Elsevier Editorial System(tm) for Chemosphere Manuscript Draft Manuscript Number: CHEM60158R2 Title: Nanopesticide based on botanical insecticide pyrethrum and its potential effects on honeybees Article Type: Research paper Section/Category: Toxicology and Risk Assessment Keywords: Nanopesticide; Biocide; Sustainable agriculture, Solid lipid nanoparticles; Bees. Corresponding Author: Dr. Leonardo Fernandes Fraceto, Ph.D Corresponding Author's Institution: State University of São Paulo First Author: Cristiane Ronchi de Oliveira, D.D. Order of Authors: Cristiane Ronchi de Oliveira, D.D.; Caio Eduardo Domingues, D.D.; Nathalie de Melo, PhD; Thaisa Roat, PhD; Osmar Malaspina, Dr.; Monica Jones-Costa, Dr.; Elaine Silva-Zacarin, Dr.; Leonardo Fraceto, Dr. Abstract: Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and nanopesticides can control agricultural pests and increase farm productivity with little environmental impact. However, it is important to evaluate their toxicity on non-target organisms, such as honeybees (Apis mellifera) that forage on crops. The aims of this study were to develop a nanopesticide that was based on solid lipid nanoparticles (SLNs) loaded with pyrethrum extract (PYR) and evaluate its physicochemical properties and short-term toxicity on a non- target organism (honeybee). SLN+PYR was physicochemically stable after 120 days. SLN+PYR had a final diameter of 260.8 ± 3.7 nm and a polydispersion index of 0.15 ± 0.02 nm, in comparison with SLN alone that had a diameter of 406.7 ± 6.7 nm and a polydispersion index of 0.39 ± 0.12 nm. SLN+PYR had an encapsulation efficiency of 99%. The survival analysis of honeybees indicated that PYR10ng presented shorter longevity than those in the control group (P ≤ 0.01). Empty nanoparticles and PYR10ng caused morphological alterations in the bees' midguts, whereas pyrethrum-loaded nanoparticles had no significant effect on digestive cells, so are considered safer, at least in the short term, for honeybees. These results are important in understanding the effects of nanopesticides on beneficial insects and may decrease the environmental impacts of pesticides. Can a nanopesticide based on solid lipid nanoparticles loaded with the botanical insecticide pyrethrum be toxic to honeybees? Cristiane R. Oliveira 1,2; Caio E. C. Domingues ³; Nathalie F. S. de Melo4; Thaisa C. Roat 3; Osmar Malaspina3; Monica Jones-Costa²; Elaine C. M. Silva-Zacarin²; Leonardo F. Fraceto¹ 1 – Universidade Estadual Paulista (UNESP), Instituto de Ciência e Tecnologia de Sorocaba, Laboratório de Nanotecnologia Ambiental, Av. Três de Março, 511, Alto da Boa Vista, 18087-180, Sorocaba, SP, Brazil. Email: cristianeronchi@hotmail.com; leonardo@sorocaba.unesp.br 2 – Universidade Federal de São Carlos (UFSCar), Campus Sorocaba, Departamento de Biologia (CCHB), Laboratório de Fisiologia da Conservação e Laboratório de Ecotoxicologia e Biomarcadores em Animais, Rodovia João Leme dos Santos km 110, Itinga, 18052-780, Sorocaba, SP, Brazil. Email: monica@ufscar.br; elaine@ufscar.br 3 –Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho”, Campus Rio Claro, Departamento de Biologia, Centro de Estudos de Insetos Sociais (CEIS),, Av. 24 A, 1515, Jardim Bela Vista, 13506-900, Rio Claro, SP, Brazil. Email: cecdomingues@gmail.com; thaisaroat@yahoo.com.br; malaspin@rc.unesp.br 4 – Faculdade de Medicina São Leopoldo Mandic, Campus Araras. Av. Dona Renata, 71, Santa Cândida, 13600-001, Araras, SP, Brazil. Email: nathaliemelo@gmail.com 01th February 2019 COVER LETTER Dear Editor of Chemosphere, I am submitting the original article “Can a nanopesticide based on solid lipid nanoparticles loaded with the botanical insecticide pyrethrum be toxic to honeybees?” (Cristiane R. OLIVEIRA et al.) for the refereeing process, in order to publish it in the Chemosphere. Aiming to minimize the effects of pesticides on non-target beneficial insects, nanoparticles that act as carrier systems for agrochemicals are being developed by means of nanotechnology. The solid lipid nanoparticles encapsulated pyrethrum biocide releases small quantities over time and thereby reduces the amount of chemical compound bioavailable in the environment. Nevertheless, it is necessary to assess the adverse effects of nanopesticides in the terrestrial environment. In this sense, our study is pioneer in evaluating the toxicity of this system on a non-target pollinator insect, the honeybees. We tried to follow precisely the journal’s author guidelines, with the title page article, Introduction, Materials and Methods, Results and Discussion and Acknowledgment. Additional Information - Total number of words of the textual elements: 6117; Total number of Tables: 1; Total number of Figures: 4. Sincerely, Dra. Elaine C. M. Silva-Zacarin Corresponding Author Dr. Leonardo Fernandes Fraceto Corresponding Author Cover Letter Sorocaba, June 30th 2019. Dear Prof. Willie J. G. M. Peijnenburg Editor Chemosphere, Ref. Chem60158 RESPONSE TO EDITOR IN CHIEF AND REVIEWER Reviewer comment: I thank you very much for submitting your revised manuscript. Having evaluated the responses to the comments made by the reviewers, there is one issue that I do not agree on and that is on the issue of the definition of nanoparticle. 100 nm is considered the upper limit of size in one dimension to allow a particle to be termed a nanoparticle. In your case, the particles are of a size of 260 nm and they should therefore not be termed 'nanoparticle' but they are 'submicron particles'. Throughout the manuscript, the term 'nano' therefore needs to be replaced by 'submicron', including in the title of the manuscript. This is depite the arguments raised in Nature Nanotechnology. Answer: The authors are very thankful to the Reviewer for his(her) valuable comment regarding the nano definition. We really respect his(her) point of view, however, we disagree to change the term nanoparticles as well as nanopesticides in the manuscript to submicron particles. Our arguments are: i) We can not use only size range to define a nanoparticle. In this way, the properties that we got with solid lipid nanoparticles in the range of size that we have in this study is totally different from the properties with bulk material. To support this statement, please look at A.D. Maynard, Don’t define nanomaterials, Nature, 2011, 475, 31–31. ii) It is clear in literature that nanoparticles prepared with polymeric and lipid materials showed a size distribution in the same range of the particles from our study and these particles are considered nanoparticles due its properties reached in the size range. Easily it is possible to find thousands of published papers in many different areas such as: cosmetics, food, medicine, pharmacy, agriculture that use particles with the same characteristics (lipid particles) and are considered by the scientific community as nanoparticles. *Response to reviewers/editor in question & answer format (word file) iii) It is stated by the editorial from Nature Nanotechnology that in the case of nanopesticides authors showed that the size range threshold is higher for this kind of systems. iv) European Food Safety Authority, a regulatory agency, described in recent guidance that nanomaterials definitions should be reconsidered for food and agriculture since they described that particles larger than 100 nm but retain properties typical of nanoparticles. v) Food and Drug Administration – USA – definition (https://www.fda.gov/regulatory- information/search-fda-guidance-documents/considering-whether-fda-regulated-product-involves- application-nanotechnology#_ftn6): “At