sucrose solution (50%
w/w, dosage: 35 µg/bee, n = 120). The test animals were kept in cages as described above. In intervals
of 24, 48 and 72 h post treatment the bees were frozen and subsequently dissected according to standard
methods [6], the intestinal parts (crop, ventriculus and rectum) being removed and transferred onto
micro-slides for pH measurement. An Inlab® Surface Electrode, which enables the pH measurement
of very small amounts of liquid (minimum of 5 µL), was carefully placed onto the different parts of the
digestive system. The electrode was connected to a FiveGoTM pH meter (limits of error: ±0.01 pH).
The sampling of the hemolymph was conducted by the removal of the front and hind wings from the
bee’s thorax. A slight pressure on the thorax enabled the extraction of the hemolymph from the wing
base. The droplet of the hemolymph was absorbed with micro-capillaries and immediately transferred
to micro-slides for pH measurement. Hemolymph samples (minimum of five bees per sample) were
pooled to gain at least 5 µL of liquid.

2.2. Computer Tomography of Honey Bee Colonies: Distribution of OAD

Internal structures of a bee hive can be demonstrated by computed tomography [7]. Two honey bee
nucleus colonies (A. m. carnica) were used for a distribution test with a macro-computed tomography
scanner (macroCT). The colonies consisted of approximately 4000 individuals and had already formed
a winter cluster. The treatments were conducted in November without brood. OAD (3.5% w/v in
sucrose solution 50% w/w) was applied in the recommended dosage (according to package instructions
for use-Oxuvar® ) by trickling onto the bees in the bee space. OAD was mixed with the water-soluble
contrast agent Unilux® (Iopamidol, 370 mg iodine/mL) in a dosage of 185 µg/bee. The contrast agent
Unilux showed no bee toxicity in a previous study [8].
For in-hive visualization of OAD distribution a macroCT scanner (Xvision, Toshiba) was used
(Figure 1). With a helical scanner, a distance of 250 mm was examined ensuring the colony was
captured completely. The CT images were reconstructed with a slice thickness of 2 mm (Table 1).
For visualization via 2D images and data analysis we used the software eFilmTM LiteTM (MergeTM
Healthcare 2008). The 2D images allowed the measuring of the density of individual bees in Hounsfield
units (HU). This density is directly related to the amount of solution applied to the surface of the bee’s
bodies. In a defined area of 100 cm2 in the central area of the comb, as well as in the boundary area of
the bee cluster, the density of single bees (n = 144) was measured over three combs. In one colony, the
measurements were conducted before application (control) and 10 min respectively thirty minutes
after applying OAD. The second colony measurements were conducted before application (control)
and 3, 7 and 14 days respectively after applying OAD (n ≤ 211/group). Only bees placed parallel to
the macroCT sectional plane were quantified. The total dosage of radiation for the scan of the entire
 Helix 250 mm
Peak X ray voltage 120 kV
X ray tube current 80 mA
Total scan time 75 s
Insects 2017, 8, 84
Matrix 512x512 4 of 17
Scan field of view large
Display field of view 480 mm
bee colony was 249.8 mGy, spread over the
Window helix distance of1000
width 250 mm (125 slices of 2 mm thickness).
Compared to the dosage of 500 mGy reported
Window level by [9] for −300 effects in Drosophila melanogaster,
biological
this dosage can be considered harmless
Total scantodosage
bees. 249.8 mGy

1. Bee colony placed in a macro-computed tomography scanner (macroCT).

Figure 1.
Figure

2.3. Statistical Analysis Table 1. Technical data for A. mellifera colony scanning.

The statistical analysis wasParameter

conducted using SigmaStat® 3.0 software. Results were tested for
Resolution
differences in the bee mortality rate using the chi2-test on values at all-time intervals. The dose
Slice thickness® 2 mm
response curves obtained with SigmaPlotPitch 3.0 software were the basis
2.5 for the probit analysis and
derivation of lethal dosage (LD) values.
Helix For the comparison of 250 pH mm values of normally distributed
data the t-test was used, alternatively in case of non-normally distributed
Peak X ray voltage 120 kV data the Mann–Whitney
X ray tube current 80 mA
rank sum test was used. The PER rates were compared between the groups for water responsiveness
Total scan time 75 s
and within the groups for the sucrose solutions using chi²-test and McNemar’s test, respectively. The
Matrix 512x512
longevity data were analyzed using
Scan fieldaof
Kaplan–Meier
view survival analysis,
large Gehan–Breslow. The density
values of the bees in the small colony
Display units
field of viewwere analyzed with480themmt-test. Regarding all statistical
Window
tests a difference was considered to bewidth
significant when the p-value1000
obtained was lower than 0.05.
Window level −300
Total scan dosage 249.8 mGy

2.3. Statistical Analysis

The statistical analysis was conducted using SigmaStat® 3.0 software. Results were tested for
differences in the bee mortality rate using the chi2 -test on values at all-time intervals. The dose response
curves obtained with SigmaPlot® 3.0 software were the basis for the probit analysis and derivation of
lethal dosage (LD) values. For the comparison of pH values of normally distributed data the t-test
was used, alternatively in case of non-normally distributed data the Mann–Whitney rank sum test
was used. The PER rates were compared between the groups for water responsiveness and within the
groups for the sucrose solutions using chi2 -test and McNemar’s test, respectively. The longevity data
were analyzed using a Kaplan–Meier survival analysis, Gehan–Breslow. The density values of the bees
in the small colony units were analyzed with the t-test. Regarding all statistical tests a difference was
considered to be significant when the p-value obtained was lower than 0.05.
Insects 2017, 8, 84 5 of 17
Insects 2017, 8, 84 5 of 16

