Comparative Sublethal Toxicity of Nine Pesticides on Olfactory Learning Performances of the Honeybee Apis mellifera

Université Bordeaux 1, Talence, Aquitaine, France
Archives of Environmental Contamination and Toxicology (Impact Factor: 1.9). 03/2005; 48(2):242-50. DOI: 10.1007/s00244-003-0262-7
Source: PubMed
Using a conditioned proboscis extension response (PER) assay, honeybees (Apis mellifera L.) can be trained to associate an odor stimulus with a sucrose reward. Previous studies have shown that observations of conditioned PER were of interest for assessing the behavioral effects of pesticides on the honeybee. In the present study, the effects of sublethal concentrations of nine pesticides on learning performances of worker bees subjected to the PER assay were estimated and compared. Pesticides were tested at three concentrations. The highest concentration of each pesticide corresponded to the median lethal dose value (48-h oral LD50), received per bee and per day, divided by 20. Reduced learning performances were observed for bees surviving treatment with fipronil, deltamethrin, endosulfan, and prochloraz. A lack of behavioral effects after treatment with lambda-cyalothrin, cypermethrin, tau-fluvalinate, triazamate, and dimethoate was recorded. No-observed-effect concentrations (NOECs) for the conditioned PER were derived for the studied pesticides. Our study shows that the PER assay can be used for estimating sublethal effects of pesticides on bees. Furthermore, comparisons of sensitivity as well as the estimation of NOECs, useful for regulatory purposes, are possible.


Available from: Axel Decourtye
Comparative Sublethal Toxicity of Nine Pesticides on Olfactory Learning
Performances of the Honeybee Apis mellifera
A. Decourtye,
J. Devillers,
E. Genecque,
K. Le Menach,
H. Budzinski,
S. Cluzeau,
M. H. Pham-Delgue
Association de Coordination Technique Agricole, Maison des Agriculteurs, La Tour de Salvagny, France
CTIS, Rillieux La Pape, France
Laboratoire de Neurobiologie Compare des Invertbrs, INRA, Bures-sur-Yvette, France
Laboratoire de Physico-Toxico-Chimie des Systmes Naturels, UMR 5472 CNRS, Universit de Bordeaux I, Talence Cedex, France
Received: 4 January 2004 /Accepted: 23 June 2004
Abstract. Using a conditioned proboscis extension response
(PER) assay, honeybees (Apis mellifera L.) can be trained to
associate an odor stimulus with a sucrose reward. Previous
studies have shown that observations of conditioned PER were
of interest for assessing the behavioral effects of pesticides on
the honeybee. In the present study, the effects of sublethal
concentrations of nine pesticides on learning performances of
worker bees subjected to the PER assay were estimated and
compared. Pesticides were tested at three concentrations. The
highest concentration of each pesticide corresponded to the
median lethal dose value (48-h oral LD50), received per bee
and per day, divided by 20. Reduced learning performances
were observed for bees surviving treatment with fipronil,
deltamethrin, endosulfan, and prochloraz. A lack of behavioral
effects after treatment with k-cyalothrin, cypermethrin, s-flu-
valinate, triazamate, and dimethoate was recorded. No-ob-
served-effect concentrations (NOECs) for the conditioned PER
were derived for the studied pesticides. Our study shows that
the PER assay can be used for estimating sublethal effects of
pesticides on bees. Furthermore, comparisons of sensitivity as
well as the estimation of NOECs, useful for regulatory pur-
poses, are possible.
The hazard assessment of pesticide toxicity to honeybees (Apis
mellifera L.) is commonly estimated from laboratory studies
(median lethal dose: LD50) and from semifield and field
experimentations when the pesticides demonstrate a hazard
quotient (application rate/LD50) over 50, or when they have a
specific mode of action (e.g., insect growth regulators), or
when there are indications of indirect effects such as delayed
action (EPPO 1992). Because behavioral effects of pesticides
in the honeybee have been shown to have the potential to
induce a significant impact on the development of colonies
(Waller et al. 1984; Bendahou et al. 1999; Decourtye et al.
2004a), such effects also could be used to better estimate the
hazard of pesticides to bees. Moreover, it is noteworthy that
the EPPO guidelines require recording all abnormal behavioral
effects observed during the experiments (EPPO 1992).
Semifield tests, representing more realistic exposure con-
ditions than in laboratory, have been cited as providing good
information for the behavioral toxicity assessment of pesti-
cides (Cluzeau 2002). However, the regulatory guidelines give
only very limited information on the type of behavioral data
that should be collected during the studies or how they should
be included and interpreted in the risk assessment scheme
(Thompson and Brobyn 2002). Moreover, the semifield tests,
even if they are well suited, are technically difficult to main-
tain and control. Their fluctuating conditions, cost, and the
necessity to have trained people to carry them out are bounds
limiting the number of facilities able to perform them in
practice. Thus, the identification of precise behavioral effects
requires additional and specific methods to make appropriate
hazard assessment (Pham-Delgue et al. 2002). Consequently,
the conditioned proboscis extension response (PER) assay
should be use to overcome these problems (Decourtye and
Pham-Delgue 2002).
The PER assay tentatively reproduces what happens in
honeybee–plant interaction: when landing on the flower, the
forager extends its proboscis as a reflex when the gustatory
receptors set on the tarsae, antennae, or mouth parts are
stimulated with nectar. This reflex leads to the uptake of nectar
and induces the memorization of the floral odors diffusing
concomitantly. Once memorized, the odors play a prominent
role in flower recognition during the next trips (Menzel et al.