this time, when considering whether an FDA-regulated product involves the application of nanotechnology, FDA will ask: 1. Whether a material or end product is engineered to have at least one external dimension, or an internal or surface structure, in the nanoscale range (approximately 1 nm to 100 nm); In addition, as we explain in more detail below, because materials or end products can also exhibit related properties or phenomena attributable to a dimension(s) outside the nanoscale range of approximately 1 nm to 100 nm that are relevant to evaluations of safety, effectiveness, performance, quality, public health impact, or regulatory status of products, we will also ask: 2. Whether a material or end product is engineered to exhibit properties or phenomena, including physical or chemical properties or biological effects, that are attributable to its dimension(s), even if these dimensions fall outside the nanoscale range, up to one micrometer (1,000 nm).” vi) Recently Nature Nanotechnology has published a series of papers that were written by worldwide specialists about the nanotechnology in agriculture (see below) and in all these papers there are a lot of citations of papers that showed size higher than 100 nm and they were considered nanomaterials/nanoparticles/nanopesticides/nanofertilizers. - https://www.nature.com/articles/s41565-019-0464-4 - https://www.nature.com/articles/s41565-019-0468-0 - https://www.nature.com/articles/s41565-019-0461-7 - https://www.nature.com/articles/s41565-019-0460-8 - https://www.nature.com/articles/s41565-019-0439-5 vii) If you look at the EU homepage below it is possible to find the definition: “Upper size limit Although 999 nm is still formally on the nanoscale, a very commonly used upper limit for nanomaterial size is 100 nm. This covers most nanomaterials, but there are exceptions. Nanomaterials clumped together can have outside dimensions larger than 100 nm, as can those which have been modified by adding a coating or an unusually large chemical group such as a long-chain organic molecule. Such materials include liposomes – small fatty globules – which can be loaded with nanoparticles for drug delivery or use in cosmetic products.” https://ec.europa.eu/health/scientific_committees/opinions_layman/nanomaterials2012/en/l-2/3.htm In this way, as our system is a solid lipid nanoparticles, this mean a structure formed by lipid covered by a surfactant it is like a liposomes, fatty globules and as mentioned below, in the area of cosmetics this is considered as nanoparticles. vii) The Chemosphere Journal has published papers aiming pest control with particles with mean size distributions higher than 400 nm and they accepted the use of the term nanoparticles. Just as example, look at: https://doi.org/10.1016/j.chemosphere.2013.11.056 Also, based on all arguments above, we do not agree to change the term nanoparticles to submicron particles. We would like thank you so much the reviewer for this discussion, but from our point of view is really more than a question of size limit (100 nm) and by properties of the material. In addition, the application of polymeric and lipid materials in agriculture are well known nowadays and the community that develop systems for this kind of application really considered sizes in the range from the particles of our study as nanoparticles. Again, thank you for your comment that we really appreciate, but in this case, we can’t agree with your suggestion to change the term in the manuscript since nowadays the scientific community has been accepted other definitions than a cut-off 100nm. Sincerely yours Dr. Leonardo Fraceto Corresponding author On-behalf of all authors. Nanopesticide based on botanical insecticide pyrethrum and its 1 potential effects on honeybees 2 Cristiane R. Oliveiraa,b; Caio E. C. Dominguesc; Nathalie F. S. de Melod; Thaisa C. Roatc; Osmar 3 Malaspinac; Monica Jones-Costab; Elaine C. M. Silva-Zacarinb and Leonardo F. Fracetoa* 4 5 a Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho”, Instituto de Ciência e Tecnologia de 6 Sorocaba, Laboratório de Nanotecnologia Ambiental, Av. Três de Março, 511, Alto da Boa Vista, 18087-180, 7 Sorocaba, SP, Brazil. 8 b Universidade Federal de São Carlos (UFSCar), Campus Sorocaba, Departamento de Biologia (CCHB), 9 Laboratório de Fisiologia da Conservação e Laboratório de Ecotoxicologia e Biomarcadores em Animais, 10 Rodovia João Leme dos Santos km 110, Itinga, 18052-780, Sorocaba, SP, Brazil. 11 c Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho”, Campus Rio Claro, Departamento de 12 Biologia, Centro de Estudos de Insetos Sociais (CEIS), Av. 24 A, 1515, Jardim Bela Vista, 13506-900, Rio 13 Claro, SP, Brazil. 14 d Faculdade de Medicina São Leopoldo Mandic, Campus Araras. Av. Dona Renata, 71, Santa Cândida, 13600-15 001, Araras, SP, Brazil. 16 17 ABSTRACT 18 Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and 19 nanopesticides can control agricultural pests and increase farm productivity with little 20 environmental impact. However, it is important to evaluate their toxicity on non-target 21 organisms, such as honeybees (Apis mellifera) that forage on crops. The aims of this study 22 were to develop a nanopesticide that was based on solid lipid nanoparticles (SLNs) loaded 23 with pyrethrum extract (PYR) and evaluate its physicochemical properties and short-term 24 toxicity on a non-target organism (honeybee). SLN+PYR was physicochemically stable after 25 120 days. SLN+PYR had a final diameter of 260.8 ± 3.7 nm and a polydispersion index of 26 0.15 ± 0.02 nm, in comparison with SLN alone that had a diameter of 406.7 ± 6.7 nm and a 27 polydispersion index of 0.39 ± 0.12 nm. SLN+PYR had an encapsulation efficiency of 99%. 28 The survival analysis of honeybees indicated that PYR10ng presented shorter longevity than 29 those in the control group (P ≤ 0.01). Empty nanoparticles and PYR10ng caused morphological 30 alterations in the bees’ midguts, whereas pyrethrum-loaded nanoparticles had no significant 31 effect on digestive cells, so are considered safer, at least in the short term, for honeybees. 32 These results are important in understanding the effects of nanopesticides on beneficial 33 insects and may decrease the environmental impacts of pesticides. 34 35 KEYWORD: Nanopesticide; Biocide; Sustainable agriculture, Solid lipid nanoparticles; 36 Bees. 37 38 Corresponding Authors 39 * Elaine C. M. Silva Zacarin - Universidade Federal de São Carlos (UFSCar), Campus Sorocaba, Departamento 40 de Biologia (Dbio, CCHB), Laboratório de Fisiologia da Conservação e Laboratório de Ecotoxicologia e 41 Biomarcadores em Animais, Rodovia João Leme dos Santos km 110, Itinga, 18052-780, Sorocaba, SP, Brazil. 42 Email: elaine@ufscar.br 43 *Leonardo Fernandes Fraceto – Universidade Estadual Paulista (UNESP), Instituto de Ciência e Tecnologia de 44 Sorocaba, Av. Três de Março, 511, Alto da Boa Vista, 18087-180, Sorocaba, SP, Brazil. Email – 45 leonardo.fraceto@unesp.br 46 47 *Revised manuscript with changes marked Click here to view linked References mailto:elaine@ufscar.br mailto:leonardo.fraceto@unesp.br http://ees.elsevier.com/chem/viewRCResults.aspx?pdf=1&docID=89035&rev=2&fileID=2095524&msid={ADFEA4FE-0F1F-4AD4-8FA4-102F5488AE22} 2 1. INTRODUCTION 48 Agri-food production and population growth are amongst the greatest challenges 49 facing humanity. Agriculture is one of the primary drivers of the economy by providing food 50 to the population and benefiting producing countries, but increased population growth has 51 significantly increased humanity’s global ecological footprint, surpassing the biocapacity of 52 the Earth (SEKHON, 2014). Human populations increase exponentially over time, whereas 53 food production increases in a linear manner. Conventional agricultural practices generally 54 have negative impacts on the environment and biodiversity, as they require many resources 55 such as energy, water, and soil, and large amounts of agrochemicals and fertilizers are used 56 to improve productivity. 