3. Results
3. Results

3.1. Laboratory Tests:

Tests: Treatment
Treatment of
of Individual
IndividualBees
Beeswith
withOAD
OAD

3.1.1. Investigation of
3.1.1. Investigation of Lethal
Lethal Effects—Acute
Effects⎯Acute Oral
Oral and
and Dermal
Dermal Toxicity
Toxicity
After
After dermal
dermal application
application of
of OAD
OAD the toxicity increased
the toxicity increased slowly
slowly during
during thethe observation
observation time time ofof 24
24
to
to 72
72 h. After 72
h. After 72 h
h the
the application
application of
of 175
175 and
and 212.5
212.5 µg/bee, respectively, showed
µg/bee,respectively, showed no no significant
significant effect.
effect.
After 2 -test, p =p 0.003)
After application
applicationof of250
250µgµgOAD
OADthethebee
beemortality
mortalitywas significantly
was higher
significantly higher(chi(chi 2-test, = 0.003)than at
than
175 andand
at 175 212.5 µg/bee.
212.5 µg/bee.In In
thethe
dosages
dosages375375
and 500500
and µg/bee thethe
µg/bee mortality increased
mortality increased to >20%
to >20%(Figure
(Figure2).
The NOAEL (72 h) for dermal application was 212.5, the LOAEL 250 µg OAD/bee.
2). The NOAEL (72 h) for dermal application was 212.5, the LOAEL 250 µg OAD/bee. The The extrapolated
LD 10 (72 h) isLD
extrapolated 256.4 µgh)OAD/bee.
10 (72 is 256.4 µgThe LD10 48The
OAD/bee. h after
LD10application reached a higher
48 h after application reachedvalue thanvalue
a higher after
72 h: after
than 467.7 72
µg/bee.
h: 467.7The LD10 24
µg/bee. h after
The LD10 application exceeded this
24 h after application value but
exceeded thiscould
valuenot butbecould
determined
not be
from the data (Table 2).
determined from the data (Table 2).

Figure 2.
Figure 2. Bee
Bee mortality
mortality rates
rates after
after dermal
dermal application
application of
of OAD
OAD during
during the
the three
three test
test intervals.
intervals. Mortality
Mortality
rates with different lower-case letters are significantly different (chi
2 2-test, p ≤ 0.05).
rates with different lower-case letters are significantly different (chi -test, p ≤ 0.05).

After oral application, bee mortality occurred at relatively low concentrations compared to the
Table 2. Toxicity parameters after dermal and oral application of OAD.
dermal treatment (Figure 3). 10 and 50 µg did not cause a mortality rate significantly different from
the control group, while 75 Dermal
µg resulted in significantly
Application (µg/bee) higher bee mortality (chi2-test,
Oral Application p = 0.027). A
(µg/bee)
total of 100 µg killed 55% of48treated
h animals after
72 h72 h. The NOAEL (72
48 h h) for oral application
72 h was
50, the LOAEL
LD10 was 75 µg OAD/bee.
467.7 The LD10 obtained
256.4 by probit analysis
68.1 was 60.3 µg/bee. 60.3The LD10
48 h after application reached
NOAEL higher
n.d. b values than212.5
after 72 h: 68.1 µg/bee.75The LD10 24 h after 50 application
LOAEL ato exceed this valueb but this could not
was expected 250
be determined from 80
the data (Table 2). 75
2
chi -test n.d. p = 0.003 p < 0.001 p = 0.027
a These values represent the lowest dosage with significant difference in bee morality compared to controls;
Table
b n.d. not defined, 2. Toxicity parameters after dermal and oral application of OAD.
no significant differences occurred after 48 h; p-values refers to the first significant increase in
mortality (LOAEL).
Dermal Application (µg/bee) Oral Application (µg/bee)
48 h 72 h 48 h 72 h
After oral application, bee mortality occurred at relatively low concentrations compared to the
LD10 467.7 256.4 68.1 60.3
dermal treatment (Figure 3). 10 and 50 µg did not cause a mortality rate significantly different from the
NOAEL n.d. b 212.5 75 50
control group, while 75aµg resulted in significantly higher bee mortality (chi2 -test, p = 0.027). A total
LOAEL 250 80 75
of 100 µg killed 55% n.d.animals
of treated b
after 72 h. The NOAEL (72 h) for oral application was 50,
chi2-test p = 0.003 p < 0.001 p = 0.027
the LOAEL was 75 µg OAD/bee. The LD10 obtained by probit analysis was 60.3 µg/bee. The LD10 48b h
a These values represent the lowest dosage with significant difference in bee morality compared to controls;
after application reached higher values than after 72 h: 68.1 µg/bee. The LD10 24 h after application
n.d. not defined, no significant differences occurred after 48 h; p-values refers to the first significant increase in
was expected to exceed this value but this could not be determined from the data (Table 2).
mortality (LOAEL).
Insects 2017, 8, 84 6 of 17
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Insects 2017, 8, 84 6 of 16

Figure 3. Bee
Bee mortality rates
rates after oral
oral application ofof OAD during
during the three
three test intervals.
intervals. Mortality
Figure 3. Bee mortality
Figure 3. mortality rates after
after oral application
application of OAD
OAD during thethe three testtest intervals. Mortality
Mortality
rates with different lower-case letters are significantly different (chi
2 2-test, p ≤ 0.05).
rates
rates with
with different
different lower-case
lower-case letters
letters are
are significantly
significantly different
different(chi -test, pp ≤
(chi2-test, 0.05).
≤ 0.05).

During the experiment, changes in behavior were observed only after oral application of dosages
During the
During the experiment, changes in
experiment, changes in behavior were observed
behavior were observed only
only after
after oral
oral application
application of
of dosages
dosages
also causing increased mortality within this period (≥75 µg/bee). The bees were less active, showed
also causing increased mortality within this period (≥75 µg/bee). The bees were less active,
also causing increased mortality within this period (≥75 µg/bee). The bees were less active, showed showed
minor movement and slowly formed bee clusters at the top of the cage. Shortly after the application
minor movement and slowly formed bee clusters at the top of the cage. Shortly after the application
minor movement and slowly formed bee clusters at the top of the cage. Shortly after the application
they showed increased self-grooming. Some bees also extended the proboscis.
they showed
they showed increased
increased self-grooming.
self-grooming. Some
Some bees
bees also
also extended
extended the
the proboscis.
proboscis.
3.1.2. Investigation of Sublethal Effects
3.1.2. Investigation of Sublethal Effects

