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Acute contact toxicity test of insecticides (Cipermetrina 25, Lorsban 48E, Thionex 35) on honeybees in the southwestern zone of Uruguay
Chemosphere 88 (2012) 439–444
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Acute contact toxicity test of insecticides (Cipermetrina 25, Lorsban 48E, Thionex 35) on honeybees in the southwestern zone of Uruguay Leonidas Carrasco-Letelier a,⇑, Yamandú Mendoza-Spina b,1, María Belén Branchiccela c a National Production and Environmental Sustainability Research Program, National Agricultural Research Institute (INIA), Experimental Station Alberto Boerger INIA La Estanzuela, Route 50 km 11, P.O. 39173, Colonia, Uruguay b Beekeeping Unit, National Agricultural Research Institute, Experimental Station Alberto Boerger INIA La Estanzuela, Route 50 km 11, P.O. 39173, Colonia, Uruguay c Laboratorio de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia 3318, CP 11600, Montevideo, Uruguay
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Article history: Received 18 August 2011 Received in revised form 23 January 2012 Accepted 17 February 2012 Available online 21 March 2012 Keywords: Insecticide Cypermethrin Chlorpyrifos Endosulfan LD50 Honeybees
a b s t r a c t Glyphosate-resistant soybean cultivation is expanding rapidly in Uruguay, with its land area having increased by 95 times during the past 10 years. Because of the region’s Neotropical conditions, insecticide use is required to ensure adequate soybean productivity. However, in areas shared by soybean crops and beekeepers – such as the southwestern zone of Uruguay (SWZU) – the use of insecticides can increase the risks of honeybee death and honey contamination. Uruguayan commercial and legal guidelines set out practices and field doses designed to prevent acute intoxication with insecticides. However, honeybees in the SWZU are predominantly a polyhybrid subspecies different from that used to set international reference values, and hence they may have a different acute toxicity response, thus rendering such precautions ineffective. The aim of this work was to assess the acute toxicity response of polyhybrid honeybees in the SWZU to cypermethrin (commercial formulation: Cipermetrina 25 AgrinÒ), chlorpyrifos (commercial formulation: Lorsban 48EÒ), and endosulfan (commercial formulation: Thionex 35Ò). Acute toxicity bioassays were conducted to determine the median lethal dose (LD50) of each insecticide for the honeybees. The results indicate that, compared with EU reference values, SWZU honeybees have a higher toxicological sensitivity to chlorpyrifos and endosulfan, and a lower toxicological sensitivity to cypermethrin, based on the commercial formulations tested. However, when these results were adjusted according to their field dose equivalents, only chlorpyrifos emerged as a potential problem for beekeeping, as the maximum recommended field dose of Lorsban 48EÒ for soybean crops in Uruguay is 23 times the corresponding LD50 for honeybees in the SWZU. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Uruguay has a total agricultural land area of 16.4 million hectares, of which 13.2 million hectares is used for cattle production and 1.16 million hectares for crops. Currently, soybean is cultivated across 849,000 hectares – an area that has increased by 95 times during the past 10 years – with soybean production reaching 1793,000 tons in 2008/9 (DIEA, 2010). This agricultural intensification is raising many questions about the potential environmental impacts (Céspedes-Payret et al., 2009), and trends in Europe indicate that such intensification may have negative collateral effects for beekeeping (Le Féon et al., 2010). Beekeeping is an economically important agricultural activity for Uruguay, as indicated by the value of annual exports of honey, ⇑ Corresponding author. Tel.: +598 45748000; fax: +598 45748012. E-mail addresses: [email protected] (L. Carrasco-Letelier), ymendoza@ inia.org.uy (Y. Mendoza-Spina), [email protected] (M.B. Branchiccela). 1 Tel.: +598 45748000; fax: +598 45748012. 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2012.02.062
which account for at least 0.5% of gross domestic product, according to the Uruguay’s Department of Agricultural Statistics (DIEA, 2005, 2006, 2007, 2008, 2009). In 2009, Uruguay exported 6484 tons of honey, produced by a total of 504,514 hives and 3180 hive owners, to the total value of US$17.6 million (DIEA, 2010; DIGEGRA, 2010). The southwestern zone of Uruguay (SWZU; Fig. 1) is historically one of the country’s most important areas for honey production, hosting 38% of its hives and 29% of the hive owners (DIGEGRA, 2010). However, the recent expansion of soybean cultivation has occurred in this same zone, increasing the risk of honeybee exposure to insecticides. The expansion of soybean cultivation also explains the increase in the volume of insecticide imports, which grew from 895 tons in 1999 to 2000 tons in 2009 (DGSSAA, 2011). This situation has raised concerns among honey producers in the SWZU concerns supported by the recent detection of honey contaminated with insecticides used in soybean cultivation (Rios et al., 2010). Of additional concern are the findings of a study by Suchail et al. (2000) on the potential toxicity of insecticides to honeybees. Their test results indicate that the same chemical compounds can have
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insecticide); and Thionex 35Ò, the commercial formulation of endosulfan (an organochlorine insecticide).
2. Materials and methods
Fig. 1. Geographic position of the southwestern zone of Uruguay (dark gray), within the Oriental Republic of Uruguay. The black cross indicates Experimental Station Alberto Boerger INIA La Estanzuela.