57 The U.S. Department of Agriculture’s (USDA) National Institute of Food and 58 Agriculture (NIFA, 2018) aims to find innovative solutions to issues related to agriculture, 59 food, the environment, and communities. NIFA’s priorities include global food security and 60 hunger, food safety, plant health and production, and animal health and production (NANO, 61 2018). Many of these issues may be resolved using nanotechnology, which has demonstrated 62 great potential in providing novel solutions to agricultural problems (SCOTT and CHEN, 63 2012; MUKHOPADHYAY, 2014). In the last few decades, nanoscience and nanotechnology 64 have been at the forefront of the development of several nanomaterials for different medical 65 and industrial purposes. Nanoparticles have been developed for a wide variety of applications 66 in the biomedical and electronic fields, while research on nanoparticles as carriers of 67 pesticides has only been conducted in the last decade, and there are still many variables to be 68 investigated before their use on crops (LIU et al., 2008; ANJALI et al., 2010; GOPAL et al., 69 2012; KAH et al., 2014; SARLAK et al., 2014; MISHRA et al., 2017; KIM et al., 2018). 70 3 Nanotechnology can deliver agricultural substances such as nanopesticides and 71 nanofertilizers that increase farm productivity, decrease the environmental impact and the 72 amount of resources used, improve pest control, and support sustainable agriculture, 73 particularly in developing countries. Furthermore, nanocarriers of pesticides and fertilizers 74 have economic advantages for agriculture, because their stability and controlled-release 75 mechanism increase efficiency and reduce the amount of chemicals required on crops 76 (PEREZ-DE-LUQUE and RUBIALES; 2009; CHEN and YADA, 2011; GRILLO et al., 77 2016; PRASAD et al., 2017; WALKER et al., 2017). 78 However, the effects of nanoparticles should be fully evaluated before they are 79 incorporated into sustainable agriculture. The U.S. National Science Foundation (NSF) and 80 Environmental Protection Agency (EPA) encourage the investigation of various aspects of 81 nanomaterials, such as their toxicity to non-target organisms, their destination, transportation, 82 and safety in the environment, and their status in terms of food legislation, and support the 83 creation of a nanomaterial database and the maintenance of food regulations (SCOTT and 84 CHEN, 2012). 85 Pyrethrum extract is a natural botanical insecticide that is extracted from 86 chrysanthemum (Chrysanthemum cinerariaefolium and Chrysanthemum cineum) flowers, is 87 composed of pyrethrin types I and II and jasmolin, and can be used on crops to control pest 88 insects (PEAY et al., 2006). Natural pyrethrum (a.i.) is highly lipophilic, photodegradable, 89 has low water solubility (<10 mg.L-1), does not exhibit biomagnification (SCHLEIER and 90 PETERSON, 2011), and leaves no toxic residues in plants. However, it is more expensive 91 than synthetic pyrethroids (PEAY et al., 2006) and is highly toxic to insects, aquatic 92 invertebrates, and fish (USEPA, 2006). Pyrethroids are insecticides that were developed to 93 improve the photodegradation of natural pyrethrin, and thus be used as an insecticide in the 94 4 field (SANTOS et al., 2007), and have great stability and target selectivity. Examples of 95 pyrethroids include deltamethrin, permethrin, and cypermethrin (MONTANHA and 96 PIMPÃO, 2012). 97 However, for the use of pyrethrum extract in the field it is necessary, at first, to load 98 it into solid lipid nanoparticles (SLNs) to prevent its fast degradation, improving its stability 99 and efficiency to allow its application on crops. Many benefits can be obtained by using 100 SLNs, such as lower large-scale production costs, greater physicochemical stability, the 101 possibility of hydrophilic and hydrophobic drug encapsulation, and the use of natural 102 products in the formulation preparation (MULLER et al., 2000; MULLER et al., 2011; 103 NASERI et al., 2015; SARANGI and PADHI et al., 2016). 104 Interactions between biological systems and nanomaterials are complex, so it is 105 important to evaluate their toxicity to non-target organisms (JACQUES et al., 2017), 106 particularly to beneficial insects such as honeybees (Apis mellifera), which play an important 107 role in pollinating agricultural crops (GIANNINI et al., 2015). Honeybee populations are 108 declining worldwide, and although multiple factors contribute to this decline (GOULSON et 109 al., 2015), it is mainly caused by agrochemicals sprayed on crops visited by bees (POTTS et 110 al., 2010). In this context, the physicochemical characterization of nanopesticides can enable 111 their future use in organic farming and contribute to sustainable agriculture, because these 112 carriers may have little effect on the environment and biodiversity (GRILLO et al., 2016; 113 PRASAD et al., 2017). However, this carrier system must have low toxicity to honeybees 114 and other beneficial insects. 115 The objectives of this study were to develop a nanopesticide that was based on SLNs 116 loaded with pyrethrum extract biocide (nanobiocide), characterize its physicochemical 117 properties, and evaluate its toxicity to honeybees (Africanized A. mellifera). We evaluated 118 5 sublethal effects on the histopathology of the bee midgut, an organ that plays a central role 119 in food digestion and nutrient absorption. It is important to emphasize the fact that there are 120 gaps of information in the literature regarding the toxicity of nanopesticides to non-target 121 organisms, such as pollinator insects including honeybees. Our results can be applied in the 122 field, can contribute to nanopesticide regulation, and can improve both environmental and 123 food security. 124 125 2. MATERIALS AND METHODS 126 2.1. Chemicals 127 The pyrethrum extract Pestanal® (biocide, CAS 8003-34-7, analytical standard), 128 polyvinyl alcohol (PVA, 30–70 kDa, CAS 9002-89-5, hydrolyzed >99%), and glyceryl 129 tripalmitate (tripalmitin, CAS 555-44-2, purity ≥99%) were purchased from Sigma-Aldrich. 130 Chloroform (CHCl3, CAS 67-66-3, purity ≥99%) was purchased from a local supplier. All 131 these products were used for the preparation of the nanoparticles. Acetone (CAS 67-64-1, 132 purity = 100%) was used as a solvent in the preparation of the pyrethrum solution. 133 134 2.1.1. Solid lipid nanoparticles 135 SLNs containing pyrethrum were prepared by the method of emulsification/solvent 136 evaporation with some modifications (VITORINO et al., 2011; de MELO et al., 2018). 137 Initially, 30 mL of an aqueous phase containing 1.25% PVA and distilled water was prepared 138 and magnetically stirred (100 rpm). An organic phase with 250 mg of glyceryl tripalmitate 139 and 5 mg of pyrethrum (active ingredient – a.i.) was then prepared, which was dissolved in 140 5 mL of chloroform. The organic phase was added to the aqueous phase, and this mixture 141 was sonicated at 40 W for 5 min producing an emulsion. The emulsion was placed in an 142 6 ULTRA-TURRAX™ homogenizer at 14,000 rpm for 7 min. The organic solvent was then 143 removed using a rotating evaporator in order to create a concentrated emulsion with 10 mL 144 of nanoparticles. The final concentration of biocide was 0.05 mg.mL-1. SLNs without 145 pyrethrum extract (control) were also prepared. 