Responsiveness to Water
Responsiveness Water and Ascending
Ascending Concentrationsof of SucroseSolution
Solution
Responsiveness toto Water andand Ascending Concentrations
Concentrations of Sucrose
Sucrose Solution
The PER on
on water increased
increased after the
the treatmentin in thetest
test (McNemar’stest,
test, p ≤ 0.001) and control
The
The PER
PER on water
water increased after
after the treatment
treatment inthe (McNemar’s test,pp≤
the test(McNemar’s 0.001) and
≤ 0.001) and control
control
bees
bees (McNemar’s
(McNemar’s test,
test, p
p ≤
≤ 0.01,
0.01,Figure
Figure 4).
4).However,
However, bees
bees treated
treated with
with OAD
OAD showed
showed significant
significant higher
higher
bees (McNemar’s test, p ≤ 0.01, Figure 4). However, bees treated with OAD showed significant higher
response rates
response rates to
to water
water than
than the
the controls
controls (chi
(chi²-test, p≤
2 -test, p ≤ 0.001).
0.001).
response rates to water than the controls (chi²-test, p ≤ 0.001).

Figure 4. PER
PER (Proboscis Extension
Extension Response) to to water prior
prior and after
after treatment: both groups show
show
Figure
Figure 4.4. PER (Proboscis
(Proboscis Extension Response)
Response) to water
water prior and
and after treatment: both groups
treatment: both groups show
increased responsiveness after the treatment (McNemar’s test, p ≤ 0.001 and p = 0.004,). Different
increased
increased responsiveness
responsiveness after
after the
the treatment
treatment (McNemar’s test, pp ≤
(McNemar’s test, ≤ 0.001 and pp == 0.004,).
0.001 and 0.004,). Different
Different
lower-case letters
lower-case lettersindicate
indicatesignificant
significantdifferences
differences between
between groups,
groups, before
before andand after
after treatment
treatment (chi2(chi²-
-test,
lower-case letters indicate significant differences between groups, before and after treatment (chi²-
test, p ≤
≤ 0.001)
ptest, 0.001) additionally;
additionally; significant
significant differences
differences are are marked
marked with with asterisks’.
asterisks’.
p ≤ 0.001) additionally; significant differences are marked with asterisks’.
Insects 2017, 8, 84 7 of 17
Insects 2017, 8, 84 7 of 16
Insects 2017, 8, 84 7 of 16
The
The sucrose
sucrose responsiveness
responsiveness was
was also
also influenced by treatments,
influenced by treatments, inin the
the control
control group
group the
the
responsiveness
The sucrose
responsiveness decreased significantly
responsiveness
decreased at
was also
significantly 3% and 10%
at 3%influenced concentration (McNemar’s
by treatments,(McNemar’s
and 10% concentration in the controltest, p ≤
test, group 0.025,
the
p ≤ 0.025,
Figure
Figure5).
responsiveness
5). decreased significantly at 3% and 10% concentration (McNemar’s test, p ≤ 0.025,
Figure 5).

Figure5.5.PER
Figure PER to ascending
to ascending sucrose
sucrose concentrations
concentrations from control
from control bees: response
bees: response is significantly
is significantly decreased
Figure 5. PER
decreased at 3%to and
ascending
10% sucrose concentrations
concentration (McNemar’s from
test,control
p = bees:
0.025 andresponse
p = is significantly
0.018), significant
at 3% and 10% concentration (McNemar’s test, p = 0.025 and p = 0.018), significant differences are
decreased at
differences 3% and 10%
with concentration (McNemar’s test, p = 0.025 and p = 0.018), significant
marked withare marked
asterisks’. asterisks’.
differences are marked with asterisks’.
Bees treated with OAD showed increased sucrose responsiveness, significantly at 0.1%
Bees
Bees treated
(McNemar’s test, pwith
treated with OAD
OAD
= 0.024, showed
showed
Figure 6). increased
increased sucrose
sucrose responsiveness, significantly atat 0.1%
responsiveness, significantly 0.1%
(McNemar’s test, p = 0.024, Figure 6).
(McNemar’s test, p = 0.024, Figure 6).

Figure 6. PER to ascending sucrose concentrations from OAD-treated bees: response after treatment
Figure
is
Figure 6.6.PER
PERtoto
significantly ascending
increased
ascending sucrose
atsucrose
0.1% concentrations
concentration fromOAD-treated
OAD-treated
(McNemar’s
concentrations from bees:response
test, p = 0.024),
bees: responseafter
significant after treatment
differences areis
treatment
is significantly
asterisks’.at 0.1% concentration (McNemar’s test, p = 0.024), significant differencesare
increased
marked withincreased
significantly at 0.1% concentration (McNemar’s test, p = 0.024), significant differences are
marked with asterisks’.
marked with asterisks’.
Insects 2017, 8, 84 8 of 17

Insects 2017, 8, 84 8 of 16

Longevity under Laboratory Conditions

Longevity under Laboratory Conditions
The highest proportional bee mortality occurred directly after the treatment (test group) and
The highest proportional bee mortality occurred directly after the treatment (test group) and
between the 20th and 21st day after hatching (control group). In the test group bees lived at least for
between the 20th and 21st day after hatching (control group). In the test group bees lived at least for
two days, for a maximum of 31 days and on average for 6.4 days. The bees in the control group lived
two days, for a maximum of 31 days and on average for 6.4 days. The bees in the control group lived
for for
an an
average of of
average 24.4 days
24.4 days(min.
(min.two
twodays,
days,max.
max. 33 days). This
33 days). Thiscan
canbebedemonstrated
demonstrated in in
thethe trend
trend of of
thethe
survival curves (Kaplan–Meier survival analysis, Gehan–Breslow, p ≤ 0.001, Figure
survival curves (Kaplan–Meier survival analysis, Gehan–Breslow, p ≤ 0.001, Figure 7). 7).

