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Acute toxicity of thiamethoxam to Apis mellifera and the use of esterases as biomarkers of exposure to
pesticides
Stephan M. Carvalho, Luc Belzunces, Osmar Malaspina
To cite this version:
Stephan M. Carvalho, Luc Belzunces, Osmar Malaspina. Acute toxicity of thiamethoxam to Apis mel- lifera and the use of esterases as biomarkers of exposure to pesticides. 42nd International Apicultural Congress, Sep 2011, Buenos Aires, Argentina. �hal-02749561�
ACUTE TOXICITY OF THIAMETHOXAM TO Apis mellifera AND THE USE OF ESTERASES
AS BIOMARKERS OF EXPOSURE TO PESTICIDES*
PESTICIDES
1Universidade Estadual Paulista, Centro de Estudo de Insetos Sociais, 13506-900 Rio Claro/SP, Brazil.
2Institut National de la Recherche Agronomique, UMR406 Abeilles et Environnement, 84914 Avignon, France.
smalfitano@uol.com.br
Several social bees was found however the speciesApis INTRODUCTION AND OBJECTIVES
Stephan M. CARVALHO1, Luc P. BELZUNCES2e Osmar MALASPINA1
Acetylcholinesterase (AChE) was assessed following Ellman et al. (1961) at 412 nm and carboxylesterases using the Several social bees was found, however, the speciesApis
spp. are the most studied due to their worldwide distribution, hive products and pollination service. For these purpose, several studies are carried out to improve the knowledge on their social organization, biology, behavior and protection against toxic compounds, like pesticides. Furthermore, honeybees can be used to assess the environmental impact caused by xenobiotics, either in mortality caused by the contact with the pesticides or by
RESULTS
method of Gomori (1953) at 568 nm for α-naphthyl acetate substrate (CaE-1), 515 nm for β-naphthyl acetate (CaE-2) and 410 nm for p-nytrophenyl acetate (CaE-3). All procedures were standardized for studies with honeybee by Carvalho (2010).
Acute toxicity assay showed that thiamethoxam was harmful to africanized honeybee, with LD50values at 24 and 48 in mortality caused by the contact with the pesticides or by
residues in their products/body (Figure 1). hours are 11.13 ng a.i./honeybee (CL95%= 9.71 - 12.55; D.F. = 16 and χ2 = 10.628) and 12.67 ng a.i./honeybee (CL95%= 9.25 - 16.08; D.F. = 18 andχ2= 17.506) (Figure 2). From the LD50 at 24h, were determinated the sublethal doses LD50/10 (1.11 ng a.i./honeybee) and LD50/20 (0.56 ng a.i./honeybee).
Among the different molecular techniques, biochemical studies may be used to evaluate the effect of different doses of
Figure 2.: Acute toxicity test (24h) of thiamethoxam on africanized honey beeApis mellifera
Figure 1. Different pathways of honeybee exposition at xenobiotics (Porrine et al., 2003).
studies may be used to evaluate the effect of different doses of thiamethoxam on carboxylesterases and acetylcholinesterase, becoming a biomarkers of exposition. Thus, the purpose of this work was determining the lethal dose 50 (LD50) of this neonicotinoid, and then assess your effect on esterase “B” from africanizesApis mellifera.
MATERIAL AND METHODS
mellifera.
After intoxication, different patterns of esterase “B” were found. AChE activity was reduced when bees were exposed at LD50/20; For CaE-1, all doses of thiamethoxam decrease the activity; and opposite CaE-2 activity were increases with the presence of this neonicotinoid. Only for CaE-3, increase/decrease behavior was found in the activity (Table 1).
Table 1 Enzymatic activity after exposition of honeybee to thiamethoxam
Firstly, the value of LD50 was determinated in dose- response assay, applying 1µL of one solution of thiamethoxam (in acetone) on the thorax of honey bee. After 24 or 48 hours, the number of dead bees per cage and concentration was subjected to statistical analysis, determining the LD50 and others sublethal doses employed on enzymatic studies (ANPP, 2003).
The enzymatic studies was carried out using a Ultrospec
Table 1. Enzymatic activity after exposition of honeybee to thiamethoxam.