146 147 2.2. Nanoparticles 148 The purpose of the formulations was to achieve greater physicochemical stability and 149 better efficiency of pyrethrum encapsulation in the nanoparticles. In order to evaluate the 150 physicochemical stability as a function of time were used the maintenance of colloidal 151 parameters in formulation. The colloidal parameters were the mean diameter, polydispersity 152 index, zeta potential, besides the nanoparticle concentration and encapsulation efficiency of 153 the pyrethrum extract. All analyses were conducted for 120 days and the results were 154 expressed (mean ± SEM). 155 156 2.2.1. Nanoparticle characterization 157 The mean diameter and polydispersion index were determined by dynamic light 158 scattering (DLS). Nanoparticle samples were diluted (10 µL:1 mL) in purified water and 159 analyzed using a Zetasizer Nano ZS90 analyzer (Malvern Panalytical, UK). Zeta potential 160 values (in mV) were also determined using the ZS90 analyzer, with the same dilution process. 161 The pH of the nanoparticles was determined using a pH meter (Tecnal®, Brazil). Further 162 details could be obtained in literature (VENKATRAMAN et al., 2005; de MELO et al., 2012; 163 OLIVEIRA et al., 2015). 164 165 2.2.2. Nanoparticle concentration 166 7 SLN size distributions and concentrations were analyzed using a nanoparticle 167 tracking analysis (NTA) instrument (NanoSight LM10). Nanoparticle samples were diluted 168 10,000 times and analyzed by injecting 1 mL of the sample into the cell (more details in 169 section 1.1 - Supplementary Material). 170 171 2.2.3. Differential Scanning Calorimetry (DSC) 172 A thermal analysis was performed to demonstrate that the pyrethrum was 173 encapsulated in the nanocarriers using a DSC Q20 differential scanning calorimeter (TA 174 Instruments). The samples of pyrethrum extract, lipid, SLNs, and SLNs loaded with 175 pyrethrum were analyzed (Section 1.2 - Supplementary Material). 176 177 2.2.4. Fourier-transform infrared spectroscopy (FTIR) 178 FTIR was performed to investigate interactions between the biocide and the SLNs 179 using an infrared spectrophotometer (Agilent). The pyrethrum extract, lipid, surfactant 180 (PVA), physical mixture, SLNs, and SLNs loaded with pyrethrum were analyzed using an 181 attenuated total reflectance accessory (POLLETO et al., 2007; WANG et al., 2010) (Section 182 1.3 - Supplementary Material). 183 184 2.3. Determination of encapsulation efficiency and quantification of pyrethrum by high-185 performance liquid chromatography (HPLC) 186 The total amount of pyrethrum extract present in the nanoparticle suspension was 187 determined by the ultrafiltration/centrifugation method. After the suspension had been 188 diluted with acetonitrile, it was filtered through a 0.22 μm Millipore™ membrane filter and 189 quantified by HPLC (Varian ProStar). The pyrethrum extract association rate was calculated 190 8 as the difference between the non-associated fraction of biocide and the total amount initially 191 added to the nanoparticles (GAMISANS et al., 1999; SCHAFFAZICK et al., 2003; KILIC 192 et al., 2005) (Table 1S- Supplementary Material). 193 194 2.4. Toxicological bioassay 195 Operculated brood combs were collected from three healthy colonies of Africanized 196 Apis mellifera located in apiaries at Sao Paulo State, Brazil. The emergence of worker bees 197 was monitored in laboratory. Following emergence, the bees were transferred to plastic pots 198 lined with filter paper and fed ad libitum sugar-aqueous solution (50%:50% water:inverted 199 sugar, v:v) to acclimatize for 24 h. 200 Subsequently, the 1-day-old bees were divided into the following experimental 201 groups in triplicate (each colony representing a replicate): I) Control (CTL) - sugar-aqueous 202 solution (syrup); II) Sublethal dose (1 ng.µL-1) of pyrethrum extract (PYR1ng); III) Sublethal 203 dose (10 ng.µL-1) pyrethrum extract (PYR10ng); IV) 1 ng.µL-1 of pyrethrum loaded in SLNs 204 (SLNP1ng); V) 10 ng.µL-1 of pyrethrum loaded in SLNs (SLNP10ng); IV) Empty SLNs; V) 205 Polyvinyl alcohol - surfactant control (PVA); VI) Acetone control (ACN) - vehicle/solvent 206 control. The dose used per bee was based on the LD5048h of pyrethrum for honeybees, i.e., 207 22 ng.bee-1 (USEPA, 1991). 208 Acute exposure was performed individually by oral administration, i.e., the 209 corresponding solution of the experimental group was administrated to the bees (1 µL) using 210 a micropipette (per os administration). Two sublethal doses of 10 ng or 1 ng of biocide per 211 bee were given of the pyrethrum extract (PYR) and pyrethrum loaded in nanoparticles 212 (SLNs). The half the LD5048h value corresponded to a 1/2 dilution (LD50/2 = 10 ng.µL-1 = 10 213 ppm), and the other dose corresponded to a 1:20 dilution of the LD5048h value (LD50/20 = 1 214 9 ng.µL-1 = 1 ppm), both being sublethal concentrations for honeybees. Concentrations of the 215 solutions, which were used for getting the sublethal doses offered to bees, were obtained by 216 serial dilution of stock solution. 217 After individually acute exposure, the bees were kept in plastic pots (cages), being 218 fed with 50% (w/w) sucrose aqueous solution, in an incubator at a relative humidity of 70% 219 ± 5 and temperature of 32 ± 2ºC, under dark conditions. Two bioassays were performed, 220 being the first one for survival analysis (N = 12 bees per pot in triplicate, per experimental 221 group, totalizing 36 individuals) and another one for histology analyzes (N = 15 bees per pot 222 in triplicate per experimental group, totalizing 45 individuals). 223 In the first bioassay (survival analysis), the bees were monitored daily until the last 224 bee has died. Specifically for survival bioassay, the deltamethrin (DLT, 10 ng.µL-1) 225 experimental group was added as positive control. In the second bioassay, the bees were 226 collected 48 h after the acute exposure (N = 6 per group) and dissected for midguts’ removal, 227 which were processed for resin embedding and histological analysis (section 2.4.1). 228 229 2.4.1. Histology procedure 230 The bee midguts were fixed in 4% buffered paraformaldehyde solution for 24 h and 231 immersed in phosphate-buffered saline (0.1 mol.L-1 phosphate buffer, pH 7.4). After, the 232 material was dehydrated in an increasing ethanol series according to Silva-Zacarin et al. 233 (2012). Subsequently, the material was embedded in historesin, and submitted to microtomy. 234 Slides containing 3-µm thick histological sections were stained with hematoxylin-eosin. 235 Posteriorly, the material was photodocumentated and both qualitative and semi-quantitative 236 histopathological analyses were performed using Leica Application Suite V3.8 coupled to 237 the light field photomicroscope (DM1000, Leica). For each bee from each experimental 238 10 group (N = 6), two slides were analyzed per individual and three non-sequential histological 239 sections were analyzed for each slide. 240 Other slides containing 3-µm thick histological sections were submitted to 241 histochemical analysis for detection of proteins, lipids and neutral glycoconjugates (SILVA-242 ZACARIN et al., 2012) (Section 1.4 - Supplementary Material and Figure 4S). 