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Arch. Environ. Contam. Toxicol. 48, 242–250 (2005)DOI: 10.1007/s00244-003-0262-7 Comparative Sublethal Toxicity of Nine Pesticides on Olfactory LearningPerformances of the Honeybee Apis mellifera A. Decourtye,1 J. Devillers,2 E. Genecque,3 K. Le Menach,4 H. Budzinski,4 S. Cluzeau,1 M. H. Pham-Del›gue3 1 Association de Coordination Technique Agricole, Maison des Agriculteurs, La Tour de Salvagny, France2 CTIS, Rillieux La Pape, France3 Laboratoire de Neurobiologie ComparØe des InvertØbrØs, INRA, Bures-sur-Yvette, France4 Laboratoire de Physico-Toxico-Chimie des Syst›mes Naturels, UMR 5472 CNRS, UniversitØ de Bordeaux I, Talence Cedex, France Received: 4 January 2004 /Accepted: 23 June 2004 Abstract. Using a conditioned proboscis extension response (Waller et al. 1984; Bendahou et al. 1999; Decourtye et al.
(PER) assay, honeybees (Apis mellifera L.) can be trainedto 2004a), such effects also couldbe usedto better estimate the associate an odor stimulus with a sucrose reward. Previous hazardof pesticides to bees. Moreover, it is noteworthy that studies have shown that observations of conditioned PER were the EPPO guidelines require recording all abnormal behavioral of interest for assessing the behavioral effects of pesticides on effects observedduring the experiments (EPPO 1992).
the honeybee. In the present study, the effects of sublethal Semifieldtests, representing more realistic exposure con- concentrations of nine pesticides on learning performances of ditions than in laboratory, have been cited as providing good worker bees subjectedto the PER assay were estimatedand information for the behavioral toxicity assessment of pesti- compared. Pesticides were tested at three concentrations. The cides (Cluzeau 2002). However, the regulatory guidelines give highest concentration of each pesticide corresponded to the only very limitedinformation on the type of behavioral data median lethal dose value (48-h oral LD50), received per bee that should be collected during the studies or how they should and per day, divided by 20. Reduced learning performances be included and interpreted in the risk assessment scheme were observedfor bees surviving treatment with fipronil, (Thompson andBrobyn 2002). Moreover, the semifieldtests, deltamethrin, endosulfan, and prochloraz. A lack of behavioral even if they are well suited, are technically difficult to main- effects after treatment with k-cyalothrin, cypermethrin, s-flu- valinate, triazamate, and dimethoate was recorded. No-ob- necessity to have trainedpeople to carry them out are bounds served-effect concentrations (NOECs) for the conditioned PER limiting the number of facilities able to perform them in were derived for the studied pesticides. Our study shows that practice. Thus, the identification of precise behavioral effects the PER assay can be usedfor estimating sublethal effects of requires additional and specific methods to make appropriate pesticides on bees. Furthermore, comparisons of sensitivity as hazardassessment (Pham-Del›gue et al. 2002). Consequently, well as the estimation of NOECs, useful for regulatory pur- the conditioned proboscis extension response (PER) assay shouldbe use to overcome these problems (Decourtye andPham-Del›gue 2002).
The PER assay tentatively reproduces what happens in honeybee–plant interaction: when landing on the flower, theforager extends its proboscis as a reflex when the gustatory The hazardassessment of pesticide toxicity to honeybees (Apis receptors set on the tarsae, antennae, or mouth parts are mellifera L.) is commonly estimatedfrom laboratory studies stimulatedwith nectar. This reflex leads to the uptake of nectar (median lethal dose: LD50) and from semifield and field and induces the memorization of the floral odors diffusing experimentations when the pesticides demonstrate a hazard concomitantly. Once memorized, the odors play a prominent quotient (application rate/LD50) over 50, or when they have a role in flower recognition during the next trips (Menzel et al.
specific mode of action (e.g., insect growth regulators), or 1993). Consequently, an individual associative learning pro- when there are indications of indirect effects such as delayed cess is important for the effective accomplishment of foraging action (EPPO 1992). Because behavioral effects of pesticides activities. The associative learning of workers, investigated in the honeybee have been shown to have the potential to with the PER assay, may therefore be regarded as having a induce a significant impact on the development of colonies high ecological significance because it is a prerequisite to theforaging success of the whole colony.