Figure 7. Survival curves under laboratory conditions: bees in the control group survived significantly
Figure 7. Survival curves under laboratory conditions: bees in the control group survived significantly
longer than bees treated with OAD (Kaplan–Meier survival analysis, Gehan–Breslow, p ≤ 0.001).
longer than bees treated with OAD (Kaplan–Meier survival analysis, Gehan–Breslow, p ≤ 0.001).

pH Values of the Digestive System and the Hemolymph

pH Values of the Digestive System and the Hemolymph
Crop: The pH values of the crops’ content after oral OAD application were subject to slight
Crop: The
variation pH 8).
(Figure values of the crops’
The average pH value content
(±SD) after oralapplication
afteroral OAD application
of OAD (24 were subject
h: 4.62 ± 0.4;to48slight
h:
variation (Figure 8). The average pH value ( ± SD) after oral application of OAD
4.64 ± 0.36; 72 h: 4.44 ± 0.43) was lower during 72 h than the control. After the dermal application, (24 h: 4.62 ± 0.4;the 48 h:
4.64pH± values
0.36; 72were at 24±
h: 4.44 h:0.43)
4.82 ±was
0.37 lower
and 48during
h: 5.02 ±72 0.4hremaining
than the control. the dermal
Afterlevel.
at the control application,
Only during the
thelast
pHinterval
values didweretheatpH 24 subside
h: 4.82 to± 4.49
0.37 ±and
0.3848 (72h:h). The±pH
5.02 0.4values
remaining
obtainedat the control
by the level.
control group Only
during the last interval did the pH subside to 4.49 ± 0.38 (72 h). The pH values obtained by the
were 24 h: 4.86 ± 0.46; 48 h: 4.80 ± 0.44; and 72 h: 4.99 ± 0.92. However the differences were not
significant
control (Mann–Whitney
group were 24 h: 4.86 ±rank 0.46;sum48 h:test,
4.80 p± ≥0.44;
0.05),
andand the4.99
72 h: standard
± 0.92. deviation
However of themeans is
differences
werecomparatively
not significant high.
(Mann–Whitney rank sum test, p ≥ 0.05), and the standard deviation of means is
Ventriculus:
comparatively The average pH values of the liquid ventriculus contents were significantly reduced
high.
between 24 and
Ventriculus: The 48 average
h after oral
pH application
values of the of liquid
OAD: 6.50 ± 0.24 (24
ventriculus h) and were
contents 6.38 ±significantly
0.32 (48 h, Mann-reduced
Whitney-rank-sum-test, p = 0.002), respectively (Figure 9). During the last interval, the pH value
between 24 and 48 h after oral application of OAD: 6.50 ± 0.24 (24 h) and 6.38 ± 0.32 (48 h,
averaged 6.59 ± 0.17 (72 h). After the dermal application of OAD, the pH value was on average 6.52 ±
Mann-Whitney-rank-sum-test, p = 0.002), respectively (Figure 9). During the last interval, the pH
0.17 (24 h). Within the following intervals the pH reduced significantly to 6.40 ± 0.21 (48 h) compared
value averaged 6.59 ± 0.17 (72 h). After the dermal application of OAD, the pH value was on average
with the control group (t-test, p = 0.002) and 6.48 ± 0.14 after 72 h (t-test, p = 0.028). The pH values
6.52 ± 0.17 (24 h). Within the following intervals the pH reduced significantly to 6.40 ± 0.21 (48 h)
obtained by the corresponding control groups remained at a constant level: 24 h: 6.55 ± 0.2; 48 h: 6.55
compared
± 0.21; 72with 0.16. group (t-test, p = 0.002) and 6.48 ± 0.14 after 72 h (t-test, p = 0.028). The pH
the ±control
h: 6.55
values obtained
Rectum: The pH corresponding
by the value of the liquid control
rectum groups remained
contents averaged at a4.98
constant
± 0.27 level: 24 h:oral
24 h after 6.55OAD± 0.2;
48 h: 6.55 ± 0.21;
application 72 h:
(Figure 6.55
10). ± 0.16.
After 48 h, the pH was significantly reduced to 4.77 ± 018 (t-test, p < 0.001); after
Rectum: The pH value
72 h, the pH reached 4.83 ± 0.16 of theand
liquid rectumsignificantly
remained contents averaged the±control
4.98
different to 0.27 24group
h after oral pOAD
(t-test, =
application (Figure 10). After 48 h, the pH was significantly reduced to 4.77
0.003). After dermal application of OAD, the pH value averaged 5.09 ± 0.16 (24 h) and 5.11 ± 0.18 ± 018 (t-test, p < 0.001);
(48
after
h).72 h, the the
During pH last
reached
interval, ± 0.16
4.83the pH wasand 5.10
remained
± 0.17 significantly different
and significantly to the
different to control group
the control (t-test,
group
p =value
0.003).(t-test,
Afterp dermal
= 0.016). The pH values
application obtained
of OAD, the pHby the corresponding
value averaged 5.09 ± 0.16
control groups
(24 h)were:
and24 ± 0.18
h: 5.05
5.11
(48 ±h).
0.31; 48 h: 5.05
During ± 0.24;
the last 72 h: 4.99
interval, the ±pH0.24.
was 5.10 ± 0.17 and significantly different to the control group
Insects 2017, 8, 84 9 of 17