243 244 2.4.2. Semi-quantitative analysis of midguts 245 Parameters for semi-quantitative analysis were defined according to the Bernet et al. 246 (1999) protocol, and histological alterations (lesions) in midgut of bees were based on 247 Soares-Lima et al. (2018) protocol. To determine alterations in the bee midguts, the lesion 248 index and the organ index, were calculated using two parameters: the importance factor and 249 the score value (BERNET et al., 1999). Alterations were classified from 0 to 3, depending 250 on their degree and extent: 0- no alteration, 1- slight alteration, 2- moderate alteration, and 251 3- severe alteration. The importance factor was established for each lesion observed (cells 252 eliminated from the epithelium, increased apocrine secretions from the digestive cells, 253 cellular vacuolization, changes in regenerative cells’ nests, and the presence of pyknotic 254 nuclei in cells of the epithelium) by a qualitative analysis based on pathological severity. This 255 factor was categorized as (1) minimal pathological importance (repairable damage), (2) 256 moderate pathological importance (damage was repairable in most cases), or (3) severe 257 pathological importance (irreparable damage) (Table 2S and section 1.4 - Supplementary 258 Material). 259 260 2.5. Statistical analysis 261 11 All data were previously subjected to homogeneity of variance (Bartlett’s) and 262 normality (Shapiro-Wilk and Kolmogorov-Smirnov) tests. The physicochemical 263 characterization data were subjected to a Student’s t-test followed by a Mann Whitney test. 264 A semi-quantitative analysis of the bee midguts was performed using a Kruskal-Wallis test 265 followed by Dunn’s multiple comparison test. The significance level was set at α = 0.05. 266 GraphPad Prism v.5.0 was used for these statistical analyses. 267 The survival curve of honeybees per each experimental group was analyzed by the 268 Log-Rank test (Kaplan-Meier method), and comparison between survival time of the groups 269 was performed by the Holm-Sidak test. The significance level was set at α = 0.05. SigmaPlot 270 13 software was used these analyze. 271 272 3. RESULTS AND DISCUSSION 273 3.1. Nanoparticle characterization 274 The SLNs were prepared using approved components that are generally recognized 275 as safe (GRAS). Tripalmitin (glyceryl tripalmitate) was used as a solid lipid and PVA was 276 used as a surfactant. Physicochemical stability of the empty and encapsulated biocide in 277 SLNs were evaluated from maintenance measurements of the colloidal parameters (mean 278 diameter, polydispersity and zeta potential), besides the concentration of nanoparticles and 279 pyrethrum encapsulation efficiency, over time (0 to 120 days). Colloidal parameter values 280 and other parameters are shown in Table 1. 281 The initial and final hydrodynamic diameters (mean ± SEM) of the empty solid lipid 282 nanoparticles (SLN) were 290.0 ± 5.0 and 406.7 ± 6.7 nm, respectively. For the SLNs loaded 283 with pyrethrum (SLN+PYR) the initial and final hydrodynamic diameters were 264.9 ± 2.8 284 and 260.8 ± 3.7 nm, respectively. There was a significant difference between the empty 285 12 nanoparticles and those loaded with pyrethrum in the initial (P ≤ 0.0001 and T = 18.18) and 286 final (P ≤ 0.0001 and T = 48.51) analyses. The hydrodynamic diameter values of empty SLNs 287 increased after 60 days of storage with significant differences between the timepoints (P ≤ 288 0.0001 and T = 54.60), while these values remained stable for SLN+PYR over the 289 experimental period (120 days) (Figure 1SA- Supplementary Material). The empty SLNs had 290 a larger mean diameter and less physicochemical stability than SLN+PYR, indicating that 291 active ingredient of pyrethrum can stabilize nanoparticle formulation and decrease aggregate 292 formation. 293 The polydispersion index at 0 and 120 days was 0.12 ± 0.01 and 0.39 ± 0.12 nm, 294 respectively, in empty SLNs, and 0.12 ± 0.01 and 0.15 ± 0.02 nm, respectively, in SLN+PYR 295 (Table 1), and values below 0.2 nm in the initial analysis were considered indicative of good 296 stability and a small distribution of particle diameters. The low values indicate that the 297 nanoparticles were of similar size and without aggregates (MASARUDIN et al., 2015). 298 Similar results were obtained by de Melo et al. (2016) in a 120-day experiment with 15d-299 PGJ2-loaded SLNs, and by González et al. (2015) at the beginning of their experiment with 300 poly (ethylene glycol)-nanoparticles containing geranium (an essential oil). However, the 301 time-based analysis revealed that the SLN polydispersion index had increased after 60 days 302 of storage (0.3 and 0.39 nm; Figure 1SB - Supplementary Material), with significant 303 differences between the timepoints (P ≤ 0.005 and T = 0.0) and significant differences 304 between SLN120 and SLN+PYR120 (P ≤ 0.005 and T = 0.0). These data indicate that there 305 was a heterogeneous distribution of particle diameters, i.e., there was a greater aggregation 306 of particles in the empty system (SLN). Particle aggregation and degradation occur in SLN 307 formulations that increase and decrease particle size, respectively, due to the loss of a 308 surfactant coating that protects the material (MULLER et al., 1996). 309 13 Both nanosystems had a negative zeta potential, with initial and final values of -13 ± 310 0.4 and -14 ± 0.3 mV, respectively, for empty SLNs and -9.7 ± 0.2 and -18.2 ± 0.3 mV, 311 respectively, for SLN+PYR. There was a significant difference between the empty 312 nanoparticles and those loaded with pyrethrum (P ≤ 0.0001, T0d = 8.989, and T120d = 24.50; 313 Table 1). After decreasing on the 30th day (-5.48 ± 0.13 mV), the zeta potential of SLN+PYR 314 increased to -12.2 ± 0.18 and -18.2 ± 0.35 mV after 90 and 120 days, respectively (Figure 315 1SD- Supplementary Material). Similarly, the empty SLN zeta potential decreased after 15 316 (-4.85 ± 0.19 mV) and 30 (-6.27 ± 0.18 mV) days, but increased on the 60th day (-15.43 ± 317 0.23 mV), indicating good stability until the end of the analysis time (Figure 1SD- 318 Supplementary Material). Zeta potential values greater than 30 mV indicate excellent 319 electrostatic stabilization (60 mV is the ideal value), while values lower 15 mV may result in 320 partial flocculation (SCHWARZ et al., 1994). Low zeta potentials were observed, but the 321 nanoparticle formulations were stable over time due to steric stabilization provided by the 322 PVA (LOURENÇO et al., 1996). Stabilizers can be used in nanoparticle formulations to 323 prevent particle aggregation (ABDELWAHED et al., 2006). In the present study, the 324 nonionic surfactant PVA was used to prepare the SLNs, which is absorbed onto surface 325 nanoparticles and promotes steric stabilization (ADITYA et al., 2013; OLIVEIRA et al., 326 2015). Therefore, unlike in previous studies, it was not superficial electrostatic repulsion that 327 provided stability to the system (PASQUOTO-STIGLIANI et al., 2017). Particles in 328 suspension are more stable if the zeta potential is greater than 20 mV, and 40 mV indicates 329 excellent stability (ADITYA et al., 2013). Similar results were obtained by Oliveira et al. 330 (2018) in zein nanoparticles loaded with the essential oil citronella (geraniol and R-331 citronellal), and by Kah et al. (2014) in a polymer-based nanoformulation of atrazine. 