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Effects of indirect contact with neonicotinoids and fipronil contaminated dusts laboratory study

In this study, the toxicity of the seed dressing formulations Poncho® (clothianidin), Gaucho® (imidacloprid), Cruiser® (thiamethoxam) and Regent® (fipronil) was assessed on forager bees. In order to do that, we considered the concentration of those active ingredients measured by CRA-ING and CRA-PAV in the experimentations carried out in 2010 (ApeNet, 2010, 2011). The a.i. concentrations found at a distance of 5 meters from the edge of the experimental field sowed with treated maize seeds, was chosen for our trials. Starting from this concentrations, we considered also treatments with 10, 100 and 1000 fold concentrated a.i..

Materials and methods

Adult forager bees were collected from a queen right colony in the farm of the University of Bologna and, after a slight anaesthesia with cold temperatures; bees were grouped by 10 in laboratory hoarding cages (13 x 6 x 11 cm). Bees were provided with sugar syrup (50% w/v sucrose) through the insertion of a no-needle syringe on the top of the cage. The experimental cages were divided into 4 treatment groups for each active ingredient, in order to test the toxicity of the pesticide concentration founded at 5 meters and further treatments 10, 100, and 1000 times more concentrated (tab. 2.2.1); an untreated control was also planned. Each treatment group consisted in three repetitions.
The contaminated dusts were obtained by a Heubach cylinder through the abrasion of treated seeds similar to those sold on the market. The active ingredient concentration was quantified by chemical analysis performed by CRA-API chemical laboratory. Afterwards, geometric dilutions in talc were performed in order to prepare different treatment concentrations that were conserved at 4°C and in darkness.
With respect to tested concentrations, clothianidin was employed at 6.25 µg/m2 instead of 5.12 µg/m2 as it has been done in the previous experime ntation (chapter 2.1.2), as a consequence of further trials performed by CRA-ING to assess the quantity and the concentration of a.i. during the experimental maize sowing (ApeNet, 2010).
Following the same treatment protocol as explained in chapter 2.1.2, a paper covered with apple leaves coming from an organic apple orchard. Treatments were administered by spreading the leaves with 0.01g of previously prepared contaminated dusts. After 3 hours from the beginning of the experience, the treatments were removed and bees were left at controlled temperature conditions of 25°C± 2°C and darkness until the end of the expe rimentation.
The number of dead bees was registered at 3, 6, 9, 12, 24, 48 and 72 hours from the beginning of
the treatment. Mortality data were statistically analyzed through ANOVA test.

Effect of indirect contact with clothianidin contaminated dusts on orientation –field study

The sublethal effects of neonicotinoids and fipronil have been largely investigated in the last years. Since these active molecules express their toxic action at a neuronal level, the most important effects are evidenced in cerebral functions. In laboratory conditions, a detrimental action in learning and memorization capacities has been evidenced, particularly for imidacloprid (Decourtye et al., 2004a) and fipronil (Aliouane Y. et al., 2009). In semi-field and field conditions, several studies have examined the effect of fipronil (Decourtye et al., 2011) and clothianidin (Schneider et al., 2012) in reducing honey bee foraging activity. The effects on orientation are mainly investigated with the evaluation of foragers homing ability, that is the capacity to find the way back to the hive. To this extent, the fist product to be assessed has been imidacloprid. Three sublethal concentrations (100, 500 and 1000 µg/L), administered via ingestion, have caused a delay in the honey bee flights between the experimental hive and an artificial feeder (Bortolotti et al., 2003), demonstrating a neonicotinoid detrimental effect on the orientation ability. More recently, the RFID technique has allowed to perform more extended studies, in which the effect of small doses of thiamethoxam on the homing flight ability has been demonstrated, as well (Henry et al., 2012).
Here, the effect of clothianidin contaminated dusts, administered via indirect contact to forager bees, was assessed.


The study of the effects of pesticides includes the assessment of the influence of such molecules at a subcellular level, on enzymes and metabolic pathways. This investigation might be pursued both with a genomic/proteomic approach and with a series of biochemical assays. The first method permits to evaluate the eventual differences in protein expression, thus considering enzymes concentration. The second approach is more focussed on the variations in enzymatic activity as a response to the exposure to several contaminants and stressors in general. A so-called multimarker approach is suitable to be employed for pesticide effects assessment. Therefore, the measure of the activity of a set of key-role metabolic enzymes provides an overview of intracellular pesticide induced modifications.
To this extent, different enzymes have been employed, particularly belonging to detoxification and oxidative stress response pathways. The use of this kind of method has been first developed in aquatic ecotoxicology, for biomonitoring purposes. In this approach, the sampling of individuals living in a polluted environment aims to individuate the traces of the exposition in significant variations of enzymes activity. This subject has been less investigated in terrestrial arthropods and pollinator insects, even though a recent studies have evidenced interesting results in honey bees (Badiou et al., 2008; Badiou-Bénéteauet al., 2012).
Here, we considered this issue with a different approach from biomonitoring. We in fact assessed, in laboratory conditions, the variation in enzymatic activities as a sublethal effect of a specific pesticide or a combination of products. Since sublethal effects are represented by the alterations of the organism physiology that don’t involve death but that, in particular conditions, may lead to a weakening of individuals and colony, the changes at an enzymatic level could constitute a valuable tool to interpret pesticide impact on honey bee organism.
We therefore chose to test different pesticides, with different modalities of treatment. We carried out the experimentations with the following scheme:
– combined exposure to sublethal doses of Bacillus thuringiensis spores and fipronil.
– combined exposure to sublethal doses of Bacillus thuringiensis spores, followed by a contact treatment with deltamethrin.
– combined exposure to sublethal doses of difenoconazole, followed by a contact treatment with deltamethrin.