1993). Consequently, an individual associative learning pro-
cess is important for the effective accomplishment of foraging
activities. The associative learning of workers, investigated
with the PER assay, may therefore be regarded as having a
high ecological significance because it is a prerequisite to the
foraging success of the whole colony.
The PER has been successfully reproduced under artificial
conditions (Kuwabara 1957; Takeda 1961), and has become a
valuable tool in studying various aspects of olfactory learning
Correspondence to: A. Decourtye; email:
Arch. Environ. Contam. Toxicol. 48, 242–250 (2005)
DOI: 10.1007/s00244-003-0262-7
Page 1
processes (Bitterman et al. 1983; Menzel et al. 1993; Sandoz
et al. 1995). The PER assay with restrained workers has also
been used to investigate the behavioral effects of pesticides
(Taylor et al. 1987; Mamood and Waller 1990; Stone et al.
1997; Abramson et al. 1999; Abramson and Boyd 2001;
Weick and Thorn 2002; Decourtye et al. 2003; Abramson
et al. 2004).
A previous work studying the behavioral toxicity of imi-
dacloprid and deltamethrin to bees indicated that a good
relationship was found between effects on olfactory responses
in free-flying foragers and in individuals subjected to the PER
paradigm (Decourtye et al. 2004a). The controlled conditions,
the relationship with field conditions, and the ability to
quantify the behavior pattern numerically led us to assume that
the use of the PER assay, as a method to evaluate the potential
effect of pesticides on the honeybees foraging behavior, could
help us to assess the toxicity of pesticides in a more compre-
hensive way than by only considering lethality as currently
made in practice (Devillers 2002). However, a survey of the
literature showed that only a limited number of chemicals had
been tested, and the studies using the PER assay were usually
not directly comparable because they were based on different
methods for the administration of chemicals, the behavioral
response, and so on. Moreover, in these works only one dose,
not necessarily sublethal, was generally tested.
To confirm the usefulness of the PER assay as a behavioral
toxicity assessment method, the goal of our study was to
compare the effects of sublethal exposure of nine pesticides on
the olfactory learning performances of worker bees subjected
to this assay.
Materials and Methods
The nine studied pesticides (Table 1) were all technical grade. Del-
tamethrin and prochloraz were obtained from Hoechst Schering Ag-
rEvo S.A. (Aventis CropScience, France). All the other compounds
were purchased from Cluzeau Info Labo (Sainte-Foy-La-Grande,
France). Their purity was at least 98%, except s-fluvalinate, which
had a purity of 76%.
The pesticides were tested at three different concentrations, with a
geometrical progression of factor 2. The highest tested concentration
corresponded to the median lethal dose value (LD50 determined 48 h
after the oral treatments) divided by 20 (Table 1). From previous
results (Decourtye et al. 2003), it was assumed that this ratio belonged
to a sublethal domain. The 48-h LD50s reported in Table 1 were
previously determined from acute oral toxicity tests for deltamethrin,
k-cyalothrin, and fipronil (Decourtye 2002), and from information
Table 1. Concentrations of agricultural chemicals applied with subchronic exposure before the conditioning procedure
Chemical Purity 48-h oral LD50 (per bee)
in stock solutions
Tested doses in
sucrose solutions
(per bee per day)
in sucrose
Deltamethrin >98% 620 ng (Decourtye 2002) 940 mg Æ L
30 ng 940 lg Æ L
15 ng 470 lg Æ L
7.5 ng 235 lg Æ L
k-Cyalothrin 98.5% 241 ng (Decourtye 2002) 360 mg Æ L
12 ng 360 lg Æ L
6 ng 180 lg Æ L
3ng 90lg Æ L
Cypermethrin 98.5% 460 ng (Vaughan 1986) 690 mg Æ L
23 ng 690 lg Æ L
-1 b
11.5 ng 345 lg Æ L
5.75 ng 172.5 lg Æ L
s-Fluvalinate 76% >200 lg (Barnavon 1987) 3 g Æ L
10 lg 300 mg Æ L
5 lg 150 mg Æ L
2.5 lg75mgÆ L
Prochloraz 97% 100 lg (Chalvet-Monfrey 1996) 1.5 g Æ L
5 lg 150 mg Æ L
2.5 lg75mgÆ L
1.25 lg 37.5 mg Æ L
Triazamate 97% 400 ng (Miniggio et al. 1990) 600 mg Æ L
20 ng 600 lg Æ L
10 ng 300 lg Æ L
5 ng 150 lg Æ L
Endosulfan 97.3% 5 lg (Stevenson 1978) 8 mg Æ L
250 ng 8 mg Æ L
(65.6% a isomer;
b isomer)
125 ng
62.5 ng
4mgÆ L
2mgÆ L
Dimethoate 98.5% 350 ng (Gough et al. 1994) 580 mg Æ L
17.5 ng 580 lg Æ L
8.7 ng 290 lg Æ L
4.3 ng 145 lg Æ L
Fipronil 98.5% 6 ng (Decourtye 2002) 9 mg Æ L
0.3 ng 9 lg Æ L
0.15 ng 4.5 lg Æ L
0.075 ng 2.2 lg Æ L
Actual concentrations of deltamethrin equal to 960, 429, and 212 lg Æ L
Actual concentrations of cypermethrin equal to 782, 388, and 207 lg Æ L
Toxicity of Pesticides on Olfactory Learning of Honeybee
Page 2
gained in the existing literature for the other chemicals. The con-
centrations were calculated for a consumption of syrup estimated to
33 ll/bee/day (Decourtye et al. 2003).