332 14 Table 1: Characterization of empty SLN and SLN loaded with pyrethrum extract over a 333 period from 0 to 120 days. 334 PARAMETERS SLN0 SLN120 SLN+PYR0 SLN+PYR120 MDDLS (NM) 290.0 ± 5.0 406.7 ± 6.7a,c 264.9 ± 2.8 260.8 ± 3.7 MDNTA (NM) 185.9 ± 4.6c 263.8 ± 18.5a,c 161.5 ± 2.7 227.0 ± 12.3b PDI 0.12 ± 0.01 0.39 ± 0.12a,c 0.12 ± 0.01 0.15 ± 0.02 ZP (-mV) 13 ± 0.4c 14 ± 0.3c 9.7 ± 0.2 18.2 ± 0.3b CT (10¹³ particles/mL) 2.7 ± 0.5 3.8 ± 0.2 5.9 ± 0.5 2.0 ± 0.1 pH 4.9 ± 0.04 5,7 ± 0.04a,c 5.0 ± 0.02 7.1 ± 0.02b EE (%) - - > 99% > 99% Legend - Mean diameter (MD) using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) 335 techniques; polydispersion index (PDI); zeta potential (ZP), concentration of nanoparticles(CT); hydrogenionic 336 potential (pH) and encapsulation efficiency (EE). The values are expressed as the mean ± standard error of six 337 measurements. a Significant difference between SLN group and times; b Significant difference between 338 SLN+PYR group and times; c Significant difference between SLN and SLN+PYR group. Paired T Test for 339 parametric test, and Mann Whitney U test for nonparametric test. 340 341 SLNs showed good stability for the encapsulated a.i, evidencing that physicochemical 342 properties not changed over time. According to Naseri et al. (2015), SLNs are good 343 nanocarriers and can be used to deliver drugs and agrochemicals. Their properties include 344 great physicochemical stability during production and storage, a good release profile, the 345 ability to solubilize lipophilic actives, and low toxicity (NASERI et al., 2015). 346 There was a significant difference in the pH of the empty SLN suspension between 0 347 and 120 days (4.9 ± 0.04 and 5.7 ± 0.04, respectively; P ≤ 0.0001 and T = 16.08), and of 348 SLN+PYR (5.0 ± 0.02 and 7.1 ± 0.02, respectively; P ≤ 0.0001 and T = 10.04; Table 1). Only 349 at 120 days was there a significant difference in pH between the treatment groups (P ≤ 0.0001 350 and T = 107.9) with SLN+PYR having a pH of 7.16 ± 0.02 (Figure 1SC - Supplementary 351 Material), indicating that hydrolytic processes occurred during this period. Similar results 352 were obtained by Oliveira et al. (2015). 353 15 The NTA revealed that the empty SLNs contained 2.7 ± 0.5 x 1013 particles per mL 354 with an initial size of 185.9 ± 4.6 nm, and SLN+PYR contained 5.9 ± 0.5 x 1013 particles per 355 mL with an initial size of 161.5 ± 2.7 nm. Table 1 shows that there was a significant 356 difference among timepoints for empty SLNs (P ≤ 0.02 and T = 3.65) and SLN+PYR (P ≤ 357 0.007 and T = 4.92), as well as between empty SLNs and SLN+PYR at 0 (P ≤ 0.0004 and T 358 = 10.68) and 120 (P ≤ 0.007 and T = 5.23) days. NTA counts the number of particles per mL 359 and is a complementary technique in the analysis of hydrodynamic diameters, and DLS and 360 NTA did not provide similar diameter values and particle concentrations. This difference 361 may have been caused by sample dilution during the NTA, which could have caused some 362 aggregates to rupture in suspension and result in smaller particles than the DLS 363 (MARUYAMA et al., 2016). 364 The encapsulation efficiency of pyrethrum into the SLNs was evaluated using an 365 analytical curve of pyrethrum determined by HPLC (Peak area (a.u.) = 4.69442 + 366 1952.15769* [pyrethrum concentration], r = 0.99341). The encapsulation efficiency was as 367 high as 99%, suggesting that the pyrethrum extract was efficiently encapsulated in this carrier 368 system. Nevertheless, is important verify the release profile of pyrethrum in field conditions 369 and it is expected that due the high encapsulation efficiency that the particles protect the a.i. 370 in order to increase its shelf life in field conditions. A high encapsulation efficiency has also 371 been reported in polymeric nanocapsules and SLNs loaded with carbendazim and 372 tebuconazole (CAMPOS et al., 2015), in chitosan nanoparticles carrying the herbicides 373 imazapic and imazapyr (MARUYAMA et al., 2016), and in microcapsules containing 374 dementholized peppermint oil (ZHAO et al., 2016). The high encapsulation value indicates 375 the affinity of the biocide to the lipid matrix (de MELO et al., 2016) due to its low solubility 376 in water (<10 mg.L-1) and high solubility in organic solvents (USEPA, 2006). 377 16 378 3.2. Differential scanning calorimetry (DSC) 379 DSC thermograms for SLN+PYR, empty SLNs, tripalmitin, and pyrethrum extract 380 are presented in Figure 1. The DSC analyzes in this study were carried out with the objective 381 of demonstrating that the pyrethrum interacts with nanocarriers components. There were no 382 endothermic peaks for the pyrethrum extract. Tripalmitin’s lowest peak was observed at 383 61ºC, which agrees with the melting point described in the literature (CHEN et al., 2006). 384 Analysis of the empty SLNs and SLN+PYR revealed that the melting points for tripalmitin 385 were 65 and 64°C, respectively, indicating that tripalmitin in the SLNs was solid, and that 386 the pyrethrum did not change the lipid core organization of the SLNs. Similar results were 387 obtained by Oliveira et al. (2015), who found that the herbicides simazine and atrazine were 388 dispersed on a nanoparticle matrix; as well as, Nasseri et al. (2016), verified that SLNs 389 containing Zataria multiflora essential oil (ZEO) not showed DSC pick of Zanataria 390 multiflora, and authors suggested that essential oil was incorporated and dissolved in the lipid 391 matrix. Analysis of the empty and encapsulated SLNs revealed two peaks, one indicating a 392 tripalmitin peak and the other possibly indicating PVA. Thermal studies of PVA have 393 reported an 88.1°C peak, probably due to moisture evaporation (GUIRGUIS; MOSELHEY, 394 2012). 395 17 396 Figure 1 - Differential scanning calorimetry evaluation of interaction between pyrethrum 397 extract and components of the SLN formulation: Thermograms for (PYR) Pyrethrum extract, 398 (TRI) Tripalmitin, (SLN) Solid lipid nanoparticles, (SLN+PYR) Pyrethrum loaded in solid 399 lipid nanoparticles. Conditions: N2 flow - 50 mL/minute, heating ramp of 10 to 300°C at a 400 rate of 10°C per minute. 401 402 3.3. Fourier Transform Infrared Spectroscopy (FTIR) 403 The physical mixture had three specific bands at 2914, 2368, and 1654 cm−1 (Figure 404 2), which corresponded with tripalmitin (2914 cm−1); and pyrethrum extract bands at 2368 405 and 1654 cm−1, corresponding with peak CO2 (OLIVEIRA and PASSOS, 2013) and a 406 stretching of the –C=C group, respectively. The infrared spectra of PVA, empty SLNs, and 407 SLN+PYR exhibited similar specific bands at 3335 cm−1 (Figure 2), which suggests the 408 presence of an –O-H group in the formulations. These groups were probably derived from 409 the water and PVA used in the preparation of the nanoparticles (ZAIN et al., 2011). The 410 18 specific bands at 2914 and 2848 cm−1 that were observed in the nanoparticles indicates a 411 stretching of the –C-H group (Figure 2), corresponding to tripalmitin (CAMPOS et al., 2015). 412 It was also possible to observe bands at 1735 cm−1, corresponding to a stretching of the –413 C=O group, at 1470 cm−1, corresponding to a bending of the –C-H2 group, and at 1178 cm−1, 414 corresponding to a stretching of the –C-O group. 415 416 19 Figure 2 - Infrared spectroscopic evaluation of interaction between pyrethrum extract and 417 components of the SLN formulation: FTIR spectra for (PM) Physical mixture (PVA) 418 Surfactant - polyvinyl alcohol; (TRI) Tripalmitin; (PYR) Pyrethrum extract; (SLN+PYR) 419 Pyrethrum loaded in solid lipid nanoparticles; (SLN) Solid lipid nanoparticles. Arrows 420 indicate the main characteristic absorption bands in each spectrum. Conditions: infrared 421 spectrophotometer with a range of 400 to 4000 cm-1, 128 scans per sample and 2 cm-1 422 resolutions. 