different median lethal doses (LDs50) for different subspecies of honeybee. These findings are particularly relevant for the SWZU, where a polyhybrid subspecies of honeybee is predominant (Diniz et al., 2003). This suggests that the acute LD50 values that apply in this region may differ from the reference values used in international guidelines, which are based on Apis mellifera mellifera (PPDB, 2010), and which constitute the toxicity reference values normally considered in defining insecticide field doses (Atkins and Kellum, 1981; Mayer et al., 1999; Sanford, 2003). Moreover, commercial formulations of insecticides include excipients, which are an unknown group of chemical compounds with the capacity to modify the ultimate toxic effect of active compounds, through undefined antagonistic and synergistic effects (Pilling et al., 1995; Rozman et al., 2010). Several genetic approaches had been employed for the characterization of the honey bee colonies (Daly et al., 1982; Rinderer et al., 1987; Del Lama et al., 1988; Estoup et al., 1995; Franck et al., 2001). Different authors had employed the analysis of the mitochondrial DNA to define the haplotype of the honeybee (Hall and Smith, 1991; Garnery et al., 1998; Franck et al., 2001). Since it is inherit by maternal via it represent half of the story of the bee, so complementary analysis should be included in order to study the drone contribution. The morphometric approach is an alternative (Daly et al., 1982; Rinderer et al., 1987). There are different methodologies in order to characterize the honey bees, but most of them are time consuming, and needs expensive equipment and qualified personal. Rinderer et al. (1987) proposed a morphometric simple methodology which allows the statistical discrimination between Africanized and European honey bees. The aim of this study was to assess the acute toxicity response of SWZU honeybees to some of the commercial formulations of insecticides normally used on soybean crops in the SWZU. To achieve this, the LD50 of the following insecticides were determined: Cipermetrina 25 AgrinÒ, the commercial formulation of cypermethrin (a synthetic pyrethroid insecticide); Lorsban 48EÒ, the commercial formulation of chlorpyrifos (an organophosphate
Honeybees used in this study were a polyhybrid subspecies of A. mellifera from SWZU. Bees were obtained from experimental apiaries kept by the Beekeeping Unit of Experimental Station Alberto Boerger INIA La Estanzuela (34° 200 22.2000 S, 57° 410 14.9300 W, Colonia, Uruguay). Colonies denominates as INIA-LE’s colonies for this study. In order to demonstrate the polyhybrid origin of the colony a genetic and morphometric analysis was employed. The mitochondrial DNA was assessed, which allow the differentiation of haplotypes A (African origin), M (West European origin), C (North Mediterranean origin), and O (Near and middle eastern) (Franck et al., 2001). The DNA extraction was carried out as described by Aguirre C., INIA La Platina, Chile (personal communication) using a modification of the protocol described by Walsh et al. (1991). The posterior leg was incubated at 37 °C for 30 min. After that Chelex 5% (Sigma) and proteinase K (Promega, 5 mg ml1) were added and incubated during 1 h at 55 °C, 15 min at 99 °C, 1 min at 37 °C and 15 min at 99 °C (T1 Biometra Thermocycler). A 2 ll of DNA were used for the amplification of the intergenic region COI–COII using the primers E2: 5‘GGCAGAATAAGTGCATTG3‘ and H2: 5‘CAATATCATTGATGACC3‘ (Garnery et al., 1998). The 20 ll reaction mixture contained 1 X buffer, 1 mM MgCl2, 0.15 lM of each primer, 0.5 mM of each dNTP and 5 U/ll of Taq polymerase (Invitrogen). The PCR cycling program consist on 5 min at 94 °C, 35 cycles of 45 s at 92 °C, 45 s at 48 °C, 2 min at 62 °C, and a final extension of 20 min at 65 °C (T1 Biometra Thermocycler) (Aguirre C., INIA La Platina, Santiago de Chile, personal communication). The size of the amplified product was determined by electrophoresis on a 1% w/v agarose gel, stained with GelRed (Biotum, USA) and visualized by UV (Biometra T13). The amplified product was digested with FastDigest DraI (Fermentas), according to manufacturer’s recommendations, and analyzed under electrophoresis on polyacrilamide gel under native conditions during 15–16 h at 80 V, and staining with GelRed (Biotium, USA). The African origin probability was calculated by a morphometric approach as described by Rinderer et al. (1987). The honeybees used in the bioassays were newborn bees (age 1–7 d), obtained from hive frames isolated with bags of plastic mesh (square cells, 1 1 mm) in hives without any treatment against varroosis. The honeybees used were closely monitored after treatment, and then observed for mortality and signs of intoxication at 48 h. The acute toxicity bioassay used in this study was developed according to the European and Mediterranean Plant Protection Organization (EPPO, 1992) and the United States Environmental Protection Agency (US-EPA, 1996) with the following criteria: 48 h in the dark, with humidity (60%) and temperature-controlled (25 °C) conditions. Five doses of each insecticide were tested, each one in triplicate. Each replicate was conducted on a group of 10 honeybees; to make each group, two honeybees were taken from each of five hives, randomly selected from an apiary of 50 hives. All insecticide’s dilutions was done in acetone. The dose of insecticide was applied to the thorax of the honeybees using a micropipette, and all the dilutions was prepared to avoid the use of volumes higher than 5 ll per bee. The honeybees were anesthetized with CO2 (g) (US-EPA, 1996) for the grouping and dose administration. Each group of 10 honeybees was kept in a glass Petri dish (i.d. 10 cm), the bottom of which was lined with clean filter paper, containing a feeder with 1 ml of sucrose 50% w/v for ad libitum consumption. For the control treatment, a similar
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procedure was performed to that described above, also in triplicate, but in which the thoracic dose of insecticide was replaced with acetone. The honeybees used were closely monitored after treatment, and then observed for mortality at 48 h. The mortality rates was obtained by the division of the number of death honeybee by the total honeybees in glass Petri dish (n = 10). All the bioassays was developed in the middle of spring season (August– November, 2008). The following commercial formulations of insecticides were assessed in the study, selected according to data on insecticides used in soybean crops: Lorsban 48EÒ (main active compound: chlorpyrifos, CAS No. 002921-88-2, 48% w/v, Dow AgroSciences Argentine S.A.); Thionex 35Ò (main active compound: endosulfan, CAS No. 115-29-7, 33% w/v, imported by Lanafil SA from Du Pont Brazil SA); and Cipermetrina 25 AgrinÒ (main active compound: cypermethrin, CAS No. 52315-07-8, 25% w/v, imported by Agro Internacional from China). These insecticides were assessed in the following concentration ranges: 0.015–0.250 lg honeybee1 for cypermethrin; 0.006–0.096 lg honeybee1 for chlorpyrifos; and 0.11–1.75 lg honeybee1 for endosulfan; these concentration ranges had been previously defined in preliminary tests (data not shown). The LD50 was determined using a generalized linear model (GLM) that employed a binomial distribution and logit or probit model (Crawley, 2007a,b,c). Statistical tests were run using the statistical package R version 2.12.0 (R Development Core Team, 2010) for the platform i486-pc-Linux-gnu (32-bit), with R Commander 1.5-4 (Fox, 2005; http://CRAN.R-project.org/package=Rcmdr) on a GNU/Linux operating system (Ubuntu 10.04, Canonical Ltd., http://www.ubuntu.com).
3. Results The genetic analysis showed that the colony has a north Mediterranean origin, since its haplotype was determined as C2 (Fig. 2). The morphometric parameters employed for the characterization of the colony allowed the determination of it African origin (Table 1). The bioassays were performed according to the requirements and criteria set by EPPO (1992), with the honeybee mortality rate less than 10% in control treatments and the monotonic mortality rates for each treatment assessed (Tables 2–4). All the estimations of the LD50 derived using the GLM satisfied the statistical conditions, with residual levels lower than the available degrees of freedom (Table 5). The LD50 determined for cypermethrin was in the range 0.097– 0.169 lg honeybee1, with a mean value of 0.124 lg honeybee1 and standard deviation of 0.039 lg honeybee1 (Table 5). The LD50 for chlorpyrifos was in the range 0.023–0.024 lg honeybee1, with a mean value of 0.024 lg honeybee1 and standard deviation of 0.0005 lg honeybee1 (Table 5). The LD50 determined for endosulfan was in the range 0.897–1.767 lg honeybee1, with a mean value of 1.240 lg honeybee1 and standard deviation of 0.463 lg honeybee1 (Table 5).