value (t-test, p = 0.016). The pH values obtained by the corresponding control groups were: 24 h:
± 0.31;
5.05 Insects 84 5.05 ± 0.24; 72 h: 4.99 ± 0.24.
488, h:
2017, 9 of 16
Hemolymph:
Insects 2017, 8, 84 OAD, 24 h after oral application, caused a significant reduction in the average 9 of 16 pH
value of theHemolymph:
hemolymph OAD,with 24 h6.72 after±oral application,
0.31 comparedcaused
to the acontrol
significant
group reduction
(6.98 ± in theMann–Whitney
0.17; average pH
rank sum test, p = 0.011, Figure 11). During the remaining intervals, the pH increased to 6.83pH
value Hemolymph:
of the OAD,
hemolymph 24withh after
6.72oral
± application,
0.31 compared caused
to the a significant
control group reduction
(6.98 ± in
0.17; the average
Mann–Whitney ± 0.22
value
rank sum of the hemolymph
test, with 6.72
p = 0.011, Figure 11).± 0.31 compared
During to the control
the remaining group
intervals, the(6.98 ± 0.17; Mann–Whitney
pH increased to 6.83 ± 0.22
(48 h) and 6.97 ± 0.27 (72 h). After dermal application, the pH values measured at the first and second
rank
(48 h)sumand test,
6.97 p± =0.27
0.011,
(72 Figure
h). After 11). During
dermal the remaining
application, the pHintervals, the pH increased
values measured toand
at the first 6.83second
± 0.22
intervals were 7.00 ± 0.12 (24 h) and 7.00 ± 0.08 (48 h), respectively, and thus similar to the control
(48 h) andwere
intervals 6.97 ±7.00
0.27± (72
0.12h).(24After dermal
h) and 7.00application, therespectively,
± 0.08 (48 h), pH values measured
and thusatsimilar
the first
to and
the second
control
groups:
intervals
groups: 6.98
± 0.17
6.98were ± 7.00
(240.12
0.17 ±(24
h);h);(24
6.96 h)±and
6.96
0.26
± 0.26
(48
7.00
(48
h).
± 0.08 After 72
(48 h),72
h). After
h, the average
respectively,
h, the averageand pH pH
thus value
similar
value was
was
to the6.88
6.88
± 0.15,
control
± 0.15,
significantly
groups:
significantlyreduced
6.98 reduced in
± 0.17 (24 comparison
h); 6.96 ± 0.26
in comparison to the control
(48control
to the group
h). After 72 h,
group 7.17
the
7.17 ± 0.21
average
± 0.21 (t-test,
pH
(t-test, p =
p =value0.006).
was 6.88 ± 0.15,
0.006).
significantly reduced in comparison to the control group 7.17 ± 0.21 (t-test, p = 0.006).

Figure 8. Average pH of the liquid contents of the honeybee crop after oral and dermal treatment (n
Figure 8. Average pH of the liquid contents of the honeybee crop after oral and dermal treatment
≤ 52/group).
Figure 8. Average pH of the liquid contents of the honeybee crop after oral and dermal treatment (n
(n ≤ 52/group).
≤ 52/group).

Figure 9. Average pH of the ventriculus contents after oral and dermal treatment (n ≤ 68/group).
Significant
Figure differences
9. Average pH are indicated
of the by asterisks
ventriculus (48after
contents h: Mann–Whitney rank
oral and dermal sum test,(np =≤ 0.002;
treatment 72 h:
68/group).
Figure 9. Average pH of the ventriculus contents after oral and dermal treatment (n ≤ 68/group).
t-test, p < 0.05);
Significant significant
differences are differences
indicated byare marked(48
asterisks with asterisks’.
h: Mann–Whitney rank sum test, p = 0.002; 72 h:
Significant differences are indicated by asterisks (48 h: Mann–Whitney rank sum test, p = 0.002; 72 h:
t-test, p < 0.05); significant differences are marked with asterisks’.
t-test, p < 0.05); significant differences are marked with asterisks’.
Insects 2017, 8, 84 10 of 17
Insects 2017, 8, 84 10 of 16
Insects 2017, 8, 84 10 of 16

Figure 10. Average

Average pHpH of the rectum
pH of rectum contents after
after oral andand dermal treatment
treatment (n≤≤ 68/group).
68/group).
Figure
Figure 10.
10. Average of the
the rectum contents
contents after oral
oral and dermal
dermal treatment (n (n ≤ 68/group).
Significant differences are indicated by asterisks (48 h: t-test, p = 0.001; 72 h: t-test, p < 0.05); significant
Significant differences are indicated by asterisks (48 h: t-test, p = 0.001; 72 h: t-test, p < 0.05); significant
Significant
differences differences
markedare
are marked withindicated by asterisks (48 h: t-test, p = 0.001; 72 h: t-test, p < 0.05); significant
asterisks’.
differences are with asterisks’.
differences are marked with asterisks’.

Figure 11. Average pH of the hemolymph after oral and dermal treatment (n ≤ 26/group). Significant
Figure 11. Average pH of the hemolymph after oral and dermal treatment (n ≤ 26/group). Significant
differences
Figure are indicated
11. Average pH of bytheasterisks’
hemolymph (24 after
h: Mann–Whitney rank
oral and dermal sum test,
treatment p =26/group).
(n ≤ 0.011; 72 h:Significant
t-test, p =
differences
0.006); are indicated
significant by asterisks’
differences are marked(24 with
h: Mann–Whitney
asterisks’. rank sum test, p = 0.011; 72 h: t-test, p =
differences are indicated by asterisks’ (24 h: Mann–Whitney rank sum test, p = 0.011; 72 h: t-test,
0.006); significant differences are marked with asterisks’.
p = 0.006); significant differences are marked with asterisks’.
3.2. Computer Tomography of Honey Bee Colonies: Distribution of OAD
3.2. Computer Tomography of Honey Bee Colonies: Distribution of OAD
3.2. Computer Tomography
The distribution of Honey
of OAD Beecolony
in the Colonies:
afterDistribution of OAD was demonstrated by macroCT
topical application
The distribution
scanning (Figure 12). Theof OAD in the
control colony after topical application
measurements was demonstrated by macroCT
The distribution of OAD in the colony afterachieved a mean density
topical application value of −219.77
was demonstrated by±macroCT
93.3 HU.
scanning
The (Figure
relatively high12). The
standardcontrol measurements
deviation was caused achieved
by the a mean density
heterogeneity of value
the of
bees’−219.77
body ± 93.3After
mass. HU.
scanning (Figure 12). The control measurements achieved a mean density value of −219.77 ± 93.3 HU.
The relatively
application high
of high standard
OADstandard deviation
(10 min) deviation
the density was caused
value by the
increased heterogeneity of the bees’ body mass. After
The relatively was caused by theto a mean valueofofthe
heterogeneity −98.97
bees’±body
87.06mass.
HU, which
After
application of OAD
was significantly (10 min) the density
different value(t-test,
increased
p ≤toto a mean value of −98.97 ± 87.06 HU, which
application of OAD (10 min)from the controls
the density value increased 0.001).
a meanThirty
valueminutes
of −98.97 after application
± 87.06 HU, whichthe
was
meansignificantly different
value was −134.98 fromHU,
± 89.5 the significantly
controls (t-test, p ≤ 0.001).
different Thirtyto
compared minutes after application
the controls the
and the values
mean value
achieved was
after 10 −134.98 ± 89.5
min (t-test, p ≤HU,
0.001,significantly
Figure 13). different compared to the controls and the values
achieved after 10 min (t-test, p ≤ 0.001, Figure 13).
 The mean value (−96.03 ± 87 HU) in the central area of the combs 10 min after treatment of the
The mean value (−96.03 ± 87 HU) in the central area of the combs 10 min after treatment of the
bees was comparable to the bees in the boundary area (−101.92 ± 86.7 HU). However, the mean
bees was comparable to the bees in the boundary area (−101.92 ± 86.7 HU). However, the mean
density values thirty min after the treatment were significantly different compared to the central area
density values thirty min after the treatment were significantly different compared to the central area
(−153.04 ± 77.8) and boundary area (−116.9 ± 97.1) of the combs (t-test, p = 0.015, Figure 14).
(−153.04 ± 77.8) and boundary area (−116.9 ± 97.1) of the combs (t-test, p = 0.015, Figure 14).
InsectsIn the8, colony
2017, 84 examined for the long-term distribution, the control measurements achieved a
11 of 17
In the colony examined for the long-term distribution, the control measurements achieved a
mean density value of −200.61 ± 86.87 HU. Three days after the application of OAD the density value
mean density value of −200.61 ± 86.87 HU. Three days after the application of OAD the density value
increased to a mean value of −128.1 ± 89.92 HU and was significantly different from the control
increased
was to a mean
significantly valuefrom
different of −128.1 ± 89.92(t-test,
the controls HU and p≤was significantly different from the control
measurement (t-test, p ≤ 0.001, Figure 15). Seven and 140.001). Thirty
days after minutes
application after application
the mean the
value was
measurement
mean value (t-test,
was − p ≤ 0.001,
134.98 ± Figure
89.5 HU, 15). Seven and
significantly 14 days
different after application
compared to the the mean
controls and value
the was
values
−151.61 ± 77.14 HU and −158.46 ± 78.32 HU, respectively, significantly different compared to the
−151.61
achieved±and 77.1410HU
after minand −158.46
(t-test, ± 78.32
p ≤ 0.001, HU, 13).
Figure respectively, significantly different compared to the
controls the values achieved after three days (t-test, p ≤ 0.001, Figure 15).
controls and the values achieved after three days (t-test, p ≤ 0.001, Figure 15).