423 424 3.4. Toxicological bioassay 425 Exposure to deltamethrin or pyrethrum extract (10 ng.µL-1) affected the longevity of 426 bees, reducing their life span. Bees exposed to pyrethrum extract (P < 0.01; 141.18 ± 21.3 427 hours) and pyrethroid (P < 0.001; 25.33 ± 0.93 h) presented shorter longevity than those in 428 the control group (257.83 ± 21.79 h). There is not significant difference between control and 429 other experimental groups (ACN; PVA; SLN; SLNP1ng; SLNP10ng and PYR1ng; P > 0.05). 430 The ACN (252.7 ± 25.03 h) data was similar to control group, as well as SLNP1ng (256.24 ± 431 21.00 h) and SLNP10ng (241.33 ± 18.81 h). The mean survival time of PVA (171.16 ± 18.09 432 h), SLN (196.54 ±11.38 h) and PYR1ng (175.33 ± 28.12 h) groups was lower than the control 433 group, but not significant (P > 0.05). The data of survival analysis were showed in 434 Supplementary Material (Figure 2S). 435 Pyrethroids can be dangerous to honeybees (JOHNSON et al., 2010), for example, 436 they interfere in the behavior (PALMQUIST et al., 2012), learning and memory performance 437 (LIAO et al., 2018). In addition, exposure to Lambda-Cyhalothrin negatively affects the life 438 span (LIAO et al., 2018; DOLEZAL et al., 2016). In line with these data, the pyrethrum 439 extract and deltamethrin also reduced survival of Africanized Apis mellifera. 440 The sublethal doses of 1 ng.µL-1 (1 ppm) and 10 ng.µL-1 (10 ppm) of biocide free or 441 encapsulated that were administered to the bees, induced short-term responses, at 442 morphological level, in the midguts of newly emerged workers. 443 20 The bee midgut is mainly responsible for food digestion and nutrient absorption, and 444 is composed of three cell types: digestive, endocrine, and regenerative cells. Digestive cells 445 are responsible for the production of digestive enzymes and nutrient absorption, endocrine 446 cells produce hormones, and regenerative cells, which are within nests, are responsible for 447 cell renewal of the epithelium (MARTINS et al., 2006). 448 Histological analysis of the bee midguts revealed morphological alterations in the 449 epithelium (Figure 3), specifically in the digestive cells, whereas the regenerative cell nests 450 maintained their normal morphological pattern. An increase in the elimination of digestive 451 cells to the intestinal lumen was observed in some treatment groups (empty SLNs, SLNP1ng, 452 and PYR10ng; Figure 3D, 3E, and 3H) in comparison to the control groups (CTL, ACN, and 453 PVA), which was significant in the empty SLN group (Figure 4A and Table 3S - 454 Supplementary Material). 455 Therefore, sublethal concentrations of pyrethrum extract in both non-encapsulated 456 and encapsulated form in nanoparticles, as well as in empty nanoparticles (SLN), caused 457 changes in digestive cells. Digestive cells have many microvilli close to the peritrophic 458 matrix in the lumen, and among these cells, nests of small regenerative cells are in the 459 intestinal epithelium (NEVES et al., 2002). These undifferentiated cells that remain in the 460 nest are a source for cell renewal in epithelium of bee midgut (CAVALCANTE and CRUZ-461 LANDIM, 2004). Thus, regenerative cells replace dead digestive cells, which were released 462 into the lumen, for new epithelial digestive cells by differentiation process (CRUZ et al., 463 2011). In this study, regenerative nests were observed in midgut epithelium, but histological 464 alterations indicative of cytotoxicity were not found in these cells, such as pyknotic nuclei. 465 If the regenerative cells from nests had presented nuclear pyknosis, which is an indicative of 466 cell death in undifferentiated cells, this alteration would have a "severe pathological 467 21 importance" because regenerative cells in adults does not suffer mitosis (CRUZ et al., 2011), 468 and consequently epithelial renewal of midgut would be compromised, resulting to partial or 469 total loss of the organ function. 470 Digestive cells are eliminated by cell degeneration under natural conditions, 471 meanwhile this process can be accelerated and/or intensified in response to xenobiotic 472 exposure (e.g., SLNs; Table 3S - Supplementary Material). Therefore, cell renewal is an 473 important process in maintaining the organ function, because the differentiation process from 474 regenerative cells can replace dead digestive cells and to renew the midgut epithelium. 475 There was less elimination of digestive cells to the intestinal lumen in bees exposed 476 to pyrethrum-loaded nanoparticles than in those exposed to empty nanoparticles (SLN). 477 Probably, the reduced cell-to-lumen liberation has been due to the interaction of the 478 pyrethrum with the active sites in the nanoparticle, providing greater stability of the colloidal 479 system over the time (0-120d) and high encapsulation efficiency (> 99% along 120d), as 480 evidenced in the physicochemical characterization data. On the contrary, empty SLNs are 481 more reactive and form aggregates more easily over time. Therefore, reactive empty SLNs 482 could interact with the epithelial cells of the midgut (oral exposure) and induce cytotoxicity 483 in digestive cells, which would trigger their elimination to the organ’s lumen. The compounds 484 used in nanoparticle formulations, and the colloidal instability of the system, can affect 485 interactions with cell membranes and trigger cytotoxicity (NAFEE et al., 2009). Whereas 486 the worker honeybee has lifetime of 45 days, and considering the acute exposure to the 487 nanopesticide during its application, probably the whole SLNP will remain stable during its 488 life span. Associating this information with the survival analysis, it can be noted that 489 encapsulated pyrethrum kept the survival time (256.24 ± 21.00 h and 241.33 ± 18.81 h, 490 SLNP1ng and SLNP1ng, respectively) of the bees similar to the control group (257.83 ± 21.79 491 22 h). Given that 10 ng of pyrethrum extract and pyrethroid (deltamethrin) reduced life span of 492 the bees, it may be noted that pyrethrum-loaded in nanoparticle is more safe for honeybees, 493 probably because of the stability of the encapsulated pyrethrum and its release as a function 494 of time. 495 Another important process that we observed was increased apocrine secretions from 496 the midgut epithelium onto the apical surfaces of midgut digestive cells (Figure 3SD and 3SE 497 - Supplementary Material). These epithelial cells secrete digestive enzymes and peritrophic 498 matrix substances normally by means of apocrine secretion. Therefore, an increase in 499 secretion may be a protective compensatory response to xenobiotic exposure. Increased 500 apocrine secretion occurred in both the empty nanoparticle-exposed and 1 ng.µL-1 of 501 pyrethrum-loaded nanoparticle-exposed groups (SLN and SLNP1ng; Table 3S and Figure 502 4B). A previous study reported an increase in apocrine secretion of midgut digestive cells in 503 bees exposed to sublethal doses of thiamethoxam insecticide (0.428 ng.µL-1 and 0.0428 504 ng.µL-1 per day for 18 days), as well as the increase in both cell vacuolization and cell 505 elimination from the epithelium to the midgut lumen over the exposure period (OLIVEIRA 506 et al., 2014). 