4. Discussion The employed methodology allowed the characterization of the honey bee colony. The results obtained by the morphometric and genetic analysis demonstrate the polyhybrid origin of the colony as the haplotype was a North Mediterranean origin and the morphometric analysis showed an African origin probability. The different values obtained considering the three parameters (forewing length, femur length and dry weight) alone and in combination could be because the grade of africanization of the colony.
Fig. 2. Genetic characterization of the colony INIA LE. A. PCR amplification of the COI–COII mitochondrial region. (1) 1 kb DNA ladder plus, Fermentas; (2) INIA-LE colony. B. Digestion of the amplified product with Fast Digest DraI, Fermentas. (1) ladder: 41pb, 47pb, 65pb, 108pb, 193pb, 420pb, 483pb; (2) INIA LE colony.
Table 1 African origin probability of the colony INIA LE by different morphometric parameters. Morphometric parameter
African origin probability
Forewing lenght Dry weight Forewing lenght and dry weight Forewing lenght and femur lenght Forewing lenght, dry weight and femur lenght
92.5 100 80 35.8 100
Table 2 Mortality rates of honeybees exposed to different doses of cypermethrin in acute toxicity bioassays. Replicate
1 2 3
Dose (lg cypermethrin honeybee1)
Control
0.015
0.030
0.060
0.130
0.250
0.0 0.0 0.1
0.0 0.2 0.2
0.0 0.2 0.4
0.3 0.6 0.5
0.8 0.8 0.8
0.0 0.0 0.0
This results are in agreement with other previous studies in our country which found hybrid colonies (based on genetic and/or morphometric analysis) (Burgett et al., 1995; Diniz et al., 2003). Similar results has been reported for neighborhood countries such as Argentina and Brazil. (Sheppard et al., 1991a,b; Diniz et al., 2003).
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Table 3 Mortality rates of honeybees exposed to different doses of chlorpyrifos in acute toxicity bioassays. Replicate
1 2 3
Dose (lg chlorpyrifos honeybee1)
Control
0.006
0.012
0.024
0.048
0.096
0.0 0.0 0.0
0.0 0.0 0.0
0.6 0.6 0.8
1.0 1.0 1.0
1.0 1.0 1.0
0.0 0.0 0.0
Table 4 Mortality rates of honeybees exposed to different doses of endosulfan in acute toxicity bioassays. Replicate
1 2 5
Dose (lg endosulfan honeybee1)
Control
0.11
0.22
0.44
0.88
1.750
0.1 0.1 0.1
0.1 0.1 0.1
0.3 0.2 0.4
0.5 0.2 0.3
0.17 0.6 0.7
Table 5 Median lethal doses (LDs50) (expressed as lg honeybee1, median lethal doses (LDs50) of reference values (PPDB, 2010),
0.1 0.1 0.1
of the commercial insecticide formulations tested with corresponding GLM residuals in brackets); and insecticides for honeybees according to international expressed as in lg honeybee1. Lorsban 48EÒ
Active compound
Cipermetrina 25 AgrinÒ Cypermethrin
Chlorpyrifos
Thionex 35Ò Endosulfan
Replicate 1
0.097 [0.48]
Replicate 2
0.169 [0.47]
Replicate 3
0.107 [1.81]
0.024 [4 1010] 0.024 [4 1010] 0.023 [5 1010]
0.897 [0.35] 1.767 [1.56] 1.056 [2.13]
Mean Standard deviation LD50-reference value
0.124 0.039 0.020
0.024 0.001 0.059
1.240 0.463 7.81
Commercial brand
The results of this study revealed notable differences from the reference values used by the EU (Table 5), as shown by the ratio of the honeybee LD50 set by the EU (PPDB, 2010; Table 5) to the LD50 determined in our study for the same commercial insecticide formulations. The comparison reveals that the LD50 values for Lorsban 48EÒ (chlorpyrifos) and Thionex 35Ò (endosulfan) were lower for SWZU honeybees than the EU reference values, implying an increased sensitivity to insecticide of 4.6 and 6.3 times, respectively. By contrast, cypermethrin differed in the opposite direction: the LD50 value for its commercial formulation Cipermetrina 25 AgrinÒ was 6.2 times higher for SWZU honeybees than the reference value set in the Pesticide Properties Database (PPDB, 2010); this suggests that SWZU honeybees have a higher tolerance to intoxication by cypermethrin than indicated by the EU reference values (Table 5). Two factors can potentially explain these differences: the polyhybridism of SWZU honeybees and the specific chemical mixture of the commercial formulations of the insecticides tested in this study. Polyhybridism gives rise to new genetic conditions that may lead to different tolerance ranges in the SWZU honeybee, as suggested by Suchail et al.’s (2000) results in their study on two different honeybee subspecies. However, this aspect may only partly explain the differences from the EU reference levels. These differences may also be the result of the chemical composition of each commercial formulation tested in our study. As mentioned previously, the specific acute toxicity of any chemical can be af-
fected by the mixture tested (Rozman et al., 2010). Although identifying the extent to which each of these mechanisms (polyhybridism, chemical mixture) affected the results constitutes an important basic scientific goal, our study design did not consider the contribution of each of these factors, because our focus was on comparing the LD50 values for SWZU honeybees with the EU reference values. Although elucidating this aspect is important, from the perspective of environmental management, a more urgent task is the definition of adequate field doses of insecticides. Defining LD50 values should be a priority in Uruguay and other countries from South America, given that the international reference values do not agree with local beekeeping conditions (genetic differences, characteristics of commercial insecticide formulation) and for the grew of soybean crops in the region (Morton et al., 2006; Viglizzo and Frank, 2006; Zak et al., 2008). The LD50 is used to determine the appropriate field doses of pesticides used near beekeeping areas. The LD50, expressed in milligrams of active compound per honeybee, is equivalent to the insecticide’s field dose expressed in pounds per hectare (kilograms per hectare, if the LD50 value is multiplied by 1.12; Atkins and Kellum, 1981). Hence, using the LD50 values determined in our study, we calculated the corresponding field doses that would kill 50% of the polyhybrid subspecies of SWZU honeybees for the chemicals tested, as follows: 0.139 kg ha1 for cypermethrin; 0.027 kg ha1 for chlorpyrifos; and 1.389 kg ha1 for endosulfan. However, as these insecticide chemical compounds are diluted in a commercial formulation, the concentration of each compound in the formulation must be considered in order to calculate the field doses of the commercial