Hazards of pesticides to bees - 14th international symposium of the ICP-PR Bee protection group, October 23 – 25 2019, Bern (Switzerland) Abstracts: Oral Presentation Julius-Kühn-Archiv, 465, 2020 47 equipped and all involved are being trained to begin the first round of testing from September 2019. The Brazilian experience will be presented during the 13th SETAC Latin America for the exchange of experiences and discussion of more species-oriented methods from the tropical and subtropical regions of the Americas, with the aim of creating a network aimed at protecting local species. 2.4 Standardization of an in vitro rearing method for the stingless bee species Scaptotrigona postica larvae and its application for determining the toxicity of dimethoate on the larval phase Annelise Rosa-Fontana 1* , Adna Dorigo 1 , Juliana Stephanie Galaschi-Teixeira 2 , Roberta C. F. Nocelli 3 , Osmar Malaspina 1 1 State University of São Paulo, 24A Avenue 1515, Rio Claro, SP, Brazil 2 Technological Institute of Vale – Boaventura da Silva St, 955, Belém, PA, Brazil 3 Federal University of São Carlos, Anhanguera Road Km 174, Araras, SP, Brazil E-mail contact: annesouzar@gmail.com DOI 10.5073/jka.2020.465.019 Abstract Currently, Brazil has a full framework for pesticide risk assessment established for Apis mellifera, based on North America’s approach. However, the use of an exotic species as model-organism as a substitute for native species of Brazil (stingless bees) has been questioned. An in vitro larval rearing method has already been described for the Brazilian native Melipona scutellaris but, Scaptotrigona postica species has shown potential to be suitable for testing, mainly because its high numer of individuals per hive comparing to the other stingless bee species and for do not belongs to the list of endangedered species, like M. scutellaris. Thus, we aimed to establish an in vitro larval rearing method for S. postica and to apply it for determining the toxicity of dimethoate on larval phase. Larvae of 24 hours old were transferred to acrylic plates and five different procedures were carried out, considering the humidity control and the required fungus Zygosaccharomyces sp. as essential for the success of larval survivorship. Each replicate consisted of 100 larvae, totalng 4,800 larvae. Mortality and emergence parameters of the individuals, as well as the progress of the larval development were assessed, in order to check the efficiency of these methods. The intertegular distance, head width and wings asymmetry were assessed from the individuals emerged from the most efficient method. The same parameters were checked on individuals emerged from in vivo brood combs. The chosen method consisted of the deposition of the pure larval food followed by adding KCl and NaCl solutions 72 and 120 hours after the larval transference, respectively. This procedure was applied to determine the lethal concentration 50% (LC50) of dimethoathe, the standard active ingredient for toxicological tests, established by OECD. The active ingredient, obtained from Sigma-Aldrich (Pestanal), was directly diluted in the larval food, and successive subsequent dilutions were performed in the food, in order to reach the following concentrations to be offered to the larvae (in ng a.i./ larva): 250, 200, 150, 100, 50 and 25. Each bioassay was carried out 4 times (20 larvae/concentration in triplicate). The negative control consisted of the pure larval food. The dose-response data were assessed with binomial generalized linear models, using the Cauchit function, for determining the LC50 for 24 and 48 hours. The analysis was performed in the R software (R Core Team). The best procedure indicated emergence/larvae, emergence/pupae and mortality/larvae of 93.44, 97.6 and 2.85%. The mean of intertegular distance for the in vitro method was 136.5 mm and for in vivo of 127.7 mm. For the head width, in vitro showed 92.58 mm and in vivo was 89.88 mm. The t test indicated no significative difference between the in vivo and in vitro methods (p >0.05). Regarding the wings asymmetry, the ANOVA Procrustes indicated a significative difference in the centroid size only in the “individual effect”, on individuals emerged from both in vitro (F = 11.33; p <0.0001) and in vivo (F = 38.35; p <0.0001) treatments, and in the wing venation pattern in the “individual effect” in vitro (F = 12.03; p <0.0001) and in vivo (F = 12.13; p <0.0001), and in the “size effect” on individuals emerged from the in vivo treatment (F = 0.50; p <0.0005). The tests with dimethoathe indicated a LC50 (in ng a.i. /larva) of 172.48 and 156.33 for 24 and 48 hours, respectively. The mais points for the success of the in vitro rearing were the humidity control, the non-use of eggs for transference, and to the use of acrylic plates manufactured which the size simulates the real dimensions of brood cells. The differences showed in some patterns of the wings asymmetry on individuals emerged from in vitro treatment are considered normal, since we can observe also on in vivo emerged individuals. These little variations in morphology are common in nature, especially because of environmental stresses. Thus, our results obtained in vitro may be used for representing in vivo conditions. According to the OECD, to be possible carry out a toxicological comparison by LC and/or LD values, is necessary that the experimental method has been