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On-line version ISSN 1678-4324
Braz. arch. biol. technol. vol.55 no.1 Curitiba Jan./Feb. 2012
AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY
Caio Fábio Stoffel Efrom*; Luiza Rodrigues Redaelli; Rafael Narciso Meirelles; Cláudia Bernardes Ourique
Departamento de Fitossanidade; Faculdade de Agronomia; Universidade Federal do Rio Grande do Sul; 91540-000; Porto Alegre - RS -Brasil
This study aimed to evaluate the effects of pesticides, used in the organic system, on Apis mellifera under laboratory conditions. Four multiple (0.25x, 0.5x, 1x and 2x) concentrations as recommended by they manufacturers of the following products: Rotenat CE®, Pironat®, Biopirol 7M®, Organic neem®, Natuneem® and lime sulfur were tested by topical application and ingestion. Of all the products and concentrations tested, only the lime sulfur (5000 ml 100L-1 and 10000 mL 100L-1 of water) by ingestion, and Rotenat CE® (1200ml 100L-1 of water) on topical application were considered slightly harmful for A. mellifera, as the classification of IOBC/WPRS for the laboratory tests.
Key words: Honeybees, rotenone, lime sulfur, neem, pyroligneous extract
The emergence of new production systems, such as organic, which aim for the sustainability, while preserving or increasing the biological diversity, and minimization of all the forms of contamination, makes it critical to search for alternative techniques of control, selective and not harmful, especially on the populations of beneficial insects, such as natural enemies and pollinators. Among the possible alternatives for the pests and diseases controlling in the organic agriculture, there are products such as oils, plant extracts and syrups. However, for most of these substances, there is no scientific evidence regarding the efficiency on the pests and sustainability to the system, especially if they are selective to beneficial insects. Knowledge of the effects that pesticides have on beneficial insects, seeking the use of selective products is crucial for the sustainability of farming systems (Croft 1990).
Pollination has great importance in agricultural ecosystems (Free 1993; Roubik 1995). Bees are the most important flower visitors; they are responsible for pollinating more plant species than any other group (Raven et al. 2001). According to FAO (2004) estimates, approximately 70% of cultivated plant species worldwide are pollinated by some the species of bee. Ollerton et al. (2011) estimated 78-94% of flowering species relying on the biotic pollination. In flowering period, in the crops of commercial value, the presence of bees promotes the increase in the production of fruits and seeds (Morgado et al. 2002). In orchards, 7580% of harvests are usually due to pollination by this group of insects (Guimarães 1989). Among the species of bees, Apis mellifera L. (Hymenoptera, Apidae) stands out for being a generalist, with excellent adaptation to the American continent, being easily found in almost all the agricultural areas, the principal responsible by the pollination of many fruit plants (Carvalho 2006; James and Pitts-Singer 2008).
In selectivity studies on the terrestrial animals, two groups are used, birds and bees (Devillers 2002). Aside from their ecological importance, the honey bees are interesting test organisms because of their rearing and manipulation facility, caused by relatively homogeneous individuals, by the general knowledge about their biology and, by presenting a life cycle, relatively short. These characteristics made A. mellifera the most widely used because of its extensive worldwide distribution (Devillers 2002). According to the author, all the insecticides used in agriculture should be tested on their bees to estimate the ecotoxicology, using different methodologies, depending on the purpose of the study.
For registration of new pesticides, the toxicological evaluation of A. mellifera, in several countries, including Brazil, is based on the protocols established by the European agencies (OECD 1998a, b; OEPP/EPPO 1992, 1993, 2001a, b) and the U.S. Environmental Protection Agency (U.S. EPA 1996a, b). Thus, this study aimed to evaluate, through the topical application and ingestion, the insecticidal effect of the pesticides used in organic production system on workers of A. mellifera under laboratory conditions.