Figure 12. MacroCT scanner image: bees on the comb, 30 min after application of OAD and Unilux.
Figure 12.
Figure MacroCT scanner
12. MacroCT scanner image:
image: bees
bees on
on the
the comb,
comb, 30
30 min
min after
after application
application of
of OAD
OAD andand Unilux.
Unilux.
Visualization of
Visualization of aa comb
comb with
with bees
bees sitting
sitting on
on it:
it: dark
dark areas
areas show
show low
low densities
densities and
and bright
bright areas
areas show
show
Visualization of a comb with bees sitting on it: dark areas show low densities and bright areas show
high densities.
high densities.
densities.
high

Figure 13. Density values before (control) and 10 and 30 min after treatment (n ≤ 144/group; t-test, p
Figure 13. Density values before (control) and 10 and 30 min after treatment (n ≤ 144/group; t-test, p
≤Figure
0.001),13. Density values
significant before
differences are(control)
marked and and 30 min after treatment (n ≤ 144/group; t-test,
with10asterisks’.
≤p 0.001), significant
≤ 0.001), differences
significant areare
differences marked
markedwith
with asterisks’.
asterisks’.

The mean value (−96.03 ± 87 HU) in the central area of the combs 10 min after treatment of
the bees was comparable to the bees in the boundary area (−101.92 ± 86.7 HU). However, the mean
density values thirty min after the treatment were significantly different compared to the central area
(−153.04 ± 77.8) and boundary area (−116.9 ± 97.1) of the combs (t-test, p = 0.015, Figure 14).
Insects 2017, 8, 84 12 of 17
Insects 2017, 8, 84 12 of 16

Insects 2017, 8, 84 12 of 16

Figure 14. Density values of the comb areas center and boundary: significant differences between
Figure 14. Density values of the comb areas center and boundary: significant differences between areas
areas occur
occur 30 min30after
mintreatment
after treatment (n ≤ 72/group;
(n ≤ 72/group; t-test,t-test, p = 0.015),
p = 0.015), significant
significant differences
differences are marked
are marked with
with asterisks’.
asterisks’.

In the colony examined for the long-term distribution, the control measurements achieved a mean
density value of −200.61 ± 86.87 HU. Three days after the application of OAD the density value
increased to a mean value of −128.1 ± 89.92 HU and was significantly different from the control
measurement
Figure 14. (t-test, ≤ 0.001,
Densityp values Figure
of the comb15). Seven
areas and
center and14 boundary:
days aftersignificant
application the mean
differences value was
between
−151.61 ± 77.14 HU and − 158.46 ± 78.32 HU, respectively, significantly different compared to the
areas occur 30 min after treatment (n ≤ 72/group; t-test, p = 0.015), significant differences are marked
controls
withand the values achieved after three days (t-test, p ≤ 0.001, Figure 15).
asterisks’.

Figure 15. Density values before (control) and 3, 7 and 14 days after treatment: different lower-case
letters indicate significant differences (n ≤ 211/group; t-test, p ≤ 0.001).