Enzyme Dose Activity (%) Cluster analysis
AChE Control 100.00 A
LD50 100.85 A
LD50/10 103.50 A
LD50/20 94.08 B
CaE-1 Control 100.00 A
LD50 82.25 B
LD50/10 83.56 B
LD50/20 75.35 B
CaE-2 Control 100.00 C
LD50 146.62 A
LD0/10 136 41 B
The enzymatic studies was carried out using a Ultrospec 2100 pro UV-vis spectrophotometer (GE Healthcare), at 25oC and 1 mL of final volume. Protein assay was made with BioRad Protein kit (Bradford) and specific activity was expressed in percentage of control treatment. Like described above, 1µL of sublethal doses (control, LD50, LD50/10 and LD50/20) of thiamethoxam was applied in dorsal thorax of bees, being kept for 24h with Candy paste and then killed to remove the head which
LD50/10 136.41 B
LD50/20 133.14 B
CaE-3 Control 100.00 B
LD50 91.35 C
LD50/10 106.98 A
LD50/20 80.82 D
Mean followed by the same letter, no differ by contrast cluster analysis (P≤0.05).
In conclusion, we demonstrated the potential of AChE and mainly CaE-1 and CaE-2 as biomarkers of exposition after the exposition of africanized honey bee to the neonicotinoid thiamethoxam Furthermore others aspects (e g time of
*Acknowledgement: We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP for the financial
support (Process 2010/00747-0). REFERENCES
ANPP (2003). Méthode CEB no. 230, 21p.
CARVALHO, S.M. (2010).Doctorat Thesis, Universidade Federal de Lavras, 105p.
ELLMAN, G.L. et al. (1961).Biochem. Pharmacol., 7, 88-95.
GOMORI, G. (1953).J. Lab. Clin. Med., 42, 445-453.
PORRINI, C. et al. (2003).Apiacta, 38, 63-70.
24h with Candy paste and then killed to remove the head which were employed on enzymatic assays.
thiamethoxam. Furthermore, others aspects (e.g. time of exposition and routes) are in development for achieve a powerful analysis in environmental assessment and honeybee protection.
Apimondia 2011
42nd International Apicultural Congress 21-25 September 2011 - Buenos Aires
ACUTE TOXICITY OF THIAMETHOXAM TO AFRICANIZED APIS MELLIFERA AND THE POSSIBLE USE OF ESTERASES AS BIOMARKERS OF EXPOSURE TO PESTICIDES*
Carvalho, S. M.1; Belzunces, L. P.2 and Malaspina, O.1
1 Universidade Estadual Paulista, Centro de Estudo de Insetos Sociais, 13506-900 Rio Claro/SP, Brazil - smalfitano@uol.com.br / malaspin@rc.unesp.br - Phone: +55 19 3526-4284 or Fax +55 19 3526-4160
2 INRA, Laboratoire de Toxicologie Environnementale, UMR 406 Abeilles & Environnement, 84914 Avignon cedex 9, France. - luc.belzunces@avignon.inra.fr – Phone: +33 (0)43272 2604 or Fax +33 (0)43272 2602
In Brazil, as well as in other countries, the use of pesticides from the recent class of neonicotinoids surpasses those from organophosphates and pyrethroids. In this class, thiamethoxam is an insecticide used in Brazil mainly to control sucking insects in several crops such as citrus, coffee, soybean, bean and cotton. However, the honey bee Apis mellifera L., 1758, which has a great value as pollinator and is required for increasing the plant production, can be exposed to residues from treatments with neonicotinoids. Thus, this work aimed to determine the acute toxicity of thiamethoxam to the honeybee expressed as LD50. This value is then used as a reference dose to assess the sublethal effects of this pesticide on type B esterases from A. mellifera. Honey bee foragers were exposed, by contact application, to different doses of thiamethoxam and mortality was measured after 24 and 48 hours. The results show that the LD50 values at 24 and 48 hours are 11.13 ng a.i./bee (CL95% = 9.71 - 12.55; D.F. = 16 and χ2 = 10.628) and 12.67 ng a.i./bee (CL95% = 9.25 - 16.08;
D.F. = 18 and χ2 = 17.506), respectively. When honeybee are exposed to sublethal doses of thiamethoxam (i.e. LD50/10 or LD50/20), acetylcholinesterase and carboxylesterase significantly decreased and represented 94% and 78% of control activity at 24 hours and 48 hours, respectively.
Thus, these enzymes could be used as biomarkers of exposure to pesticides to help for a better honey bee management and to follow the environmental health.
Key-words: honeybee, néonicotinoïde insecticides, sublethal effect, enzyme, biomarkers.
* Acknowledgement: We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP for the financial support (Process 2010/00747-0).