formulations. Therefore, the above doses correspond to 0.556 kg ha1 of Cipermetrina 25 AgrinÒ, 0.056 kg ha1 of Lorsban 48EÒ, and 4.209 kg ha1 of Thionex 35Ò. By comparison, the current maximum doses recommended for use in Uruguay are 0.080–0.200 L ha1 for Cipermetrina 25 AgrinÒ, 0.75–1.3 L ha1 for Lorsban 48EÒ, and 1.25–1.5 L ha1 for Thionex 35Ò (Modernel, 2009). This reveals important differences between the recommended doses and the LD50 determined in this study. For cypermethrin (Cipermetrina 25 AgrinÒ) and endosulfan (Thionex 35Ò), the recommended field doses are lower than the determined LD50. However, for chlorpyrifos (Lorsban 48EÒ), the field doses suggested by Modernel (2009) for use on soybean crops in Uruguay are higher than the LD50 values determined in this study. Indeed, according to our findings, the maximum recommended field dose of Lorsban 48EÒ is 23 times the LD50 value for SWZU honeybees found in this study. By contrast, the maximum recommended field doses of Cipermetrina 25 AgrinÒ and Thionex 35Ò are 50% of the respective LD50 values for SWZU honeybees. These results highlight the need to adjust the recommended field doses of the commercial formulations of the insecticides assessed in this study. The findings also suggest there is reason to conduct similar reviews of other insecticides authorized for use on soybean crops in Uruguay, in order to ensure sustainable development and management of beekeeping in the SWZU. Furthermore, these findings should be studied in relation to natural pollinators, in order to understand how the current trend of agricultural intensification could be affecting both existing ecosystem services and future agriculture, especially in terms of pollination capacity (Aizen et al., 2009). The situation found for Uruguayan soybean crop region can be considered as one with the neighboring countries, Argentine and Brazil. In this wider region, it is possible that the beekeeping production is undergoing a non-adequate exposure risk to insecticides such as SWZU. The first evidence to consider these three countries as a region are the results that showed that polyhybrid honeybee studied in Uruguay are similar to those honeybees from these neighboring countries; and second, because insecticides utilized and their recommended doses are almost the same. In some cases
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the only difference between these countries are the commercial brand names, but these insecticides belong to the same international companies. For example Cypermetrina 25, is imported from the same Chinese companies (Hangzhou Ruijiang Chemical Co., Ltd.; Shenzen Qinfeng Pesticide Co. Ltd., Dalian Winyard Chemical Co. Ltd.) to Uruguay, Argentine and Brazil; or the case of Lorsban 48 E made by Dow Agro Science in Argentine and Brazil. Current doses used in Brazil for Cipermetrine 25, Lorsban 480Ò (Dow AgroSciences Industrial Ltda., Brazil), and Thionex 35Ò (DuPont, Brazil) are 0.050–0.200 L ha1, 0.50–0.75 L ha1, and 1.0–1.5 L ha1, respectively. In Argentine, doses for the first two products are the same as in Brazil and Uruguay, while Thionex is used at a lower rate (0.9–1.0 L ha1) than in the other two countries. Regional similarities with the SWZU beekeeping and soybean cultivation suggests the need to check if the current maximum insecticides recommended doses for Argentine and Brazil are secure for the beekeeping production. 5. Conclusions The results of this study indicate that the acute toxicity response of polyhybrid honeybees in the SWZU to chlorpyrifos, cypermethrin, and endosulfan differs from the international reference values used in the European Union, which are based on A. m. mellifera. The findings indicate the need to adjust the recommended field doses of insecticides with respect to beekeeping both for honeybees other than A. m. mellifera and for commercial insecticide formulations with different chemical compositions. Finally, the current recommended doses of chlorpyrifos (commercial formulation: Lorsban 48EÒ) may be endangering beekeeping activity near soybean cultivation areas in the SWZU. Acknowledgments This work was financed by the INIA-Sa07 Grant ‘‘Tools for production and sustainability of watershed with forest aptitude’’ (INIA-Uruguay). We thank K. Antunez and Silvina Stewart for her collaboration with this study, and to the technicians G. Ramallo, S. Díaz, M. Vera and P. Ojeda for theirs assistances in laboratory and field works. We also thank the organizations that share their free software for GNU/linux (Ubuntu, GNOME, GIMP, KDE, LibreOffice, R-CRAN, and Quantum GIS). References Aizen, M.A., Garibaldi, L.A., Cunningham, S.A., Klein, A.M., 2009. How much does agriculture depend on pollinators? Lessons from long-term trends in crop production. Ann. Bot. 103, 1579–1588. Atkins, E.L., Kellum, D., 1981. Reducing Pesticide Hazard to honey bees: mortality prediction techniques and integrated management strategies. Dept. of Enthomology, Division of Agricultural Sciences. University of California, USA. Burgett, M., Shorney, S., Codara, J., Gardiol, G., Sheppard, W., 1995. The present status of the Africanized honey bees in Uruguay. Am. Bee J. 135, 328–330. Cespedes-Payret, C., Pineiro, G., Achkar, M., Gutierrez, O., Panario, D., 2009. The irruption of new agro-industrial technologies in Uruguay and their environmental impacts on soil, water supply and biodiversity: a review. IJEnvH 3, 175–197. Crawley, M.J., 2007a. Generalized linear models. In: Crawley, M.J. (Ed.), The R Book. John Wiley & Sons, Ltd., pp. 511–526. Crawley, M.J., 2007b. Proportion data. In: Crawley, M.J. (Ed.), The R Book. John Wiley & Sons, Ltd., pp. 569–591. Crawley, M.J., 2007c. Binary response variables. In: Crawley, M.J. (Ed.), The R Book. John Wiley & Sons, Ltd., pp. 593–609. Daly, H.V., Hoelmer, K., Norman, P., Allen, T., 1982. Computer-assisted measurement and identification of honey bees (Hymenoptera: Apidae). Ann. Entomol. Soc. Amer. 75, 591–594. Del Lama, M.A., Figueiredo, R.A., Soares, A.E.E., Del Lama, S.N., 1988. Hexokinase polymorphism in Apis mellifera and its use for africanized honeybee identification. Rev. Brazil Genet. 11, 287–297. DGSSAA, 2011. Resumen Estadístico. Importación de productos fitosanitarios. División de Análisis y Diagnóstico. Dirección de Servicios Agrícolas. Ministerio de Ganadería Agricultura y Pesca, Montevideo, Uruguay. (in Spanish).