Stock solutions with a given concentration of each chemical were
prepared in acetone (Table 1). Acetone was chosen following the
EPPO guidelines, because it is a rather generalist solvent (EPPO
1992). Aliquots of the stock solutions were used to make each test
solution at a specific concentration. The chemicals were added to a
500 g L
sucrose solution. The final concentration of acetone in the
sucrose solutions was 1% (vol/vol). The effects of insecticide-added
solutions were compared with that of an untreated sucrose solution
(with 1% acetone vol/vol). Fresh dosing solutions were prepared for
each test.
Samples of contaminated sucrose solutions of deltamethrin and
cypermethrin delivered to bees were analyzed by gas chromatography/
mass spectrometry (K. Le Menach and H. Budzinski, unpublished).
Experiments were carried out with worker bees of Apis mellifera
ligustica L. They were conducted with bees collected from outdoor
hives. Emerging worker bees were caged in groups of 60 individuals.
They were provided with sugar food (mixture of sugar and honey),
and water ad libitum during the 2 first days and with pollen for the
next 8 days. After 2 days, bees were continuously fed with sucrose
solution contaminated or not during 11 consecutive days. The feeders
were changed daily with fresh sucrose solutions. The bees were kept
in an incubator (33 2°C, 40 10% relative humidity, darkness)
until 14–15 days old, and were used in the PER assay. It has been
shown that on average, worker bees become foragers at that age
(Sakagami 1953; Seeley 1982) and give the most consistent perfor-
mances in the conditioned proboscis extension assay (Pham-Delgue
et al. 1990).
For bees from 2 to 14–15 days old, the quantity of the contaminated
sugar solution provided daily was adjusted to the number of survivors.
The mortality and consumption of syrup were recorded daily, and
dead bees were discarded. Every testing day was organized as follows:
bees previously exposed to three concentrations of each chemical
were tested, as well as untreated control bees, leading to a total of 60–
80 bees tested per day, with 16–20 bees for each treatment. Experi-
ments were replicated at least three times, until about 50–60 bees per
treatment were obtained.
After treatment, the bees were mounted individually in glass tubes
with only their antennae and mouth parts left free. They were starved
for 4 h prior to conditioning. They were selected for showing a pro-
boscis extension reflex after stimulation of the antennae with a su-
crose solution (300 g Æ L
). The number of individuals exhibiting the
reflex response was recorded. The ability to produce the reflex re-
sponse reflects the state of the sensory-motor pathway underlying the
PER. The general stimulation conditions as well as the conditioning
and testing procedures were adapted from the work of Bitterman et al.
(1983) and are detailed in Sandoz et al. (1995). Bees were then placed
in an airflow (main airflow of 50 ml Æ s
added to a secondary airflow
of 2.5 ml Æ s
) for 15 s, to be familiarized with the mechanical
stimulation and with the experimental background. For the condi-
tioning trials, the conditioned stimulus (10 ll of pure linalool, a
standard floral odor, soaked on a filter paper strip inserted in a Pasteur
pipette cartridge; Sigma, 95–97% purity) was delivered through the
secondary flow (2.5 ml Æ s
) for 6 s. During odor delivery, the PER
was elicited after 3 s by contacting the antennae with a sucrose
solution (300 g Æ L
) as the unconditioned stimulus, and the same
solution was immediately given as a reward, before the odor delivery
ended. Three successive conditioning trials (Cond1–Cond3) were
carried out, followed by five test trials (Test1–Test5). The time
interval between trials was 20–30 min. Conveniently, the positive
responses at T1 of the individuals are scaled to 100 in order to better
characterize the extinction slope. During a test trial, the conditioned
stimulus (pure linalool) was delivered for 6 s. The conditioned PER
was recorded as a yes-or-no response (i.e., 0 or 1) when the odor alone
was delivered during the 6 s of the test trial.
Data Analysis
For each chemical, the mortality accumulated over 11 days of expo-
sure was compared between each concentration and the control by
multiple two-by-two v
tests with 1 df. To ensure that the experiment
error rate was a = 0.05, each comparison was carried out according to
the Dunn-Sidak method (Sokal and Rohlf 1995) at a critical proba-
bility of a'=1–(1–a)
, where k was the number of intended tests.
The significance level was 0.0085 for two-by-two comparisons of the
responses to three concentrations of each chemical and one control
The number of initial reflex responses and the number of condi-
tioned responses in each trial were compared between the three
concentrations of each chemical and the control by multiple two-by-
two v
tests with 1 df, with a critical probability level of 0.0085,
according to the Dunn-Sidak correction of the standard probability
level. When conditions of application of the v
test were not fulfilled
according to the Cochran's rule, the Fisher's exact method was applied
(Sokal and Rohlf 1995).
Syrup Consumption
During the treatment period (i.e., 11 days) for the nine tested
pesticides, the volumes of syrup consumed for control (from
22.0 to 45.2 ll/bee/day) and pesticide-treated groups (from
23.6 to 44.7 ll/bee/day) are not significantly different
(ANOVA, 3 df, P > 0.05, in all cases). The geometrical pro-
gression of factor 2 between the different concentrations of
chemicals was respected on the whole. These results suggest
that the tested concentrations for all pesticides do not have
antifeedant effect on honeybees. The volumes of syrup con-
sumed are in agreement with the consumption initially esti-
mated (33 ll/bee/day; Decourtye et al. 2003). Consequently,
the quantities of chemicals actually ingested by bees are close
to wanted quantities.