The PER has been successfully reproduced under artificial conditions (Kuwabara 1957; Takeda 1961), and has become a Correspondence to: A. Decourtye; email: axel.decourtye@acta.asso.fr valuable tool in studying various aspects of olfactory learning Toxicity of Pesticides on Olfactory Learning of Honeybee Table 1. Concentrations of agricultural chemicals applied with subchronic exposure before the conditioning procedure a Actual concentrations of deltamethrin equal to 960, 429, and 212 lg Æ L)1.
b Actual concentrations of cypermethrin equal to 782, 388, and207 lg Æ L)1.
processes (Bitterman et al. 1983; Menzel et al. 1993; Sandoz To confirm the usefulness of the PER assay as a behavioral et al. 1995). The PER assay with restrainedworkers has also toxicity assessment method, the goal of our study was to been usedto investigate the behavioral effects of pesticides compare the effects of sublethal exposure of nine pesticides on (Taylor et al. 1987; MamoodandWaller 1990; Stone et al.
the olfactory learning performances of worker bees subjected 1997; Abramson et al. 1999; Abramson andBoyd2001; Weick andThorn 2002; Decourtye et al. 2003; Abramsonet al. 2004).
A previous work studying the behavioral toxicity of imi- dacloprid and deltamethrin to bees indicated that a goodrelationship was foundbetween effects on olfactory responsesin free-flying foragers and in individuals subjected to the PER paradigm (Decourtye et al. 2004a). The controlledconditions,the relationship with fieldcond The nine studied pesticides (Table 1) were all technical grade. Del- quantify the behavior pattern numerically ledus to assume that tamethrin andprochloraz were obtainedfrom Hoechst Schering Ag- the use of the PER assay, as a methodto evaluate the potential rEvo S.A. (Aventis CropScience, France). All the other compounds effect of pesticides on the honeybees foraging behavior, could were purchasedfrom Cluzeau Info Labo (Sainte-Foy-La-Grand help us to assess the toxicity of pesticides in a more compre- France). Their purity was at least 98%, except s-fluvalinate, which hensive way than by only considering lethality as currently made in practice (Devillers 2002). However, a survey of the The pesticides were tested at three different concentrations, with a geometrical progression of factor 2. The highest testedconcentration literature showedthat only a limitednumber of chemicals had corresponded to the median lethal dose value (LD50 determined 48 h been tested, and the studies using the PER assay were usually after the oral treatments) divided by 20 (Table 1). From previous not directly comparable because they were based on different results (Decourtye et al. 2003), it was assumedthat this ratio belonged methods for the administration of chemicals, the behavioral to a sublethal domain. The 48-h LD50s reported in Table 1 were response, andso on. Moreover, in these works only one dose, previously determined from acute oral toxicity tests for deltamethrin, not necessarily sublethal, was generally tested.
k-cyalothrin, andfipronil (Decourtye 2002), andfrom information gainedin the existing literature for the other chemicals. The con- was elicitedafter 3 s by contacting the antennae with a sucrose centrations were calculatedfor a consumption of syrup estimatedto solution (300 g Æ L)1) as the unconditioned stimulus, and the same 33 ll/bee/day (Decourtye et al. 2003).
solution was immediately given as a reward, before the odor delivery Stock solutions with a given concentration of each chemical were ended. Three successive conditioning trials (Cond1–Cond3) were preparedin acetone (Table 1). Acetone was chosen following the carriedout, followedby five test trials (Test1–Test5). The time EPPO guidelines, because it is a rather generalist solvent (EPPO interval between trials was 20–30 min. Conveniently, the positive 1992). Aliquots of the stock solutions were usedto make each test responses at T1 of the individuals are scaled to 100 in order to better solution at a specific concentration. The chemicals were added to a characterize the extinction slope. During a test trial, the conditioned 500 g L-1sucrose solution. The final concentration of acetone in the stimulus (pure linalool) was delivered for 6 s. The conditioned PER sucrose solutions was 1% (vol/vol). The effects of insecticide-added was recorded as a yes-or-no response (i.e., 0 or 1) when the odor alone solutions were comparedwith that of an untreatedsucrose solution was delivered during the 6 s of the test trial.
(with 1% acetone vol/vol). Fresh dosing solutions were prepared foreach test.