MATERIALS AND METHODS
General experimental procedures
Bioassays to assess the action of the pesticides on A. mellifera were performed according to the methods of testing for exposure through oral and contact protocols established by the "Organisation for Economic Cooperation and Development (OECD 1998a, b) and the results were classified according to criteria of the IOBC/WPRS (International Organization for Biological Control) for laboratory tests on beneficial organisms (Hassan et al. 1985; Hassan et al. 1992) into: 1) harmless (mortality <30%), 2) slightly harmful (> 30% and <79%), 3) moderately harmful (> 80% and <99%), and 4) harmful (>99%). The experiment was conducted in an environmental room at 29 ± 1ºC, 70 ± 10% R.H., without photophase. For each product the concentrations described in Table 1 were used, and as a control, distilled water and fenthion (50 g 100L-1) were used. Bioassays were conducted under a completely randomized design with three replicates per treatment/concentration.
Colony of A. mellifera
The workers of A. mellifera used in the experiments were collected from the central comb of a rational beehive American type, with no evidence of contamination by the pathogens, maintained at the experimental site of the Department of Phytosanity of UFRGS. The hive was kept as recommended for honey production (Pereira et al. 2003). A protein paste comprising soy flour, granulated sugar and honey (3:1:1, respectively) and a sugar and water based syrup (1:1) were fed for the colony weekly (directly into coverage feeder).
Contact action in Apis mellifera
To assess the topical action by contact of the products, about 30 honeybees workers were removed and placed directly in the cages for the test made of PVC pipe (12 cm diameter x 8 cm in height), closed at one end with wire screen of 16 mesh /cm² and on the other, a foam (density 33 and 4 cm in height), according to a methodology proposed by Sattler et al. (1990). In the laboratory, the honeybees workers were anesthetized by cold at -12ºC for up to three minutes and then subjected to topical application using 1µL of test solution on the dorsum of the thorax employing 1mL microsyringe coupled to a manual microapplicator (Burkard Manufacturing Co. Ltd.). The products were applied in four concentrations (Table 1). The workers bees were fed with distilled water, soaked in cotton wool and a piece of crystallized honey (10 g), provided in a circular plastic container (3 x 2 cm). The cages were kept in an environmental room.
Ingestion action in A. mellifera
The bioassay to evaluate the action by ingestion of the products was carried out following the protocols of OECD (1998b). About 30 honeybee's workers per cage were deprived of food for two hours before the treatments. The products were offered at a solution of 50% sucrose, at the concentrations used for testing topical application (Table 1), through a strip of fabric Spontex Resist® inserted into a glass tube of 20 mL. After four hours, the treatment solution was removed and replaced, ad libtum, by an aqueous solution only of sucrose (50%).
Evaluation of bioassays on A. mellifera
The percentage of surviving individuals was recorded in each treatment after 24 and 48 h, considering those bees as dead which didn't show any movement when touched. The survival values were corrected by the formula of Abbott (1925) and the variation in the percentage of surviving insects were subjected to variance analyses of repeated measures, using SPSS® (Statistical Package for Social Science release 15.0 - SPSS Inc., Chicago, IL). The averages were compared for the significance by Tukey test, with 5% significance of error. Finally, the values were evaluated as parameters of toxicity of the IOBC (International Organisation for Biological Control) (Hassan et al. 1985; Bakker et al. 1992; Hassan 1994, Reis et al. 1998).
RESULTS AND DISCUSSION
The product Rotenat CE® (1200 ml 100L-1) was the only one to cause significant mortality by topical application, resulting in survival of 80.2 ± 7.70% of A. mellifera 24 h after the treatment. However, this product was significantly less toxic to the bees than fenthion control (Table 2). Within 48 h after application, this product also showed significant effects, with a survival of 69.5 ± 6.67% of the bees. This value despite being significantly higher than the control with fenthion, was very close to the threshold than the IOBC considered as a product slightly harmful (mortality between > 30% and < 79%) (Hassan et al. 1985; Bakker et al. 1992; Hassan 1994; Reis et al. 1998).
There was a toxic effect of lime sulfur at 5000 ml 100L-1 and 10000mL 100L-1 on the workers of A. mellifera after 48 h (Table 2). The effect at the highest dose despite being below to the IOBC regarded as slightly harmful, also was a value very close to the threshold, and therefore, the solution would be to follow the sequential schedule of evaluation, conducting the tests in semi-field conditions.