Chronic Mortality
Cumulative mortality in bees significantly increases from that
of the control groups only with dimethoate and fipronil (Ta-
ble 2). A significant increase in mortality occurs with
dimethoate at concentration of 580 lg Æ L
(28% versus 9.3%
mortality after 11 days, in the treated and control groups,
respectively; v
= 9.3, 1 df, P = 0.002). The number of dead
A. Decourtye et al.
Page 3
bees in the groups exposed to fipronil at concentrations
ranging from 2.2 to 9 lg Æ L
(40.6–91.1% mortality) are
significantly different (v
,1df, P < 0.0083, in both cases) from
that of the control group (6.6% mortality). Consequently,
feeding honeybees with the sucrose solutions with added del-
tamethrin, prochloraz, endosulfan, k-cyalothrin, cypermethrin,
s-fluvalinate, or triazamate might be considered as sublethal,
contrary to fipronil and dimethoate treatments, which are le-
Reflex Response
The comparison of the number of reflex responses obtained
when the antennae were contacted with a sucrose solution, in
treated and control bees, was used to evaluate the effects of the
pesticides on the gustatory and motor functions of the PER. At
least 66% of bees show a clear PER. For all chemicals, the
same level of reflex response in treated and untreated bees is
found (v
,1df, P > 0.0083, in all cases; Table 2). This sug-
gests that the exposure to pesticides tested does not disrupt the
sensory and motor components controlling the PER.
Learning Performances
Table 3 shows the olfactory learning performances represented
as the percentage of conditioned PER obtained during the
training (Cond1–Cond3) and testing (Test1–Test5) phases, in
bees feeding the three concentrations of each pesticide and in
the control bees feeding only sucrose. Different letters indicate
significantly different response levels (v
test or Fishers exact
method, 1 df, P < 0.0083). The results for deltamethrin are
provided in Figure 1, as an illustrative example of the learning
curves that can be drawn in the PER assay.
The percentage of bees treated with the highest dose of
dimethoate (580 lg Æ L
) extending their proboscis in response
to the first presentation of odor (spontaneous responses ob-
served at Cond1) is significantly higher than is observed with
untreated bees (36% versus 6%; v
= 7.8, 1 df, P = 0.0052).
The feeding of workers with sucrose solution contaminated
with deltamethrin, prochloraz, endosulfan, or fipronil induces
significantly lower responses compared to the untreated bees,
considering Cond2–Cond3 for deltamethrin and Test4 for the
others pesticides. A reduction of the olfactory learning per-
formances is noted during conditioning trials in bees treated
with the highest concentration of deltamethrin (nominal and
actual concentrations of 940 and 960 lg Æ L
, respectively):
48% and 60% conditioned responses at Cond2 and Cond3,
respectively, versus 60% and 84% in the control (v
,1df, P<
0.0083, in both cases). At the testing trial Test4, lower levels
of responses are obtained with the highest dose of: prochloraz
(150 mg Æ L
), reaching 36% of conditioned responses versus
73% in the control group (v
= 8.2, 1 df, P = 0.0048); endo-
sulfan (8 mg Æ L
), reaching 6% of conditioned responses,
versus 45% in the control group (v
= 7.8, 1 df, P = 0.0037);
fipronil (4.5 lg Æ L
), reaching 7% of conditioned responses,
versus 56% in the control group (v
= 12.5, 1 df, P<0.001).
Conversely, for the above pesticides, no behavioral effect is
observed in the last training trial (Test5) (Table 3).
Table 2. Effects of subchronic exposures of nine agricultural chem-
icals on the survey and reflex responses of the honeybees
Chemical % Mortality % Reflex responses
940 lg Æ L
12.0 81.6
470 lg Æ L
10.6 86.7
235 lg Æ L
9.3 86.9
Control 18.0 90.4
300 98115
360 lg Æ L
15.8 (b)
180 lg Æ L
8.5 (ab) 84.2
90 lg Æ L
6.7 (a) 91.6
Control 10.3 (ab) 88.4
N 165 3438
690 lg Æ L
10.5 95.2
345 lg Æ L
13 96.8
172.5 lg Æ L
18.5 95.6
Control 15 95.6
N 200 4663
300 mg Æ L
4.2 70.7
150 mg Æ L
9.0 76.3
75 mg Æ L
10.3 76.7
Control 8.7 75.0
N 150 2843
150 mg Æ L
7.3 78.4
75 mg Æ L
4.3 79.7
37.5 mg Æ L
9.6 76.8
Control 8.6 78.2
N 210 3350
600 lg Æ L
2.0 88.2
300 lg Æ L
3.3 91.6
150 lg Æ L
7.3 91.6
Control 4.0 93.9
N 150 33-36
8mgÆ L
5.2 (a) 85.0
4mgÆ L
15.1 (b) 75.0
2mgÆ L
9.0 (ab) 67.5
Control 7.6 (ab) 71.4
N 150 3642
580 lg Æ L
28.0 (b) 73.1
290 lg Æ L
12.6 (a) 68.0
145 lg Æ L
13.3 (a) 66.0
Control 9.3 (a) 66.0
N 150 3944
9 lg Æ L
91.1 (c)
4.5 lg Æ L
87.3 (c) 85.0
2.2 lg Æ L
40.6 (b) 67.5
Control 6.6 (a) 66.6
N 150 1142
N, number of bees per treatment group.
For each chemical, the number of the cumulated mortality in treated
groups and in the control group were compared using v
test or
Fishers exact method with 1 df (P < 0.0083). Different letters indicate
significantly different response levels.