4. Discussion
We found unequal results of OAD toxicity after dermal and oral application—ingestion of OAD
is much more toxic to bees than application on the cuticula, which was relatively well tolerated. This
corresponds with our first report of bee tolerability concerning OAD as a single compound [10].
Investigations of toxicity are often conducted with combined substances. This makes it difficult
Figure 15. Density values before (control) and 3, 7 and 14 days after treatment: different lower-case
to compare15.
Figure Density
these values
results withbefore (control) and
our findings. 3, 7 and 14the
Concerning days after treatment:
dermal different
application of the lower-case
toxic effects of
letters indicate
letters indicate significant
significant differences
differences (n
(n ≤
≤ 211/group;
211/group; t-test,
t-test,pp≤≤0.001).
0.001).
different dosages are reported in the literature. Dosages <100 µg/bee did not cause significant
mortality
4. after 48 h, this is less than half the dosage of 212.5 µg/bee with no observed mortality in our
Discussion
test. The described LD10 (48 h) of 176.68 µg/bee was derived from a combinatory effect of OAD and
We found unequal results of OAD toxicity after dermal and oral application—ingestion of OAD
is much more toxic to bees than application on the cuticula, which was relatively well tolerated. This
corresponds with our first report of bee tolerability concerning OAD as a single compound [10].
Insects 2017, 8, 84 13 of 17

4. Discussion
We found unequal results of OAD toxicity after dermal and oral application—ingestion of OAD
is much more toxic to bees than application on the cuticula, which was relatively well tolerated. This
corresponds with our first report of bee tolerability concerning OAD as a single compound [10].
Investigations of toxicity are often conducted with combined substances. This makes it difficult
to compare these results with our findings. Concerning the dermal application of the toxic effects
of different dosages are reported in the literature. Dosages <100 µg/bee did not cause significant
mortality after 48 h, this is less than half the dosage of 212.5 µg/bee with no observed mortality in
our test. The described LD10 (48 h) of 176.68 µg/bee was derived from a combinatory effect of OAD
and acetone on anaesthetized worker bees (CO2 ) [11]. It was lower than the LD10 (48 h) of OAD as
a single agent in our trial, which reached a level of 467.7 µg/bee. A combinatory effect of OAD and
acetone on anaesthetized worker bees (CO2 ) may explain the explicitly higher responsiveness of the
tested animals in the cited trials compared to our findings. Other tests with a polyhybrid subspecies of
A. mellifera resulted in a level without observed effect of 400 µg/bee and a lowest observed effect level
of 600 µg/bee after 48 h [12].
In our experiments 175 µg/bee, corresponding to the 3.5% solution (30–50 mL depending on
colony size) used in beekeeping practice, did not cause mortality for individual bees, different from
controls 72 h after dermal application. Oral application resulted in high bee mortality at relatively
low concentrations compared to the dermal treatment. Bees reacted much more sensitively to the
oral application of OAD. The LOAEL for dermal application is higher than for oral application by a
factor >3.
To assess risk of an applied substance, not only mortality but also the effects on physiological
processes and behavior after pesticide exposition must be considered as described [13]. Physiological
effects concerning acetylcholinesterase and glutathione S-transferase activities, as described for
exposure to fluvalinate, are not documented after OAD treatment (3% in 32% sucrose solution w/w,
50 mL/colony) on pupae, newly emerged, nursery and forager bees [14,15]. Honey bee larvae,
treated with OAD by spraying (about 121 µg solution/larvae) undergo histologic changes—accidental
cell death leading to necrosis [16]. These effects may be causally responsible for the brood death
after application of OAD (3% in 50% sucrose solution, two treatments during summer) in breeding
colonies [17]. In beekeeping practice, acute damages to the brood can be excluded when honeybee
colonies are treated with OAD during the brood free period. However, long term effects are possible;
OAD has been found in bee colonies even six months after topical application following regular
treatment [18]. Long-term effects (up to four months after application) like a reduced amount of brood
in treated colonies (3% OAD w/v, 4 mL per comb side, spraying) have been reported [19].
After dermal and oral application, respectively, of high dosages up to 1320 µg/bee, OAD was
recovered in the internal organs and the hemolymph of adult bees [20,21]. It is assumed that after
dermal application the cuticula can be penetrated by the acid [21]. Also, pathological repercussions
e.g., degeneration of rectal epithelium, malphigian tubules and ventriculus, have been described after
dermal application [20]. C14 -marked OAD could be found in the abdominal structures of worker bees
after topical application into the colony [22].
The treatment with OAD caused sublethal effects on A. mellifera. First indications of changes
in the pH of internal organs and the hemolymph after a single dermal application of OAD with a
dosage of 175 µg/bee have been provided [23]. The individual treatment of honeybees with OAD in
our experiments changed the pH-value of the intestinal parts and the hemolymph. The pH values of
the crop contents reflect the acid application, specifically after oral ingestion. Significant differences
could be found in the pH values of the ventriculus 48 h after oral and 72 h after dermal treatment:
These deferred reductions in the tested time intervals could be caused by the slower penetration of
the acid through the cuticula compared to the direct oral intake. Differences in the pH structure of
the rectum and the hemolymph were also found, the pH reduction occurs faster after oral application.
The proof of a pH reduction in the digestive system even after 72 h indicates a long disposition of OAD
Insects 2017, 8, 84 14 of 17