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Acute contact toxicity test of insecticides (Cipermetrina 25, Lorsban 48E, Thionex 35) on honeybees in the southwestern zone of Uruguay Leonidas Carrasco-Letelier a,⇑, Yamandú Mendoza-Spina b,1, María Belén Branchiccela c a National Production and Environmental Sustainability Research Program, National Agricultural Research Institute (INIA), Experimental Station Alberto Boerger INIA La Estanzuela, Route 50 km 11, P.O. 39173, Colonia, Uruguay b Beekeeping Unit, National Agricultural Research Institute, Experimental Station Alberto Boerger INIA La Estanzuela, Route 50 km 11, P.O. 39173, Colonia, Uruguay c Laboratorio de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia 3318, CP 11600, Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 23 January 2012 Accepted 17 February 2012 Available online 21 March 2012 Keywords: Insecticide Cypermethrin Chlorpyrifos Endosulfan LD50 Honeybees
a b s t r a c t Glyphosate-resistant soybean cultivation is expanding rapidly in Uruguay, with its land area having increased by 95 times during the past 10 years. Because of the region’s Neotropical conditions, insecticide use is required to ensure adequate soybean productivity. However, in areas shared by soybean crops and beekeepers – such as the southwestern zone of Uruguay (SWZU) – the use of insecticides can increase the risks of honeybee death and honey contamination. Uruguayan commercial and legal guidelines set out practices and field doses designed to prevent acute intoxication with insecticides. However, honeybees in the SWZU are predominantly a polyhybrid subspecies different from that used to set international reference values, and hence they may have a different acute toxicity response, thus rendering such precautions ineffective. The aim of this work was to assess the acute toxicity response of polyhybrid honeybees in the SWZU to cypermethrin (commercial formulation: Cipermetrina 25 AgrinÒ), chlorpyrifos (commercial formulation: Lorsban 48EÒ), and endosulfan (commercial formulation: Thionex 35Ò). Acute toxicity bioassays were conducted to determine the median lethal dose (LD50) of each insecticide for the honeybees. The results indicate that, compared with EU reference values, SWZU honeybees have a higher toxicological sensitivity to chlorpyrifos and endosulfan, and a lower toxicological sensitivity to cypermethrin, based on the commercial formulations tested. However, when these results were adjusted according to their field dose equivalents, only chlorpyrifos emerged as a potential problem for beekeeping, as the maximum recommended field dose of Lorsban 48EÒ for soybean crops in Uruguay is 23 times the corresponding LD50 for honeybees in the SWZU. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Uruguay has a total agricultural land area of 16.4 million hectares, of which 13.2 million hectares is used for cattle production and 1.16 million hectares for crops. Currently, soybean is cultivated across 849,000 hectares – an area that has increased by 95 times during the past 10 years – with soybean production reaching 1793,000 tons in 2008/9 (DIEA, 2010). This agricultural intensification is raising many questions about the potential environmental impacts (Céspedes-Payret et al., 2009), and trends in Europe indicate that such intensification may have negative collateral effects for beekeeping (Le Féon et al., 2010). Beekeeping is an economically important agricultural activity for Uruguay, as indicated by the value of annual exports of honey, ⇑ Corresponding author. Tel.: +598 45748000; fax: +598 45748012. E-mail addresses: [email protected] (L. Carrasco-Letelier), ymendoza@ inia.org.uy (Y. Mendoza-Spina), [email protected] (M.B. Branchiccela). 1 Tel.: +598 45748000; fax: +598 45748012. 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2012.02.062
which account for at least 0.5% of gross domestic product, according to the Uruguay’s Department of Agricultural Statistics (DIEA, 2005, 2006, 2007, 2008, 2009). In 2009, Uruguay exported 6484 tons of honey, produced by a total of 504,514 hives and 3180 hive owners, to the total value of US$17.6 million (DIEA, 2010; DIGEGRA, 2010). The southwestern zone of Uruguay (SWZU; Fig. 1) is historically one of the country’s most important areas for honey production, hosting 38% of its hives and 29% of the hive owners (DIGEGRA, 2010). However, the recent expansion of soybean cultivation has occurred in this same zone, increasing the risk of honeybee exposure to insecticides. The expansion of soybean cultivation also explains the increase in the volume of insecticide imports, which grew from 895 tons in 1999 to 2000 tons in 2009 (DGSSAA, 2011). This situation has raised concerns among honey producers in the SWZU concerns supported by the recent detection of honey contaminated with insecticides used in soybean cultivation (Rios et al., 2010). Of additional concern are the findings of a study by Suchail et al. (2000) on the potential toxicity of insecticides to honeybees. Their test results indicate that the same chemical compounds can have
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insecticide); and Thionex 35Ò, the commercial formulation of endosulfan (an organochlorine insecticide).
2. Materials and methods
Fig. 1. Geographic position of the southwestern zone of Uruguay (dark gray), within the Oriental Republic of Uruguay. The black cross indicates Experimental Station Alberto Boerger INIA La Estanzuela.