Toxicity of Pesticides on Olfactory Learning of Honeybee
Page 4
Table 3. Effects of subchronic exposures of nine agricultural chemicals on the learning performances of the honeybee
% Conditioned responses
Chemical Cond1 Cond2 Cond3 Test1 Test2 Test3 Test4 Test 5
940 lg Æ L
20.7 48.2 (b)
60.3 (b) 100
79.4 87.1 74.3 58.9
470 lg Æ L
19.3 68.4 (a) 71.9 (ab) 100 91.4 78.7 80.8 74.4
235 lg Æ L
6.7 55.9 (ab) 74.5 (ab) 100 86.0 79.0 74.4 74.4
Control 19.3 59.6 (a) 84.2 (a) 100 84.3 84.3 82.3 74.5
5759 4351
360 lg Æ L
7.1 60.7 75.0 100 75.0 50.0 43.8 18.8
180 lg Æ L
6.2 81.2 75.0 100 68.4 52.6 42.1 36.8
90 lg Æ L
0.0 81.8 78.7 100 56.0 48.0 32.0 12.0
Control 6.6 66.6 73.3 100 81.0 52.4 23.8 14.3
N2833 1625
690 lg Æ L
12.5 60.0 80.0 100 92.1 92.1 89.4 76.3
345 lg Æ L
30.0 85.0 92.5 100 88.2 76.4 76.4 70.5
172.5 lg Æ L
27.5 77.5 75.0 100 96.9 93.9 81.8 87.8
Control 20.0 75.0 82.5 100 97.1 94.2 91.4 82.8
N40 3338
300 mg Æ L
20.6 58.6 65.5 100 75.0 66.7 41.7 33.3
150 mg Æ L
10.3 68.9 75.8 100 100 55.6 50.0 33.3
75 mg Æ L
12.1 72.7 78.7 100 90.9 72.7 54.5 36.4
Control 9.5 71.4 71.4 100 63.6 45.5 27.3 9.1
N2133 1122
150 mg Æ L
22.8 80.0 88.5 100 87.8 63.6 36.3 (b) 33.3 (b)
75 mg Æ L
25.7 85.7 80.0 100 85.7 87.5 78.5 (a) 46.4 (ab)
37.5 mg Æ L
23.5 85.2 91.1 100 90.0 80.0 73.3 (a) 63.3 (ab)
Control 25.0 86.1 88.8 100 93.3 86.5 73.3 (a) 70.0 (b)
N3436 2833
600 lg Æ L
13.3 73.3 73.3 100 77.7 55.5 48.1 40.7
300 lg Æ L
12.1 75.7 72.7 100 80.7 50.0 46.1 26.9
150 lg Æ L
39.3 87.8 87.8 100 93.3 66.6 63.3 50.0
Control 9.6 80.6 80.6 100 73.0 53.8 38.4 26.9
N3033 2630
8mgÆ L
4.0 52.0 68.0 100 64.7 35.3 5.9 (b) 5.9
4mgÆ L
27.7 61.1 77.7 100 83.3 66.7 58.3 (a) 41.7
2mgÆ L
4.7 33.3 47.6 100 83.3 41.7 33.3 (ab) 16.7
Control 8.3 62.5 70.8 100 80.0 55.0 45.0 (a) 10.0
N1825 1220
580 lg Æ L
36.6 (a) 66.6 73.3 100 80.0 55.0 30.0 25.0
290 lg Æ L
34.3 (ab) 68.5 60.0 100 64.7 47.0 35.2 17.6
145 lg Æ L
31.2 (ab) 71.8 68.7 100 78.2 43.4 13.0 17.3
Control 6.0 (b) 69.7 72.7 100 88.8 62.9 33.3 18.5
N2633 1727
9 lg Æ L
4.5 lg Æ L
18.0 35.2 76.4 100 71.4 21.4 7.1 (b) 14.2
2.2 lg Æ L
14.8 51.8 66.6 100 68.1 50.0 27.2 (ab) 13.6
Control 10.7 46.4 64.2 100 68.7 56.2 56.2 (a) 31.2
N1728 1422
N, number of bees per treatment group.
For each chemical, the number of the conditioned responses in treated groups and those in the control group were compared using v
test or
Fishers exact method with 1 df (P < 0.0083). Different letters indicate significantly different response levels.
Positive responses at T1 are scaled to 100.
A. Decourtye et al.
Page 5
In all trials, the level of responses of bees exposed to
k-cyalothrin, cypermethrin, s-fluvalinate, and triazamate is
equivalent to that obtained with control bees (v
,1df, P >
0.0083, in all cases). For these four chemicals, 66–93% of
conditioned responses are obtained in treated bees at the last
conditioning trial (Cond3) and 71–83% in the untreated bees.
It is of interest to characterize honeybee behaviors that can be
routinely used as indicators of sublethal exposure to pesticides.
The possible long-term exposure to a toxic agent by contam-
ination of stored food has been established by studying the
transfer into the colony of pesticides sprayed on a crop (Fries
and Wibran 1987; Koch and Weisser 1997; Russel et al. 1998;
Villa et al. 2000). Thus, it is necessary to evaluate the viability
of worker bees newly involved in foraging duties based on
their learning ability, after being fed with a contaminated food
within the hive. The preconditioning treatment applied in the
present study leads to determining whether or not a pesticide
exposure applied prior to a learning task may affect the bees'
performances. Among the nine pesticides tested, only fipronil,
deltamethrin, endosulfan, and prochloraz yielded behavioral
effects during the PER assay. This is consistent with previous
works reporting that the PER assay was adapted to the
screening of the adverse effects of various pesticides to bees
(Taylor et al. 1987; Mamood and Waller 1990; Stone et al.
1997; Abramson et al. 1999; Abramson and Boyd 2001;
Weick and Thorn 2002; Decourtye et al. 2003; Abramson et
al. 2004). Conversely, our results clearly indicate that the
range of tested concentrations of k-cyalothrin, cypermethrin,
s-fluvalinate, and triazamate does not affect the learning per-
formances of bees. However, cypermethrin and s-fluvalinate
are less toxic to honeybees than k-cyalothrin and triazamate.