in the bee. The returned balance of the pH measured in the hemolymph 48 h after oral application also
indicates a possible buffer capacity of the hemolymph.
We could demonstrate that OAD moving through the digestive system or penetrating the cuticula
modified the pH structure of the honeybee’s intestinal parts and the hemolymph. The shift towards
stronger acidity after OAD treatment supports that damage to the epithelial tissue and organs [20]
may be caused by hyperacidity. It also corresponds to the timescale of recovered OAD after oral
application [21]. The increased acidity can cause chemical burns which eventually lead to necrosis [16],
however a threshold cannot be derived from our tests due to the low dose applied.
Further sublethal effects could be demonstrated in a decreased longevity under laboratory
conditions and increased responsiveness to water. Bees treated with OAD died much sooner than
bees treated with sucrose solution. The increased responsiveness to water 24 h after OAD treatment
indicated an acidosis of the bees, which they may compensate with an increased uptake of water.
This assumption is supported by the shift towards stronger acidity, found—after OAD treatment
conducted—in the measurements of the pH-values. These results indicate a general impairment of
the bees after treatment. The treatment in autumn/winter affected primarily long-living winter bees
which are essential for winter survival and successful colony development in the spring. Treatment
during the summer with brood can lead to substantial brood damage [15] as described above. Even by
treating artificial swarms or nucleus colonies, it cannot be certain that damages will not occur due to
the long-term exposure to OAD in the colony [18,19].
It has been described that after dermal administration bees carry white deposits primarily on the
body [11,20] and later also on the third pair of legs [24]. Concerning sublethal effects in the colony
after individual dermal application of 175 µg/bee, changes in behavior were found [24]: the bees were
less active, showed reduced brood care, increased grooming behavior, and they also had a reduced
life span.
In beekeeping practice OAD is mostly applied topically. The distribution in the colony may occur
on two pathways: (1) through oral intake and distribution via trophallaxis; and/or (2) contact between
bees and also contaminated hive material. OAD vapors in the colony seem to be of minor importance
as OAD has a low volatility [25]. However, the colony treatment using 3.5% OAD solution (30–50 mL
per colony, depending on colony strength, applied by trickling) corresponds to an individual dosage of
approximately 175 µg OAD/bee. This dosage is well tolerated by individual bees in the laboratory and
is well below the lowest observed adverse effect level concerning mortality after dermal application in
our test.
OAD applied orally in the laboratory causes high bee mortality. As the mortality observed in the
treated colonies was not highly increased, OAD was probably ingested in relatively low amounts by
grooming and/or trophallaxis. The oral intake of OAD after treatment at the colony level seems to
be of little importance. The threshold value derived in the laboratory was therefore not exceeded for
most of the target animals. This corresponds with the findings that when OAD is applied in a solution
with high sugar content the bee mortality increases [26]. This suggests that high sugar content leads
to more ingestion. In laboratory trials, significantly higher bee mortality was found when sugar was
added to the OAD solution [27].
In practical beekeeping, appropriate use of OAD (on average 175 µg/bee) by topical application
in the field is relatively safe for A. mellifera on the colony level, even when some individuals die.
Based on our findings in the laboratory, the threshold with first adverse effects (LOAEL) could be
reached in the colony when an overdose of 43% for the individual bee is applied. However, due to the
attractiveness of sucrose solutions to bees they ingest the solution even if it contains toxic substances.
A sugar substitute e.g., glycerol, with a high viscosity and desirable distribution can prohibit oral
uptake by the bees [9]. It could optimize the application of OAD, but so far it has not been approved
as a veterinary drug ingredient for bees in combination with OAD.
High acaricidal efficacy after topical application was only found when bee to bee contact took
place [11]. In further trials, when body contact between the bees was prevented, but trophallaxis
Insects 2017, 8, 84 15 of 17

allowed, it has been shown that trophallactic interaction did not lead to mite mortality; bee to bee
contact seemed to be the primary route of distribution of OAD in acaricidal relevant dosages [28].
Due to only minor oral intake, systemic efficacy against V. destructor seems to be improbable.
A systemic effect requires a transfer of the acid into the hemolymph of the bee. OAD was found in
the hemolymph only after application of very high dosages [20,21]. After application of the 3.5%
OAD solution (175 µg/bee), as used in beekeeping, changes in the pH structure were found, but OAD
was not recovered in the hemolymph of single bees at a detection limit of 2.5 µg [24]. This amount
used in beekeeping practice seems to be too low to reach a systemic toxicity to V. destructor through
ingestion. Therefore, we conclude that the mode of action in the colony must be contact poisoning
against V. destructor.
In order to reach high efficacy, the ingredient acting by contact must be distributed in the colony.
The distribution of OAD was shown by macroCT. The results of the roentgenoscopy showed high
density values for the individual bees in the test, much higher than in the control measurement. A good
distribution was already achieved after 10 min; this could be documented in the central and boundary
areas of the combs. Lower density values in the central comb areas compared to the boundary regions
obtained after thirty minutes reflected the movement of the bees. After thirty minutes the density
was generally lower, which led us to the assumption that OAD was now also spread to the material,
e.g., the wall of the hive. Bees have constant contact with the hive material; therefore, OAD can be
distributed again onto the bees, maintaining a long-term contact with the acid. OAD on hive material
can be found even several months after application [18]. The macroCT analysis demonstrated a rapid
and consistent distribution of OAD involving a reduction of the individual dosage over time. Even
after 14 days, the density of the bees was still significantly higher than prior to treatment, indicating
a potential efficacy of at least up to 14 days. The results from the field trials, where the maximum
efficacy against mites was reached ten days after treatment, support this assumption [29].

5. Conclusions
OAD used to treat varroosis of A. mellifera shows a rapid and consistent distribution in the colony
for at least up to 14 days, and high efficacy against the mite, but also lethal and sublethal effects.
In practical beekeeping, appropriate use of OAD (one topical application, on average 175 µg/bee)
is relatively safe for A. mellifera at the colony level, even when some individuals are lost. However,
ingestion leads to high mortality. The reported sublethal effects are highly decreased longevity,
a reduction in pH-values in the digestive system and the hemolymph, and an increased responsiveness
to water. The shift towards stronger acidity after treatment confirms that damage to the epithelial
tissue and organs is likely to be caused by hyperacidity. Pathological repercussions e.g., degeneration
of rectal epithelium, malpighian tubules and ventriculus may also occur.
These results indicate a general impairment of the bees after treatment. The treatment in autumn
or winter affects primarily long-living winter bees which are essential for winter survival and successful
colony development in the spring. Treatment during summer with brood can cause substantial brood
damage. Even when treating artificial swarms or nucleus colonies it cannot be certain that damages
will not occur due to the extensive exposure to OAD in the colony. Long-term effects such as reduced
amount of brood in treated colonies have been reported.
OAD is one of the most important organic acids used for the control of V. destructor. It is
indispensable but must be dosed precisely and applied as seldom as possible to prevent sublethal
damages which eventually lead to the loss of bees. Long disposition in the bee hive can cause
accumulation of the acid and therefore induce further damage.

Acknowledgments: We would like to thank the Tierärztliche Klinik für Pferde Seeburg, Germany for
their cooperation.
Author Contributions: Eva Rademacher, Marika Harz and Saskia Schneider conceived and designed the
experiments; Marika Harz and Saskia Schneider performed the experiments; Eva Rademacher, Marika Harz and
Saskia Schneider analyzed the data; Eva Rademacher, Marika Harz and Saskia Schneider wrote the paper.
Insects 2017, 8, 84 16 of 17

Conflicts of Interest: The authors declare no conflict of interest.

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