different median lethal doses (LDs50) for different subspecies of honeybee. These findings are particularly relevant for the SWZU, where a polyhybrid subspecies of honeybee is predominant (Diniz et al., 2003). This suggests that the acute LD50 values that apply in this region may differ from the reference values used in international guidelines, which are based on Apis mellifera mellifera (PPDB, 2010), and which constitute the toxicity reference values normally considered in defining insecticide field doses (Atkins and Kellum, 1981; Mayer et al., 1999; Sanford, 2003). Moreover, commercial formulations of insecticides include excipients, which are an unknown group of chemical compounds with the capacity to modify the ultimate toxic effect of active compounds, through undefined antagonistic and synergistic effects (Pilling et al., 1995; Rozman et al., 2010). Several genetic approaches had been employed for the characterization of the honey bee colonies (Daly et al., 1982; Rinderer et al., 1987; Del Lama et al., 1988; Estoup et al., 1995; Franck et al., 2001). Different authors had employed the analysis of the mitochondrial DNA to define the haplotype of the honeybee (Hall and Smith, 1991; Garnery et al., 1998; Franck et al., 2001). Since it is inherit by maternal via it represent half of the story of the bee, so complementary analysis should be included in order to study the drone contribution. The morphometric approach is an alternative (Daly et al., 1982; Rinderer et al., 1987). There are different methodologies in order to characterize the honey bees, but most of them are time consuming, and needs expensive equipment and qualified personal. Rinderer et al. (1987) proposed a morphometric simple methodology which allows the statistical discrimination between Africanized and European honey bees. The aim of this study was to assess the acute toxicity response of SWZU honeybees to some of the commercial formulations of insecticides normally used on soybean crops in the SWZU. To achieve this, the LD50 of the following insecticides were determined: Cipermetrina 25 AgrinÒ, the commercial formulation of cypermethrin (a synthetic pyrethroid insecticide); Lorsban 48EÒ, the commercial formulation of chlorpyrifos (an organophosphate
Honeybees used in this study were a polyhybrid subspecies of A. mellifera from SWZU. Bees were obtained from experimental apiaries kept by the Beekeeping Unit of Experimental Station Alberto Boerger INIA La Estanzuela (34° 200 22.2000 S, 57° 410 14.9300 W, Colonia, Uruguay). Colonies denominates as INIA-LE’s colonies for this study. In order to demonstrate the polyhybrid origin of the colony a genetic and morphometric analysis was employed. The mitochondrial DNA was assessed, which allow the differentiation of haplotypes A (African origin), M (West European origin), C (North Mediterranean origin), and O (Near and middle eastern) (Franck et al., 2001). The DNA extraction was carried out as described by Aguirre C., INIA La Platina, Chile (personal communication) using a modification of the protocol described by Walsh et al. (1991). The posterior leg was incubated at 37 °C for 30 min. After that Chelex 5% (Sigma) and proteinase K (Promega, 5 mg ml1) were added and incubated during 1 h at 55 °C, 15 min at 99 °C, 1 min at 37 °C and 15 min at 99 °C (T1 Biometra Thermocycler). A 2 ll of DNA were used for the amplification of the intergenic region COI–COII using the primers E2: 5‘GGCAGAATAAGTGCATTG3‘ and H2: 5‘CAATATCATTGATGACC3‘ (Garnery et al., 1998). The 20 ll reaction mixture contained 1 X buffer, 1 mM MgCl2, 0.15 lM of each primer, 0.5 mM of each dNTP and 5 U/ll of Taq polymerase (Invitrogen). The PCR cycling program consist on 5 min at 94 °C, 35 cycles of 45 s at 92 °C, 45 s at 48 °C, 2 min at 62 °C, and a final extension of 20 min at 65 °C (T1 Biometra Thermocycler) (Aguirre C., INIA La Platina, Santiago de Chile, personal communication). The size of the amplified product was determined by electrophoresis on a 1% w/v agarose gel, stained with GelRed (Biotum, USA) and visualized by UV (Biometra T13). The amplified product was digested with FastDigest DraI (Fermentas), according to manufacturer’s recommendations, and analyzed under electrophoresis on polyacrilamide gel under native conditions during 15–16 h at 80 V, and staining with GelRed (Biotium, USA). The African origin probability was calculated by a morphometric approach as described by Rinderer et al. (1987). The honeybees used in the bioassays were newborn bees (age 1–7 d), obtained from hive frames isolated with bags of plastic mesh (square cells, 1 1 mm) in hives without any treatment against varroosis. The honeybees used were closely monitored after treatment, and then observed for mortality and signs of intoxication at 48 h. The acute toxicity bioassay used in this study was developed according to the European and Mediterranean Plant Protection Organization (EPPO, 1992) and the United States Environmental Protection Agency (US-EPA, 1996) with the following criteria: 48 h in the dark, with humidity (60%) and temperature-controlled (25 °C) conditions. Five doses of each insecticide were tested, each one in triplicate. Each replicate was conducted on a group of 10 honeybees; to make each group, two honeybees were taken from each of five hives, randomly selected from an apiary of 50 hives. All insecticide’s dilutions was done in acetone. The dose of insecticide was applied to the thorax of the honeybees using a micropipette, and all the dilutions was prepared to avoid the use of volumes higher than 5 ll per bee. The honeybees were anesthetized with CO2 (g) (US-EPA, 1996) for the grouping and dose administration. Each group of 10 honeybees was kept in a glass Petri dish (i.d. 10 cm), the bottom of which was lined with clean filter paper, containing a feeder with 1 ml of sucrose 50% w/v for ad libitum consumption. For the control treatment, a similar
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procedure was performed to that described above, also in triplicate, but in which the thoracic dose of insecticide was replaced with acetone. The honeybees used were closely monitored after treatment, and then observed for mortality at 48 h. The mortality rates was obtained by the division of the number of death honeybee by the total honeybees in glass Petri dish (n = 10). All the bioassays was developed in the middle of spring season (August– November, 2008). The following commercial formulations of insecticides were assessed in the study, selected according to data on insecticides used in soybean crops: Lorsban 48EÒ (main active compound: chlorpyrifos, CAS No. 002921-88-2, 48% w/v, Dow AgroSciences Argentine S.A.); Thionex 35Ò (main active compound: endosulfan, CAS No. 115-29-7, 33% w/v, imported by Lanafil SA from Du Pont Brazil SA); and Cipermetrina 25 AgrinÒ (main active compound: cypermethrin, CAS No. 52315-07-8, 25% w/v, imported by Agro Internacional from China). These insecticides were assessed in the following concentration ranges: 0.015–0.250 lg honeybee1 for cypermethrin; 0.006–0.096 lg honeybee1 for chlorpyrifos; and 0.11–1.75 lg honeybee1 for endosulfan; these concentration ranges had been previously defined in preliminary tests (data not shown). The LD50 was determined using a generalized linear model (GLM) that employed a binomial distribution and logit or probit model (Crawley, 2007a,b,c). Statistical tests were run using the statistical package R version 2.12.0 (R Development Core Team, 2010) for the platform i486-pc-Linux-gnu (32-bit), with R Commander 1.5-4 (Fox, 2005; http://CRAN.R-project.org/package=Rcmdr) on a GNU/Linux operating system (Ubuntu 10.04, Canonical Ltd., http://www.ubuntu.com).
3. Results The genetic analysis showed that the colony has a north Mediterranean origin, since its haplotype was determined as C2 (Fig. 2). The morphometric parameters employed for the characterization of the colony allowed the determination of it African origin (Table 1). The bioassays were performed according to the requirements and criteria set by EPPO (1992), with the honeybee mortality rate less than 10% in control treatments and the monotonic mortality rates for each treatment assessed (Tables 2–4). All the estimations of the LD50 derived using the GLM satisfied the statistical conditions, with residual levels lower than the available degrees of freedom (Table 5). The LD50 determined for cypermethrin was in the range 0.097– 0.169 lg honeybee1, with a mean value of 0.124 lg honeybee1 and standard deviation of 0.039 lg honeybee1 (Table 5). The LD50 for chlorpyrifos was in the range 0.023–0.024 lg honeybee1, with a mean value of 0.024 lg honeybee1 and standard deviation of 0.0005 lg honeybee1 (Table 5). The LD50 determined for endosulfan was in the range 0.897–1.767 lg honeybee1, with a mean value of 1.240 lg honeybee1 and standard deviation of 0.463 lg honeybee1 (Table 5).