Samples of contaminatedsucrose solutions of d cypermethrin delivered to bees were analyzed by gas chromatography/ mass spectrometry (K. Le Menach and H. Budzinski, unpublished).
For each chemical, the mortality accumulatedover 11 days of expo-sure was comparedbetween each concentration andthe control bymultiple two-by-two v2 tests with 1 df. To ensure that the experiment error rate was a = 0.05, each comparison was carriedout according tothe Dunn-Sidak method (Sokal and Rohlf 1995) at a critical proba-bility of a' = 1 – (1 – a)1/k, where k was the number of intended tests.
Experiments were carriedout with worker bees of Apis mellifera The significance level was 0.0085 for two-by-two comparisons of the ligustica L. They were conducted with bees collected from outdoor responses to three concentrations of each chemical andone control hives. Emerging worker bees were caged in groups of 60 individuals.
They were provided with sugar food (mixture of sugar and honey),andwater ad libitum during the 2 first days and with pollen for the The number of initial reflex responses andthe number of condi- tionedresponses in each trial were comparedbetween the three next 8 days. After 2 days, bees were continuously fed with sucrose concentrations of each chemical andthe control by multiple two-by- solution contaminated or not during 11 consecutive days. The feederswere changeddaily with fresh sucrose solutions. The bees were kept two v2 tests with 1 df, with a critical probability level of 0.0085,according to the Dunn-Sidak correction of the standard probability in an incubator (33 € 2°C, 40 € 10% relative humidity, darkness) level. When conditions of application of the v2 test were not fulfilled until 14–15 days old, and were used in the PER assay. It has been according to the Cochran's rule, the Fisher's exact method was applied shown that on average, worker bees become foragers at that age(Sakagami 1953; Seeley 1982) andgive the most consistent perfor- mances in the conditioned proboscis extension assay (Pham-Del›gueet al. 1990).
For bees from 2 to 14–15 days old, the quantity of the contaminated During the treatment period(i.e., 11 days) for the nine tested sugar solution provided daily was adjusted to the number of survivors.
pesticides, the volumes of syrup consumed for control (from The mortality and consumption of syrup were recorded daily, and 22.0 to 45.2 ll/bee/day) and pesticide-treated groups (from dead bees were discarded. Every testing day was organized as follows: 23.6 to 44.7 ll/bee/day) are not significantly different bees previously exposedto three concentrations of each chemical (ANOVA, 3 df, P > 0.05, in all cases). The geometrical pro- were tested, as well as untreated control bees, leading to a total of 60– gression of factor 2 between the different concentrations of 80 bees testedper day, with 16–20 bees for each treatment. Experi-ments were replicatedat least three times, until about 50–60 bees per chemicals was respectedon the whole. These results suggest that the tested concentrations for all pesticides do not have After treatment, the bees were mounted individually in glass tubes antifeedant effect on honeybees. The volumes of syrup con- with only their antennae andmouth parts left free. They were starved sumedare in agreement with the consumption initially esti- for 4 h prior to conditioning. They were selected for showing a pro- mated(33 ll/bee/day; Decourtye et al. 2003). Consequently, boscis extension reflex after stimulation of the antennae with a su- the quantities of chemicals actually ingestedby bees are close crose solution (300 g Æ L)1). 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 thePER. The general stimulation conditions as well as the conditioningand testing procedures were adapted from the work of Bitterman et al.
(1983) and are detailed in Sandoz et al. (1995). Bees were then placedin an airflow (main airflow of 50 ml Æ s)1 added to a secondary airflow Cumulative mortality in bees significantly increases from that of 2.5 ml Æ s)1) for 15 s, to be familiarizedwith the mechanical of the control groups only with dimethoate and fipronil (Ta- stimulation and with the experimental background. For the condi-tioning trials, the conditioned stimulus (10 ll of pure linalool, a ble 2). A significant increase in mortality occurs with standard floral odor, soaked on a filter paper strip inserted in a Pasteur dimethoate at concentration of 580 lg Æ L)1 (28% versus 9.3% pipette cartridge; Sigma, 95–97% purity) was delivered through the mortality after 11 days, in the treated and control groups, secondary flow (2.5 ml Æ s)1) for 6 s. During odor delivery, the PER respectively; v2 = 9.3, 1 df, P = 0.002). The number of dead Toxicity of Pesticides on Olfactory Learning of Honeybee Table 2. Effects of subchronic exposures of nine agricultural chem- bees in the groups exposedto fipronil at concentrations icals on the survey andreflex responses of the honeybees ranging from 2.2 to 9 lg Æ L)1 (40.6–91.1% mortality) aresignificantly different (v2, 1 df, 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 anddimethoate treatments, which are le- The comparison of the number of reflex responses obtained when the antennae were contactedwith a sucrose solution, in treatedandcontrol bees, was usedto 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 treatedanduntreatedbees is found(v2, 1 df, P > 0.0083, in all cases; Table 2). This sug- gests that the exposure to pesticides tested does not disrupt the sensory andmotor components controlling the PER.