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Effect of three Bt toxins on honey bee mortality and feeding behaviour

The effect of Bt toxins Cry1Ab-2, Cry1C-1 and Cry3Aa on adult bees was assessed through a chronic 10-day administration. Cry1Ab and Cry1C toxins are mostly used against Lepidopteran pests in maize and rice (Hofte and Whiteley, 1989; Martinez et al., 2004), whereas Cry3Aa is mainly used against Coleoptera in potato (Hussein et al., 2006). The tested concentrations were chosen to be consistent with environmental realistic exposure. Thus, 0.01, 0.1, 1 and 10 µg/kg of purified toxins were employed as tested concentration. The cumulative mortality of the control remained under 10% and was significantly lower than all other treatment groups (p<0.001). For the Cry1Ab toxin, 10 µg/L dose caused the highest morta lity (43.3%) even though no significant differences were shown between treatments (fig. 3.1.1 a). The Cry1C-1 toxin expressed the most important effect, as honey bees died up to 56% as a consequence of the 0.1 µg/L treatment, though statistical analysis showed no significant difference between doses (fig. 3.1.1 b). Honey bees treated with Cry3Aa toxin died at a higher rate than the control (p=0.016), and the highest dose (10 µg/L) caused the lowest mortality among treatments (p=0.001)(fig. 3.1.1 c).
No influence on syrup consumption can be attributed to treatment or dose for every tested toxin (fig. 3.1.1 d, e, f).

Combined effect of Bt spores and Fipronil on honey bee mortality and feeding behaviour

As Bt strains, Bt 4Q2 and Bt 4D1 were tested, the first being a modified strain that does not express any Cry toxin and the second representing a kurstaki strain and expressing Cry1Aa, Cry1Ab, Cry1Ac, Cry2A and Cry2B toxins. The tested concentrations were 100 and 1000 µg/kg. Fipronil was tested at 1 µg/L. This concentration i s lower than the average residue amount found in pollen samples (Mullin et al., 2010) and therefore consistent with environmental levels.
The joint effect of 4Q2/4D1 spores and fipronil on survival of emerging honey bees was investigated performing a 10-day treatment followed by a 15-day mortality assessment. The cumulated mortality remained under 2% for all the treatment groups at 15 days and did not exceed 15% at 25 days (fig. 3.1.2 a, b). No significant differences between Bt treated groups and control can be found at 25 days (p=0.566). The ANOVA analysis on all data revealed en effect of the treatments on feeding behaviour (p=0.012), as the combined administration of Bt 4Q2 and Fipronil resulted in a lower syrup consumption (p=0.03). Daily syrup intake was also influenced by time, thus by honey bee age (p <1× 10-16) (fig. 3.1.2 c, d).

Table of contents :

1.1 The honey bee
1.2 Honey bees and the environment: a double-sided relationship
1.3 Routes of exposure of honey bees to pesticides
1.3.1 Exposure via direct contact
1.3.2 Exposure via indirect contact
1.3.3 Exposure via ingestion
1.3.4 Combined exposure to multiple pesticides
1.4. Pesticide effects and toxicity
1.4.1 Lethal effects
1.4.2 Sublethal effects
1.4.3 Effects on biochemical-physiological changes
1.5 Aims of the research
2.1 Effects of clothianidin contaminated dusts – laboratory and semi-field study
2.1.1 Introduction
2.1.2 Materials and methods
2.1.3 Results
2.1.4 Discussion and conclusions
2.2. Effects of indirect contact with neonicotinoids and fipronil contaminated dusts – laboratory study
2.2.1 Materials and methods
2.2.2 Results and discussion
2.3 Effect of indirect contact with clothianidin contaminated dusts on orientation –field study
2.3.1 Materials and methods
2.3.2 Results and discussion
3.1 Chronic effect of three Cry toxins and combined effect of Bt and fipronil on adult honey bees: toxicity and physiological changes
3.1.1 Introduction
3.1.2 Materials and methods
3.1.3 Results
3.1.4 Discussion and conclusions
3.2 Honey bees combined exposure to Bt spores and deltamethrin: toxicity and physiological changes
3.2.1 Introduction
3.2.2 Materials and methods
3.2.3 Results
3.2.4 Discussion and conclusions
3.3 Physiological changes induced by a combined treatment with difenoconazole and deltamethrin
3.3.1 Materials and methods
3.3.2 Results and discussion
6.1 Protein extraction
6.2 Glutathione-S-Transferase (GST) activity assay
6.3 Catalase (CAT) activity assay
6.4 Superoxyde dismutase (SOD) activity assay
6.5 Alkaline phosphatase (ALP) activity assay
6.6 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity assay
6.7 Glucose-6-Phosphate dehydrogenase (G6PDH) activity assay
6.8 List of abbreviations


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