4. Discussion The employed methodology allowed the characterization of the honey bee colony. The results obtained by the morphometric and genetic analysis demonstrate the polyhybrid origin of the colony as the haplotype was a North Mediterranean origin and the morphometric analysis showed an African origin probability. The different values obtained considering the three parameters (forewing length, femur length and dry weight) alone and in combination could be because the grade of africanization of the colony.
Fig. 2. Genetic characterization of the colony INIA LE. A. PCR amplification of the COI–COII mitochondrial region. (1) 1 kb DNA ladder plus, Fermentas; (2) INIA-LE colony. B. Digestion of the amplified product with Fast Digest DraI, Fermentas. (1) ladder: 41pb, 47pb, 65pb, 108pb, 193pb, 420pb, 483pb; (2) INIA LE colony.
Table 1 African origin probability of the colony INIA LE by different morphometric parameters. Morphometric parameter
African origin probability
Forewing lenght Dry weight Forewing lenght and dry weight Forewing lenght and femur lenght Forewing lenght, dry weight and femur lenght
92.5 100 80 35.8 100
Table 2 Mortality rates of honeybees exposed to different doses of cypermethrin in acute toxicity bioassays. Replicate
1 2 3
Dose (lg cypermethrin honeybee1)
Control
0.015
0.030
0.060
0.130
0.250
0.0 0.0 0.1
0.0 0.2 0.2
0.0 0.2 0.4
0.3 0.6 0.5
0.8 0.8 0.8
0.0 0.0 0.0
This results are in agreement with other previous studies in our country which found hybrid colonies (based on genetic and/or morphometric analysis) (Burgett et al., 1995; Diniz et al., 2003). Similar results has been reported for neighborhood countries such as Argentina and Brazil. (Sheppard et al., 1991a,b; Diniz et al., 2003).
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Table 3 Mortality rates of honeybees exposed to different doses of chlorpyrifos in acute toxicity bioassays. Replicate
1 2 3
Dose (lg chlorpyrifos honeybee1)
Control
0.006
0.012
0.024
0.048
0.096
0.0 0.0 0.0
0.0 0.0 0.0
0.6 0.6 0.8
1.0 1.0 1.0
1.0 1.0 1.0
0.0 0.0 0.0
Table 4 Mortality rates of honeybees exposed to different doses of endosulfan in acute toxicity bioassays. Replicate
1 2 5
Dose (lg endosulfan honeybee1)
Control
0.11
0.22
0.44
0.88
1.750
0.1 0.1 0.1
0.1 0.1 0.1
0.3 0.2 0.4
0.5 0.2 0.3
0.17 0.6 0.7
Table 5 Median lethal doses (LDs50) (expressed as lg honeybee1, median lethal doses (LDs50) of reference values (PPDB, 2010),
0.1 0.1 0.1
of the commercial insecticide formulations tested with corresponding GLM residuals in brackets); and insecticides for honeybees according to international expressed as in lg honeybee1. Lorsban 48EÒ
Active compound
Cipermetrina 25 AgrinÒ Cypermethrin
Chlorpyrifos
Thionex 35Ò Endosulfan
Replicate 1
0.097 [0.48]
Replicate 2
0.169 [0.47]
Replicate 3
0.107 [1.81]
0.024 [4 1010] 0.024 [4 1010] 0.023 [5 1010]
0.897 [0.35] 1.767 [1.56] 1.056 [2.13]
Mean Standard deviation LD50-reference value
0.124 0.039 0.020
0.024 0.001 0.059
1.240 0.463 7.81
Commercial brand
The results of this study revealed notable differences from the reference values used by the EU (Table 5), as shown by the ratio of the honeybee LD50 set by the EU (PPDB, 2010; Table 5) to the LD50 determined in our study for the same commercial insecticide formulations. The comparison reveals that the LD50 values for Lorsban 48EÒ (chlorpyrifos) and Thionex 35Ò (endosulfan) were lower for SWZU honeybees than the EU reference values, implying an increased sensitivity to insecticide of 4.6 and 6.3 times, respectively. By contrast, cypermethrin differed in the opposite direction: the LD50 value for its commercial formulation Cipermetrina 25 AgrinÒ was 6.2 times higher for SWZU honeybees than the reference value set in the Pesticide Properties Database (PPDB, 2010); this suggests that SWZU honeybees have a higher tolerance to intoxication by cypermethrin than indicated by the EU reference values (Table 5). Two factors can potentially explain these differences: the polyhybridism of SWZU honeybees and the specific chemical mixture of the commercial formulations of the insecticides tested in this study. Polyhybridism gives rise to new genetic conditions that may lead to different tolerance ranges in the SWZU honeybee, as suggested by Suchail et al.’s (2000) results in their study on two different honeybee subspecies. However, this aspect may only partly explain the differences from the EU reference levels. These differences may also be the result of the chemical composition of each commercial formulation tested in our study. As mentioned previously, the specific acute toxicity of any chemical can be af-
fected by the mixture tested (Rozman et al., 2010). Although identifying the extent to which each of these mechanisms (polyhybridism, chemical mixture) affected the results constitutes an important basic scientific goal, our study design did not consider the contribution of each of these factors, because our focus was on comparing the LD50 values for SWZU honeybees with the EU reference values. Although elucidating this aspect is important, from the perspective of environmental management, a more urgent task is the definition of adequate field doses of insecticides. Defining LD50 values should be a priority in Uruguay and other countries from South America, given that the international reference values do not agree with local beekeeping conditions (genetic differences, characteristics of commercial insecticide formulation) and for the grew of soybean crops in the region (Morton et al., 2006; Viglizzo and Frank, 2006; Zak et al., 2008). The LD50 is used to determine the appropriate field doses of pesticides used near beekeeping areas. The LD50, expressed in milligrams of active compound per honeybee, is equivalent to the insecticide’s field dose expressed in pounds per hectare (kilograms per hectare, if the LD50 value is multiplied by 1.12; Atkins and Kellum, 1981). Hence, using the LD50 values determined in our study, we calculated the corresponding field doses that would kill 50% of the polyhybrid subspecies of SWZU honeybees for the chemicals tested, as follows: 0.139 kg ha1 for cypermethrin; 0.027 kg ha1 for chlorpyrifos; and 1.389 kg ha1 for endosulfan. However, as these insecticide chemical compounds are diluted in a commercial formulation, the concentration of each compound in the formulation must be considered in order to calculate the field doses of the commercial formulations. Therefore, the