These results corroborate those of Taylor et al. (1987) showing
that among a set of six pyrethroids, cypermethrin and s-flu-
valinate yielded the least impact on the honeybee learning.
The originality of our approach consists in taking into ac-
count different concentrations in the PER assays. The deter-
mination of the threshold toxicity concentrations is also
possible. Thus, the no-observed-effect concentration (NOEC)
for the conditioned PER is set to 2.2 lg Æ L
, 470 lg Æ L
(actual concentration equals 429 lg Æ L
), 4 mg Æ L
, and
75 mg Æ L
for fipronil, deltamethrin, endosulfan, and pro-
chloraz, respectively. Considering the consumption of con-
taminated syrup and the number of bees, we can estimate that
the no-observed-effect dose of pesticide received per bee and
per day is 0.07 ng for fipronil (LD50 divided by 80), 15 ng for
deltamethrin (LD50 divided by 40), 125 ng for endosulfan
(LD50 divided by 40), and 2.5 lg for prochloraz (LD50 di-
vided by 40). Thus, fipronil is the most effective of the above
pesticides tested to induce learning performances impairment.
Under similar experimental conditions, the NOECs for imi-
dacloprid and hydroxy-imidacloprid were estimated to 6 and
60 lg Æ L
, corresponding to the DL50 value divided by 160
and 80, respectively (Decourtye et al. 2003). As regards k-
cyalothrin, cypermethrin, s-fluvalinate, and triazamate, we can
only say that the NOECs of these pesticides are superior to
360 lg Æ L
, 690 lg Æ L
(actual concentration of 782 lg Æ
), 300 mg Æ L
, and 600 lg Æ L
, respectively.
To evaluate the usefulness of PER as a measure for toxicity
assessment, it is necessary to compare these responses to
standard toxicity endpoints such as mortality. Learning per-
formances after treatment with the highest concentration of
deltamethrin, endosulfan, or prochloraz are decreased, in
contrast to survival, which is not affected. The NOEC of hy-
droxy-imidacloprid for the mortality was estimated to be
120 lg Æ L
, whilst the NOEC for the conditioned responses
was established at 60 lg Æ L
(Decourtye et al. 2003). From
this study, it appears that most often the impairments in
olfactory learning abilities are shown for chemical concen-
trations at which no additional mortality occurred.
The choice of sublethal concentrations of pesticides is a
crucial problem when an attempt is made to estimate the ef-
fects of pesticides on bee behaviors. In this study, for each
chemical, the highest tested dose was the 48-h oral LD50 value
Fig. 1. Learning performances of deltamethrin-treated bees during conditioning (A) and testing (B) procedures of PER assay
Toxicity of Pesticides on Olfactory Learning of Honeybee
Page 6
divided by 20. Considering the low mortality observed for
most of the tested pesticides, it appears that this choice was
In case of lethal treatment, the exposure to insecticide can
result in a selection of worker bees staying alive because they
are less sensitive to this pesticide than the other congeners.
Such tolerant bees can give an intact conditioned response
level in the PER assay. For example, bees treated with Decis
(0.5% a.i. deltamethrin) at a high dose exhibited similar pat-
tern of learning performances than control bees (Abramson
et al. 1999). In the current study, an adverse effect of
dimethoate at its highest concentration (580 lg Æ L
) is shown
on survival of honeybees, but not on their learning perfor-
mances. Previous studies have assessed the effect of chlor-
pyrifos ethyl, an organophosphorus insecticide like
dimethoate, on the behavior of parasitoids (Leptopilina het-
erotoma). Females of parasitoids were conditioned to associate
an odor with the oviposition in host larvae of Drosophila
(Rafalimanana et al. 2002). Parasitoids exposed to the LD20
value of chlorpyrifos oviposited the host larvae more quickly
than controls did. In our experiment, higher levels of sponta-
neous responses were obtained in bees treated with the highest
concentration of dimethoate (580 lg Æ L
). Thus, current re-
sults and those found in the literature suggest that the high
behavioral response levels in organophosphorus-treated insects
were probably linked to pharmacological action. These
chemicals act by inhibiting acetylcholinesterase and conse-
quently by prolonging activity of synapses (Padilla 1995). We
assume that the increase in spontaneous responses in dimeth-
oate-treated bees may be due to amplification of stimulus
perception or of response motricity. To confirm this hypoth-
esis, further experiments should be necessary. Works by
Abramson et al. (1999) should provide some insight to per-
form them.
The adverse effects are observed during a conditioning or
testing procedure according to the chemical tested. Ingestion
of deltamethrin significantly reduces the level of conditioned
responses in the conditioning procedure. This result suggests
an adverse effect of deltamethrin on the ability of treated
animals to learn the temporal relation between the uncondi-
tioned stimulus and the conditioned one. In addition to con-
ditioning procedure, the testing procedure points out the
resistance of bees to extinguish the response to a conditioned
stimulus no longer associated with a reward. Abramson et al.
(1999) reported that endosulfan tested at the concentration
recommended to control the cotton boll weevil influenced
extinction of the conditioned response. The authors suggested
that motor system disruption was responsible for this event
rather than an effect on the learning process. Our results
clearly indicate that endosulfan, as well as fipronil and pro-
chloraz, do not affect either the reflex response or the condi-
tioned response level in the conditioning procedure, but the
decrease of response level in the testing procedure occurs more
rapidly compared to the control group.