The other products tested by the topical application did not affect the honeybees survival at any dose and any evaluation period (Table 2). The absence of deleterious effect of neem on A. mellifera at high concentrations had been reported by Schmutterer (1990), with commercial Margosan-O®, through direct contact. A similar result was obtained by González-Gómez et al. (2006) evaluating the toxicity of neem seed extract and a commercial product (Neem PHC®) on A. mellifera. However, this product could have an effect on the immature stages, as azadirachtin, the main component of the extract acted by interfering in the functioning of the endocrine glands that control metamorphosis in the insects (Viegas Jr. 2003).
A significant increase in the mortality of A. mellifera at different concentrations of the Natuneem®, Organic neem®, lime sulfur and Pironat® (Table 2) were recorded in the present study between 24 and 48 h. This indicated that if they were followed for a longer period, these products could cause toxicity on A. mellifera. However, because of the difficulty of keeping the bees alive for long periods outside the hive, the assessments after 48 h were not done.
Results on the action by ingestion of the products at 24 h showed that only lime sulfur (10000mL 100L-1) differed from the control with water (Table 3). However, after 48 h, several products and doses differed from the control, especially the lime sulfur (5000mL 100L-1 - 69.9±2.99%; and 10000 mL 100L-1 - 31.7±1.51%) and Rotenat CE® (1200 mL 100L-1 - 76.6±2.89%), which presented higher toxicity level, with the exception of control with fenthion. This was similar to what occurred in the topical test, but with smaller percentages of survival. This could be due to a differentiated action of the exposure method, with a higher toxicity when the product was ingested than by the topical contact. Hoskins and Gordon (1956) considered that the cuticle penetration and the effectiveness of the insecticides decreases. Croft (1990) reported that in general the contamination by ingestion was higher.
The Biopirol 7M® and products based on neem caused significant mortality at the doses recommended by the manufacturers, which could be due to some antifeeding effect, mainly from neem whose main component (azadirachtin) showed a phagodeterrent effect on the insects (Godfrey 1994; Martinez 2002; Viegas Jr. 2003; Isman 2006).
There was a significant decrease in the survival between 24 and 48 h for most products and doses, suggesting that the toxic effects for ingestion was over a longer period of time. The control reference insecticide (fenthion -50g 100L-1) caused the death of 100% of the honeybees at first evaluation 24 h after the application for both the methods of exposure tested., Aguiar-Menezes (2005) found rotenone as non-toxic for the bees. However, the results of this study and classification of IOBC demonstrated the need for more tests on A. mellifera, using other methods of exposure and especially in conditions of semi-field.
Hunt et al. (2003) found the products based on rotenone, azadirachtin and sulfur, such as lime sulfur relatively non-toxic to A. mellifera. However, to Silva (2005) and Dal Soglio et al. (2007) found lime sulfur causing a great impact on this insect's fauna in the area of fruit plants such as citrus. The authors found a quantitative reduction in the taxa and a decrease in the abundance of those less frequently caught in McPhail trap in citrus orchards and considered that this reduction could be related to the number of applications of the lime sulfur, since at higher intensities higher impacts were observed.
The results obtained in this study raised issues concerning the marketing and correct use of pesticides which have been used routinely in the organic production system, because it could be harmful to the pollinators such as A. mellifera. The results indicated that it was appropriate to avoid the application of the products such as lime sulfur and rotenone at the times of flowering in the culture, native vegetation and surrounding areas with the presence of bees, as they might be toxic to them. Also, further studies would be needed on the effects of the products on the immature stages of A. mellifera, and field tests by observing the behavior of the bee's orientation in the hive, in collecting nectar and in the presence of residues in the honey.
The following products were slightly harmful to A. mellifera, as the classification of IOBC/WPRS under laboratory tests: lime sulfur (5000mL 100L-1 and 10000 mL 100L-1 of water) by ingestion, and Rotenat CE® (1200 mL 100L-1 of water) on topical application.
Authors thank to the National Council for Scientific and Technological Development (CNPq) for scholarships granted to authors.
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Received: October 05, 2010; Revised: May 17, 2011; Accepted: November 10, 2011