above doses correspond to 0.556 kg ha1 of Cipermetrina 25 AgrinÒ, 0.056 kg ha1 of Lorsban 48EÒ, and 4.209 kg ha1 of Thionex 35Ò. By comparison, the current maximum doses recommended for use in Uruguay are 0.080–0.200 L ha1 for Cipermetrina 25 AgrinÒ, 0.75–1.3 L ha1 for Lorsban 48EÒ, and 1.25–1.5 L ha1 for Thionex 35Ò (Modernel, 2009). This reveals important differences between the recommended doses and the LD50 determined in this study. For cypermethrin (Cipermetrina 25 AgrinÒ) and endosulfan (Thionex 35Ò), the recommended field doses are lower than the determined LD50. However, for chlorpyrifos (Lorsban 48EÒ), the field doses suggested by Modernel (2009) for use on soybean crops in Uruguay are higher than the LD50 values determined in this study. Indeed, according to our findings, the maximum recommended field dose of Lorsban 48EÒ is 23 times the LD50 value for SWZU honeybees found in this study. By contrast, the maximum recommended field doses of Cipermetrina 25 AgrinÒ and Thionex 35Ò are 50% of the respective LD50 values for SWZU honeybees. These results highlight the need to adjust the recommended field doses of the commercial formulations of the insecticides assessed in this study. The findings also suggest there is reason to conduct similar reviews of other insecticides authorized for use on soybean crops in Uruguay, in order to ensure sustainable development and management of beekeeping in the SWZU. Furthermore, these findings should be studied in relation to natural pollinators, in order to understand how the current trend of agricultural intensification could be affecting both existing ecosystem services and future agriculture, especially in terms of pollination capacity (Aizen et al., 2009). The situation found for Uruguayan soybean crop region can be considered as one with the neighboring countries, Argentine and Brazil. In this wider region, it is possible that the beekeeping production is undergoing a non-adequate exposure risk to insecticides such as SWZU. The first evidence to consider these three countries as a region are the results that showed that polyhybrid honeybee studied in Uruguay are similar to those honeybees from these neighboring countries; and second, because insecticides utilized and their recommended doses are almost the same. In some cases
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the only difference between these countries are the commercial brand names, but these insecticides belong to the same international companies. For example Cypermetrina 25, is imported from the same Chinese companies (Hangzhou Ruijiang Chemical Co., Ltd.; Shenzen Qinfeng Pesticide Co. Ltd., Dalian Winyard Chemical Co. Ltd.) to Uruguay, Argentine and Brazil; or the case of Lorsban 48 E made by Dow Agro Science in Argentine and Brazil. Current doses used in Brazil for Cipermetrine 25, Lorsban 480Ò (Dow AgroSciences Industrial Ltda., Brazil), and Thionex 35Ò (DuPont, Brazil) are 0.050–0.200 L ha1, 0.50–0.75 L ha1, and 1.0–1.5 L ha1, respectively. In Argentine, doses for the first two products are the same as in Brazil and Uruguay, while Thionex is used at a lower rate (0.9–1.0 L ha1) than in the other two countries. Regional similarities with the SWZU beekeeping and soybean cultivation suggests the need to check if the current maximum insecticides recommended doses for Argentine and Brazil are secure for the beekeeping production. 5. Conclusions The results of this study indicate that the acute toxicity response of polyhybrid honeybees in the SWZU to chlorpyrifos, cypermethrin, and endosulfan differs from the international reference values used in the European Union, which are based on A. m. mellifera. The findings indicate the need to adjust the recommended field doses of insecticides with respect to beekeeping both for honeybees other than A. m. mellifera and for commercial insecticide formulations with different chemical compositions. Finally, the current recommended doses of chlorpyrifos (commercial formulation: Lorsban 48EÒ) may be endangering beekeeping activity near soybean cultivation areas in the SWZU. Acknowledgments This work was financed by the INIA-Sa07 Grant ‘‘Tools for production and sustainability of watershed with forest aptitude’’ (INIA-Uruguay). We thank K. Antunez and Silvina Stewart for her collaboration with this study, and to the technicians G. Ramallo, S. Díaz, M. Vera and P. Ojeda for theirs assistances in laboratory and field works. We also thank the organizations that share their free software for GNU/linux (Ubuntu, GNOME, GIMP, KDE, LibreOffice, R-CRAN, and Quantum GIS). References Aizen, M.A., Garibaldi, L.A., Cunningham, S.A., Klein, A.M., 2009. How much does agriculture depend on pollinators? Lessons from long-term trends in crop production. Ann. Bot. 103, 1579–1588. Atkins, E.L., Kellum, D., 1981. Reducing Pesticide Hazard to honey bees: mortality prediction techniques and integrated management strategies. Dept. of Enthomology, Division of Agricultural Sciences. University of California, USA. Burgett, M., Shorney, S., Codara, J., Gardiol, G., Sheppard, W., 1995. The present status of the Africanized honey bees in Uruguay. Am. Bee J. 135, 328–330. Cespedes-Payret, C., Pineiro, G., Achkar, M., Gutierrez, O., Panario, D., 2009. The irruption of new agro-industrial technologies in Uruguay and their environmental impacts on soil, water supply and biodiversity: a review. IJEnvH 3, 175–197. Crawley, M.J., 2007a. Generalized linear models. In: Crawley, M.J. (Ed.), The R Book. John Wiley & Sons, Ltd., pp. 511–526. Crawley, M.J., 2007b. Proportion data. In: Crawley, M.J. (Ed.), The R Book. John Wiley & Sons, Ltd., pp. 569–591. Crawley, M.J., 2007c. Binary response variables. In: Crawley, M.J. (Ed.), The R Book. John Wiley & Sons, Ltd., pp. 593–609. Daly, H.V., Hoelmer, K., Norman, P., Allen, T., 1982. Computer-assisted measurement and identification of honey bees (Hymenoptera: Apidae). Ann. Entomol. Soc. Amer. 75, 591–594. Del Lama, M.A., Figueiredo, R.A., Soares, A.E.E., Del Lama, S.N., 1988. Hexokinase polymorphism in Apis mellifera and its use for africanized honeybee identification. Rev. Brazil Genet. 11, 287–297. DGSSAA, 2011. Resumen Estadístico. Importación de productos fitosanitarios. División de Análisis y Diagnóstico. Dirección de Servicios Agrícolas. Ministerio de Ganadería Agricultura y Pesca, Montevideo, Uruguay. (in Spanish).
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