The conditioning and testing phases are two independent
processes that could be differentially affected by a toxic
exposure. This may rely on the fact that different steps of the
memorization are involved. If we refer to the model of
memory temporal schedule in the honeybee as described by
Menzel (1999), the conditioning covers the information stor-
age in the short-term memory, whilst long-term memory is
already established when the testing phase occurs. Delta-
methrin would affect the first step of information storage,
whereas endosulfan, fipronil, and prochloraz would interfere
with the retrieval process resulting in the capacity to restore
the conditioned response. However, further work is still nee-
ded to investigate more precisely the effects of these chemicals
on the different parameters of the memory (acquisition, re-
trieval, short-, medium- and long-term memory) during an
olfactory conditioning of the PER, as investigated with imi-
dacloprid (Decourtye et al. 2004b).
Besides behavioral effects of fipronil, an increase in the
mortality after 11 days appears in bees treated with this pes-
ticide. The lowest lethal dose of fipronil (0.1 ng per bee per
day corresponding to a concentration of 2.2 lg Æ L
times lower than the LD50 value. At the same time, a lethal
effect is significantly observed for bees exposed to the highest
dose of dimethoate (20 ng per bee per day corresponding to a
concentration of 580 lg Æ L
). Although the long-term lethal
effect of dimethoate was previously demonstrated (Waller et
al. 1984), we have determined for the first time the chronic
toxicity of fipronil to the honeybee. Using a similar laboratory
chronic oral test with bees fed with contaminated syrup,
chronic toxicity can be found even at low concentrations of
imidacloprid (Suchail et al. 2000; Decourtye et al. 2003; De-
chaume-Moncharmont et al. 2003). In chronic toxicity studies,
imidacloprid reacts at doses 60 to 6000 times lower than those
required to produce the same effect in acute intoxication
studies (Suchail et al. 2001). Thus, the acute toxicity tests,
performed according to the EPPO guidelines (EPPO 1993),
appear to give only a partial measure of the lethal effects
because of the short duration of these tests (1 to 3 days in most
cases). Now, when the acute lethal effect is not obvious,
additional testing could give information on the long-term
lethal effects possibly induced by the toxic, as that was proved
with dimethoate and fipronil.
Although spraying of dimethoate-based formulations is
prohibited on flowering crops, the field application of formu-
lations containing deltamethrin, fipronil, or imidacloprid is
allowed. In the current study, concentrations of 455 and
227.5 lg Æ L
of deltamethrin (actual concentrations of 429
and 212 lg Æ L
) were tested. They are realistic because
500 lg Æ L
corresponds to the maximum concentration
measured in oilseed rape flowers after spraying of De-
Micro (CETIOM unpublished data). We noted the absence
of lethal and behavioral effects after administration of these
concentrations. In an outdoor flight cage, representing more
realistic exposure conditions than those performed in a labo-
ratory, a sugar solution containing 500 lg Æ L
of delta-
methrin offered to a colony had no effects on an olfactory
learning discrimination task in free-flying foragers and in the
PER procedure of restrained individuals (Decourtye et al.
2004a). Thus, the intact learning performances in treated bees
at a realistic concentration of deltamethrin during a PER assay
are in agreement with those obtained in semifield conditions,
at the colony level. This suggests that in field conditions,
foraging bees could not suffer from behavioral effects of
deltamethrin after visiting of flowering crops treated with
Micro. Contrary to the current study, the impact of
deltamethrin has been shown on survival of worker bees in the
flight cage. This discrepancy might result in differences be-
tween the dose received per bee and per day. In the flight cage
A. Decourtye et al.
Page 7
study, we can estimate that the highest dose of deltamethrin
received per bee and per day was about equal to the LD50
value, while the value of 30 ng obtained in the current labo-
ratory study corresponds to the LD50 divided by 20.
Fipronil and imidacloprid, being the active ingredient of the
and Gaucho
formulations, respectively, are author-
ized as a sunflower seed coating. In France, imidacloprid and
fipronil were accused of being a cause for the decline of sun-
flower honey production. It is suspected that these products or
theirs metabolites could migrate into nectar or pollen of treated
sunflowers and induce deleterious effects in foraging bees after
ingestion of contaminated food. Despite the fact that several
semifield and field tests indicated that seed dressing with imi-
dacloprid posed no risk during sunflower flowering (Cur et al.
2000; Schmuck et al. 2001), a behavioral effect in the labora-
tory can be found at a concentration potentially encountered in
plant tissues (Decourtye et al. 2003). From 1999 onwards, the
French Ministry of Agriculture decided to suspend the regis-
tration of the seed treatment product Gaucho
in sunflowers
according to the precautionary principle. As with imidacloprid,
our study shows that an effect of fipronil can be observed on the
learning performances of bees in the range of 2.2 to 4.5 lg Æ
. Additional experiments are needed to establish the
threshold concentration of fipronil or of its metabolites from
which the forager bees could be exposed and possibly induces
drastic bee population losses, as observed by French beekeepers
in colonies foraging on sunflowers treated with Regent
Our application of the PER assay for pesticide toxicity
assessment led to characterization of effects on a behavioral
endpoint related to ability to associate an odor stimulus with
sucrose reward. In general, a detailed basic knowledge on
behavior relevant for honeybee assays in ecotoxicology is still
relatively scarce, and this is especially true for the influence of
principal general test variables on foraging behavior. The
crucial problem in behavioral toxicology is the lack of stan-
dardization for the tests. Therefore, the PER assay could be a
useful tool in the studies on behavioral effects of pesticides,
especially on foraging behavior, since it guarantees a good
control of bee-rearing conditions and of exposure to chemicals.
During bee-rearing under laboratory conditions, the quality of
the food and the olfactory environment of the individuals must
be strictly controlled because these factors can later influence
pesticide sensitivity (Wahl and Ulm 1983) and learning per-
formances in the PER assay (Sandoz et al. 2000), respectively.