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 (v2 test or FisherÕs 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 treatedwith the highest d dimethoate (580 lg Æ L)1) extending their proboscis in response to the first presentation of odor (spontaneous responses ob- servedat Cond1) is significantly higher than is observedwith untreatedbees (36% versus 6%; v2 = 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 comparedto the untreatedbees, 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 and960 lg Æ L)1, respectively): 48% and60% conditioned responses at Cond2 and Cond3, respectively, versus 60% and84% in the control (v2, 1 df, P < 0.0083, in both cases). At the testing trial Test4, lower levels of responses are obtainedwith the highest dose of: prochloraz (150 mg Æ L)1), reaching 36% of conditioned responses versus N, number of bees per treatment group.
73% in the control group (v2 = 8.2, 1 df, P = 0.0048); endo- For each chemical, the number of the cumulatedmortality in treated sulfan (8 mg Æ L)1), reaching 6% of conditioned responses, groups andin the control group were comparedusing v2 test or versus 45% in the control group (v2 = 7.8, 1 df, P = 0.0037); FisherÕs exact methodwith 1 df (P < 0.0083). Different letters indicatesignificantly different response levels.
fipronil (4.5 lg Æ L)1), reaching 7% of conditioned responses,versus 56% in the control group (v2 = 12.5, 1 df, P < 0.001).
Conversely, for the above pesticides, no behavioral effect isobservedin the last training trial (Test5) (Table 3).
Table 3. Effects of subchronic exposures of nine agricultural chemicals on the learning performances of the honeybee a N, number of bees per treatment group.
b For each chemical, the number of the conditioned responses in treated groups and those in the control group were compared using v2 test orFisherÕs exact methodwith 1 df (P < 0.0083). Different letters indicate significantly different response levels.
c Positive responses at T1 are scaledto 100.
Toxicity of Pesticides on Olfactory Learning of Honeybee Fig. 1. Learning performances of deltamethrin-treated bees during conditioning (A) and testing (B) procedures of PER assay In all trials, the level of responses of bees exposedto The originality of our approach consists in taking into ac- k-cyalothrin, cypermethrin, s-fluvalinate, andtriazamate is count different concentrations in the PER assays. The deter- equivalent to that obtainedwith control bees (v2, 1 df, P > mination of the thresholdtoxicity concentrations is also 0.0083, in all cases). For these four chemicals, 66–93% of possible. Thus, the no-observed-effect concentration (NOEC) conditioned responses are obtained in treated bees at the last for the conditioned PER is set to 2.2 lg Æ L)1, 470 lg Æ L)1 conditioning trial (Cond3) and 71–83% in the untreatedbees.
(actual concentration equals 429 lg Æ L)1), 4 mg Æ L)1, and75 mg Æ L)1 for fipronil, deltamethrin, endosulfan, and pro-chloraz, respectively. Considering the consumption of con-taminatedsyrup andthe number of bees, we can estimate that the no-observed-effect dose of pesticide received per bee andper day is 0.07 ng for fipronil (LD50 divided by 80), 15 ng for It is of interest to characterize honeybee behaviors that can be deltamethrin (LD50 divided by 40), 125 ng for endosulfan routinely used as indicators of sublethal exposure to pesticides.
(LD50 divided by 40), and 2.5 lg for prochloraz (LD50 di- The possible long-term exposure to a toxic agent by contam- vided by 40). Thus, fipronil is the most effective of the above ination of storedfoodhas been establishedby studying the pesticides tested to induce learning performances impairment.
transfer into the colony of pesticides sprayed on a crop (Fries Under similar experimental conditions, the NOECs for imi- andWibran 1987; Koch andWeisser 1997; Russel et al. 1998; dacloprid and hydroxy-imidacloprid were estimated to 6 and Villa et al. 2000). Thus, it is necessary to evaluate the viability 60 lg Æ L)1, corresponding to the DL50 value divided by 160 of worker bees newly involvedin foraging duties basedon and80, respectively (Decourtye et al. 2003). As regards k- their learning ability, after being fedwith a contaminatedfood cyalothrin, cypermethrin, s-fluvalinate, andtriazamate, we can within the hive. The preconditioning treatment applied in the only say that the NOECs of these pesticides are superior to present study leads to determining whether or not a pesticide 360 lg Æ L)1, 690 lg Æ L)1 (actual concentration of 782 lg Æ exposure appliedprior to a learning task may affect the bees' L)1), 300 mg Æ L)1, and600 lg Æ L)1, respectively.
performances. Among the nine pesticides tested, only fipronil, To evaluate the usefulness of PER as a measure for toxicity deltamethrin, endosulfan, and prochloraz yielded behavioral assessment, it is necessary to compare these responses to effects during the PER assay. This is consistent with previous standard toxicity endpoints such as mortality. Learning per- works reporting that the PER assay was adapted to the formances after treatment with the highest concentration of screening of the adverse effects of various pesticides to bees deltamethrin, endosulfan, or prochloraz are decreased, in (Taylor et al. 1987; MamoodandWaller 1990; Stone et al.
contrast to survival, which is not affected. The NOEC of hy- 1997; Abramson et al. 1999; Abramson andBoyd2001; droxy-imidacloprid for the mortality was estimated to be Weick andThorn 2002; Decourtye et al. 2003; Abramson et 120 lg Æ L)1, whilst the NOEC for the conditioned responses al. 2004). Conversely, our results clearly indicate that the was establishedat 60 lg Æ L)1 (Decourtye et al. 2003). From range of testedconcentrations of k-cyalothrin, cypermethrin, this study, it appears that most often the impairments in s-fluvalinate, andtriazamate does not affect the learning per- olfactory learning abilities are shown for chemical concen- formances of bees. However, cypermethrin and s-fluvalinate trations at which no additional mortality occurred.
are less toxic to honeybees than k-cyalothrin andtriazamate.
The choice of sublethal concentrations of pesticides is a These results corroborate those of Taylor et al. (1987) showing crucial problem when an attempt is made to estimate the ef- that among a set of six pyrethroids, cypermethrin and s-flu- fects of pesticides on bee behaviors. In this study, for each valinate yielded the least impact on the honeybee learning.
chemical, the highest testeddose was the 48-h oral LD50 value divided by 20. Considering the low mortality observed for already established when the testing phase occurs. Delta- most of the testedpesticides, it appears that this choice was methrin wouldaffect the first step of information storage, whereas endosulfan, fipronil, and prochloraz would interfere In case of lethal treatment, the exposure to insecticide can with the retrieval process resulting in the capacity to restore result in a selection of worker bees staying alive because they the conditioned response. However, further work is still nee- are less sensitive to this pesticide than the other congeners.