Our study shows that the observations on conditioned PER
can be of interest in the assessment of behavioral effects of
pesticides to the honeybee, because this endpoint can be used
to compare different pesticides by accounting for various
concentrations for increasing the accuracy of the results and
for deriving NOECs. In a previous study, we showed that the
behavioral toxicity of imidacloprid observed in laboratory
conditions at the individual level (conditioned PER assay) was
consistent with results obtained in semifield experiments at the
colony level (Decourtye et al. 2004a). For the pesticides tested
in the current study, further studies are still needed to establish
similar relationships.
Acknowledgments. We wish to thank M. Charreton and B. Roger for
technical assistance in bee rearing. This work benefited from a grant
by the French Ministry of the Environment (MATE-01133-Evaluation
et rduction des risques lies l'utilisation des pesticides).
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  • Source
    • "The pollen paste was provided for 18 d. Proboscis extension response (PER) was the standard method used to assess effects of Cry toxin on honey bees learning performance (Decourtye et al. 2005, Ramirez-Romero et al. 2008). After 15 d of exposure to the diets, the bees were subjected to this test. "
    [Show abstract] [Hide abstract] ABSTRACT: The honey bee (Apis mellifera L.) is a key nontarget insect in environmental risk assessments of insect-resistant genetically modified crops. In controlled laboratory conditions, we evaluated the potential effects of Cry1Ie toxin on survival, pollen consumption, and olfactory learning of young adult honey bees. We exposed worker bees to syrup containing 20, 200, or 20,000 ng/ml Cry1Ie toxin, and also exposed some bees to 48 ng/ml imidacloprid as a positive control for exposure to a sublethal concentration of a toxic product. Results suggested that Cry1Ie toxin carries no risk to survival, pollen consumption, or learning capabilities of young adult honey bees. However, during oral exposure to the imidacloprid treatments, honey bee learning behavior was affected and bees consumed significantly less pollen than the control and Cry1Ie groups.
    Full-text · Article · Apr 2016 · Journal of Economic Entomology
  • Source
    • "Neonicotinoids act on the insect nervous system through agonistic actions on nicotinic acetylcholine receptors (Tan et al., 2007). For European honeybees, the LD50 for oral administration of thiamethoxam over 48 h is 5 ng, and LD50 for contact administration over 24 h is 29 ng (Decourtye et al., 2005). Furthermore, the LD50 of thiamethoxam for newly emerged AHBs is 4.28 ng bee À1 (Oliveira et al., 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: Multiple stressors, such as chemicals and pathogens, are likely to be detrimental for the health and lifespan of Apis mellifera, a bee species frequently exposed to both factors in the field and inside hives. The main objective of the present study was to evaluate comparatively the health of Carniolan and Africanized honey bees (AHB) co-exposed to thiamethoxam and Nosema ceranae (N. ceranae) spores. Newly-emerged worker honey bees were exposed solely with different sublethal doses of thiamethoxam (2% and 0.2% of LD50 for AHB), which could be consumed by bees under field conditions. Toxicity tests for the Carniolan bees were performed, and the LD50 of thiamethoxam for Carniolan honey bees was 7.86 ng bee−1. Immunohistological analyses were also performed to detect cell death in the midgut of thiamethoxam and/or N. ceranae treated bees. Thiamethoxam exposure had no negative impact on Nosema development in experimental conditions, but it clearly inhibited cell death in the midgut of thiamethoxam and Nosema-exposed bees, as demonstrated by immunohistochemical data. Indeed, thiamethoxam exposure only had a minor synergistic toxic effect on midgut tissue when applied as a low dose simultaneously with N. ceranae to AHB and Carniolan honey bees, in comparison with the effect caused by both stressors separately. Our data provides insights into the effects of the neonicotenoid thiamethoxam on the AHB and Carniolan honey bee life span, as well as the effects of simultaneous application of thiamethoxam and N. ceranae spores to honey bees.
    Full-text · Article · Mar 2016 · Chemosphere
  • Source
    • "The odors applied in appetitive PER conditioning are generally floral-like, such as hexanal or nonanal [12]. However, training can also be carried out successfully with non-floral odors such as the bee´s Nasonov pheromone components geraniol and citral [13,14] and some pesti- cides151617. For example, A. mellifera can be trained to respond with proboscis extension even when the learned odor is the sting pheromone, indicating that workers can override their innate response to sting pheromone and learn to associate this odor with a food reward [18,19]. "
    [Show abstract] [Hide abstract] ABSTRACT: In Southeast Asia the native honey bee species Apis cerana is often attacked by hornets (Vespa velutina), mainly in the period from April to November. During the co-evolution of these two species honey bees have developed several strategies to defend themselves such as learning the odors of hornets and releasing alarm components to inform other mates. However, so far little is known about whether and how honey bees modulate their olfactory learning in the presence of the hornet predator and alarm components of honey bee itself. In the present study, we test for associative olfactory learning of A. cerana in the presence of predator odors, the alarm pheromone component isopentyl acetate (IPA), or a floral odor (hexanal) as a control. The results show that bees can detect live hornet odors, that there is almost no association between the innately aversive hornet odor and the appetitive stimulus sucrose, and that IPA is less well associated with an appetitive stimulus when compared with a floral odor. In order to imitate natural conditions, e.g. when bees are foraging on flowers and a predator shows up, or alarm pheromone is released by a captured mate, we tested combinations of the hornet odor and floral odor, or IPA and floral odor. Both of these combinations led to reduced learning scores. This study aims to contribute to a better understanding of the prey-predator system between A. cerana and V. velutina.
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