ded to investigate more precisely the effects of these chemicals Such tolerant bees can give an intact conditioned response on the different parameters of the memory (acquisition, re- level in the PER assay. For example, bees treatedwith DecisÒ trieval, short-, medium- and long-term memory) during an (0.5% a.i. deltamethrin) at a high dose exhibited similar pat- olfactory conditioning of the PER, as investigated with imi- tern of learning performances than control bees (Abramson et al. 1999). In the current study, an adverse effect of Besides behavioral effects of fipronil, an increase in the dimethoate at its highest concentration (580 lg Æ L)1) is shown mortality after 11 days appears in bees treated with this pes- on survival of honeybees, but not on their learning perfor- ticide. The lowest lethal dose of fipronil (0.1 ng per bee per mances. Previous studies have assessed the effect of chlor- day corresponding to a concentration of 2.2 lg Æ L)1) is 60 times lower than the LD50 value. At the same time, a lethal dimethoate, on the behavior of parasitoids (Leptopilina het- effect is significantly observedfor bees exposedto the highest erotoma). Females of parasitoids were conditioned to associate dose of dimethoate (20 ng per bee per day corresponding to a an odor with the oviposition in host larvae of Drosophila concentration of 580 lg Æ L)1). Although the long-term lethal (Rafalimanana et al. 2002). Parasitoids exposed to the LD20 effect of dimethoate was previously demonstrated (Waller et value of chlorpyrifos ovipositedthe host larvae more quickly al. 1984), we have determined for the first time the chronic than controls did. In our experiment, higher levels of sponta- toxicity of fipronil to the honeybee. Using a similar laboratory neous responses were obtainedin bees treatedwith the highest chronic oral test with bees fedwith contaminatedsyrup, concentration of dimethoate (580 lg Æ L)1). Thus, current re- chronic toxicity can be foundeven at low concentrations of sults andthose foundin the literature suggest that the high imidacloprid (Suchail et al. 2000; Decourtye et al. 2003; De- behavioral response levels in organophosphorus-treatedinsects chaume-Moncharmont et al. 2003). In chronic toxicity studies, were probably linkedto pharmacological action. These imidacloprid reacts at doses 60 to 6000 times lower than those chemicals act by inhibiting acetylcholinesterase andconse- uce the same effect in acute intoxication quently by prolonging activity of synapses (Padilla 1995). We studies (Suchail et al. 2001). Thus, the acute toxicity tests, assume that the increase in spontaneous responses in dimeth- performed according to the EPPO guidelines (EPPO 1993), appear to give only a partial measure of the lethal effects perception or of response motricity. To confirm this hypoth- because of the short duration of these tests (1 to 3 days in most esis, further experiments shouldbe necessary. Works by cases). Now, when the acute lethal effect is not obvious, Abramson et al. (1999) shouldprovide some insight to per- additional testing could give information on the long-term lethal effects possibly induced by the toxic, as that was proved The adverse effects are observed during a conditioning or testing procedure according to the chemical tested. Ingestion Although spraying of dimethoate-based formulations is of deltamethrin significantly reduces the level of conditioned prohibitedon flowering crops, the fieldapplication of formu- responses in the conditioning procedure. This result suggests lations containing deltamethrin, fipronil, or imidacloprid is an adverse effect of deltamethrin on the ability of treated allowed. In the current study, concentrations of 455 and animals to learn the temporal relation between the uncondi- 227.5 lg Æ L)1 of deltamethrin (actual concentrations of 429 tioned stimulus and the conditioned one. In addition to con- and212 lg Æ L)1) were tested. They are realistic because ditioning procedure, the testing procedure points out the 500 lg Æ L)1 corresponds to the maximum concentration resistance of bees to extinguish the response to a conditioned measuredin oilseedrape flowers after spraying of De- stimulus no longer associatedwith a reward. Abramson et al.
cisÒMicro (CETIOM unpublisheddata). We notedthe absence of lethal andbehavioral effects after administration of these recommended to control the cotton boll weevil influenced concentrations. In an outdoor flight cage, representing more extinction of the conditioned response. The authors suggested realistic exposure conditions than those performed in a labo- that motor system disruption was responsible for this event ratory, a sugar solution containing 500 lg Æ L)1 of delta- rather than an effect on the learning process. Our results methrin offeredto a colony hadno effects on an olfactory clearly indicate that endosulfan, as well as fipronil and pro- learning discrimination task in free-flying foragers and in the chloraz, do not affect either the reflex response or the condi- PER procedure of restrained individuals (Decourtye et al.
tioned response level in the conditioning procedure, but the 2004a). Thus, the intact learning performances in treatedbees decrease of response level in the testing procedure occurs more at a realistic concentration of deltamethrin during a PER assay rapidly compared to the control group.
are in agreement with those obtainedin semifieldconditions, The conditioning and testing phases are two independent at the colony level. This suggests that in fieldcond foraging bees couldnot suffer from behavioral effects of exposure. This may rely on the fact that different steps of the deltamethrin after visiting of flowering crops treated with memorization are involved. If we refer to the model of DecisÒMicro. Contrary to the current study, the impact of memory temporal schedule in the honeybee as described by deltamethrin has been shown on survival of worker bees in the Menzel (1999), the conditioning covers the information stor- flight cage. This discrepancy might result in differences be- age in the short-term memory, whilst long-term memory is tween the dose received per bee and per day. In the flight cage Toxicity of Pesticides on Olfactory Learning of Honeybee study, we can estimate that the highest dose of deltamethrin by the French Ministry of the Environment (MATE-01133-Evaluation receivedper bee andper day was about equal to the LD50 et rØduction des risques lies à l'utilisation des pesticides).
value, while the value of 30 ng obtainedin the current labo-ratory study corresponds to the LD50 divided by 20.
Fipronil and imidacloprid, being the active ingredient of the RegentÒ andGauchoÒ formulations, respectively, are author-izedas a sunflower seedcoating. In France, imidaclopridand Abramson CI, Aquino IS, Ramalho FS, Price JM (1999) The effect of fipronil were accusedof being a cause for the decline of sun- insecticides on learning in the Africanized honey bee (Apis mel- flower honey production. It is suspected that these products or lifera L.). Arch Environ Contam Toxicol 37:529–535 theirs metabolites couldmigrate into nectar or pollen of treated Abramson CI, BoydBJ (2001) An automatedapparatus for condi- sunflowers and induce deleterious effects in foraging bees after tioning proboscis extension in honey bees (Apis mellifera L.).
ingestion of contaminatedfood. Despite the fact that several Abramson CI, Squire J, Sheridan A, Mulder PG Jr (2004) The effect semifield and field tests indicated that seed dressing with imi- of insecticides considered harmless to honey bees (Apis mellifera dacloprid posed no risk during sunflower flowering (CurØ et al.
L.): Proboscis conditioning studies using the insect growth reg- 2000; Schmuck et al. 2001), a behavioral effect in the labora- ulators ConfirmÒ2F (Tebufenozide) and DimilinÒ2L (Dif- tory can be foundat a concentration potentially encounteredin lubenzuron). Environ Entomol 33:378–388 plant tissues (Decourtye et al. 2003). From 1999 onwards, the Barnavon M (1987) ExpØrimentation en laboratoire et en plein champ French Ministry of Agriculture decided to suspend the regis- du fluvalinate. Principes pour un insecticide. DØfense VØgØtaux tration of the seedtreatment product GauchoÒ in sunflowers according to the precautionary principle. As with imidacloprid, Bendahou N, FlØchØ C, Bounias M (1999) Biological and biochemical our study shows that an effect of fipronil can be observed on the effects of chronic exposure to very low levels of dietary cyper- learning performances of bees in the range of 2.2 to 4.5 lg Æ methrin (Cymbush) on honeybee colonies (Hymenoptera: Api-dae). Ecotoxicol Environ Saf 44:147–153 L)1. Additional experiments are needed to establish the Bitterman ME, Menzel R, Fietz A, Schäfer S (1983) Classical con- thresholdconcentration of fipronil or of its metabolites from ditioning proboscis extension in honeybees (Apis mellifera).
which the forager bees couldbe exposedandpossibly induces drastic bee population losses, as observed by French beekeepers Chalvet-Monfrey K (1996) Synergie entre la deltamØthrine et le in colonies foraging on sunflowers treatedwith RegentÒ.
prochloraze chez l'abeille (Apis mellifera L.): Hypoth›ses de Our application of the PER assay for pesticide toxicity mØcanismes dÕaction testØes par modØlisation. PhD Thesis, Uni- assessment ledto characterization of effects on a behavioral endpoint related to ability to associate an odor stimulus with Cluzeau S (2002) Risk assessment of plant protection products on sucrose reward. In general, a detailed basic knowledge on honey bees. In: Devillers J, Pham-Del›gue MH (eds) Honey bees: behavior relevant for honeybee assays in ecotoxicology is still estimating the environmental impact of chemicals. Taylor andFrancis, London, pp 42–55 relatively scarce, andthis is especially true for the influence of CurØ G, Schmidt HW, Schmuck R (2000) Results of a comprehensive principal general test variables on foraging behavior. The field research programme with the systemic insecticide imida- crucial problem in behavioral toxicology is the lack of stan- cloprid(Gaucho). In: Belzunces LP, PØlissier C, Lewis GB (eds) dardization for the tests. Therefore, the PER assay could be a Hazards of pesticides to bees. INRA Edition, Avignon, pp 49–59 useful tool in the studies on behavioral effects of pesticides, Dechaume-Moncharmont FX, Decourtye A, Hennequet C, Pham- especially on foraging behavior, since it guarantees a good Del›gue MH, Pons O (2003) Statistical analysis of the honeybee control of bee-rearing conditions and of exposure to chemicals.
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