507 Higher frequency of eliminated digestive cells and release of apocrine secretion 508 (Figure 4) were considered reversible alterations in the bee midgut and that did not affect 509 survival of bees in empty SLNs or encapsulated pyrethrum (SLNPs) groups. In normal 510 physiological situations, there is low frequency of senescent or dead cells eliminated to the 511 lumen (CAVALCANTE; CRUZ-LANDIM, 1999), and releasing of digestive enzymes from 512 cells to the peritrophic matrix in the lumen, usually by apocrine secretion (TERRA; 513 FERREIRA, 2012). Therefore, these alterations were classified as importance factor 1 in the 514 semi-quantitative analysis, because normally they are reversible, i.e., damage recovery in 515 23 epithelium occurs through the differentiation of regenerative cells from their nests in order 516 to have new digestive cells. Thus, there is a compensatory response to the potential 517 physiological stress triggered by agrochemicals or nanocarriers that can lead to the 518 elimination of cells and/or intensification of apocrine secretion. Soares et al. (2012) reported 519 an elimination of cells into the lumen, increased apocrine secretion, and pyknotic nuclei in 520 the epithelial cells of the Scaptotrigona postica midgut after applying sublethal doses of the 521 insecticide imidacloprid. Similarly, Rossi et al. (2011) exposed Africanized A. mellifera to 522 sublethal doses of imidacloprid and observed an increase in both cell elimination and 523 apocrine secretion in the midgut. 524 Aljedani (2017) evaluated the effects of acute exposure to deltamethrin on foraging 525 worker honeybees (A. mellifera jemenatica). The bees that were fed a sugary solution 526 containing 2.5 ppm of pyrethroid presented morphological changes in the midgut. In our 527 study, sublethal concentrations of pyrethrum extract (1 and 10 ng.µL-) did not induce 528 histopathological effects on midguts’ honeybees when the cell biomarkers were analyzed 529 separately, but the total organ index analysis showed alterations in 10 ng.µL-1 pyrethrum 530 extract that could potentially impair midgut function, since there was a decrease in the 531 longevity of the bees, demonstrating the relevance of evaluation of total organ index in bees 532 exposed to pesticides coupled to survival analysis. 533 24 534 25 Figure 3 – Honeybees (Africanized A. mellifera) midguts after 48 h of acute exposure. A) 535 CTL - syrup control; B) ACN –acetone control; C) PVA - surfactant control; D) SLN – Solid 536 lipid nanoparticles; E) SLNP1ng– 1 ng.µL-1 of pyrethrum loaded in solid lipid nanoparticles; 537 F) SLNP10ng – 10 ng.µL-1 of pyrethrum loaded in solid lipid nanoparticles G) PYR1ng – 1 538 ng.µL-1 of pyrethrum extract; H) PYR10ng – 10 ng.µL-1 of pyrethrum extract. Legend: dc = 539 digestive cell; ec = eliminated cell in the lumen; lu = lumen; n = nucleus, v = vacuolization; 540 as = apocrine secretion; Black arrow = Regenerative cell; TM = Malpighi's tubes; m = 541 muscle. Staining: Hematoxylin-Eosin. Bars: 50 µm. 542 543 Although vacuolization can be present in bee midgut cells as a physiological process 544 of autophagy for intracellular turnover, their increased level frequently has been associated 545 to side-effects of xenobiotics, especially in bees exposed to pesticides. For example, Cruz et 546 al. (2010) reported cytoplasmic vacuolization and cell elimination in A. mellifera larvae 547 midguts exposed to fipronil (0.1 and 1 μg.g-1) and boric acid (1.0, 2.5, and 7.5 mg.g-1). 548 Kakamand et al. (2008) observed an increase in the vacuolization of midgut cells in 549 honeybees exposed to deltamethrin (1, 2.5, 5, and 10 mg.L-1) and the degeneration of the 550 midgut epithelium of bees exposed to the highest concentration of this compound. 551 Histochemical analysis of vacuolization areas in digestive cells (Figure 4S - 552 Supplementary Material) showed that they are negative for proteins or neutral 553 glycoconjugates, but had positive labelling for lipids that could indicate multivesicular 554 bodies, because newly emerged honeybees have no spherocrystals yet. Multivesicular bodies 555 are frequently found in midgut cells of insects (SERRAO; CRUZ-LANDIM, 1996), and are 556 formed from early endosomes due to an inward budding of its membrane resulting in 557 intralumenal vesicles whose main function is “collecting” plasma membrane receptors to be 558 degraded into the lysosomes. Multivesicular bodies and autophagy are closely related 559 (FADER; COLOMBO, 2009). 560 At the present study, intensification of cytoplasm vacuolization was considered a 561 morphological alteration indicative of cytoplasmic loss, which is of greater pathological 562 26 importance than the other alterations analyzed because, especially in insects, autophagy may 563 act as a pro-death process at the cellular/organ level (MALAGOLI et al., 2010), although its 564 effects at the organismal level can still be considered as fundamental for survival. 565 Cell vacuolization increased in both groups exposed to pyrethrum extract (Figure 3G 566 and 3H, Figure 3SG and 3SH, and Table 3S), but there was no significant difference due to 567 the highly variable degree of vacuolization among individuals exposed to pyrethrum extract 568 (Figure 4C). However, when the organ index was calculated, vacuolization accounted for a 569 higher total index under 10 ng.µL-1 of pyrethrum extract (Figure 4D), as this alteration was 570 classified as importance factor 2 in the semi-quantitative analysis (Table 3S) because of the 571 loss of cytoplasmic material and the severity level. 572 In the total organ index analysis, the empty nanoparticles and 10 ng.µL-1 of pyrethrum 573 extract caused more significant changes than the other experimental groups (Table 3S). In 574 contrast, nanoparticles loaded with 1 ng.µL-1 pyrethrum extract did not increase cell 575 alterations more than the other groups (nanoparticles and pyrethrum extract). The SLNP 576 groups exhibited a decrease in short-term cell alterations, so in this respect was considered 577 safer for bees over short exposure times. 578 27 579 Figure 4 – Alterations and organ index in honeybee (Africanized A. mellifera) midguts. a) 580 Eliminated cell index; b) Apocrine secretion index; c) Vacuolization index; d) Total organ 581 index. Legend: CTL – syrup control; ACN – acetone control; PVA - surfactant control; SLN 582 – Solid lipid nanoparticles; SLNP1ng – 1 ng.µL-1 of pyrethrum loaded in solid lipid 583 nanoparticles; SLNP10ng – 10 ng.µL-1 of pyrethrum loaded in solid lipid nanoparticles PYR1ng 584 – 1 ng.µL-1 of pyrethrum extract; PYR10ng – 10 ng.µL-1 of pyrethrum extract. Kruskal Wallis 585 One-way ANOVA, followed by Dunn's multiple comparison test. *represent significant 586 differences between groups. 587 28 At the lowest sublethal doses (1 ng.µL-1), the biocide did not evidence significant 588 histopathological changes in the total lesion index, indicating that could be applied on crops. 589 A carrier system could be developed to improve pyrethrum extract stability, thus allowing its 590 use as nanopesticides. Besides, when the pyrethrum extract was encapsulated in nanocarriers 591 and demonstrated lower toxicity when compared with pyrethrum only. Therefore, 592 nanocarriers are an alternative to conventional pesticide applications. Nanotechnology 593 applied in the agricultural sector could increase agricultural production and crop protection, 594 contribute to sustainable agriculture and eco-friendly carrier systems, and reduce 595 environmental effects and toxicity to organisms (GRILLO et al., 2016). Oliveira et al. (2018) 596 found that zein nanoparticles loaded with citronella effectively controlled the pest species 597 Tetranychus urticae with low toxicity. 598 The empty SLNs showed effects onto honeybee, for example, in the total lesion index, 599 with the increase the eliminated cells and apocrine secretion. Therefore, nanocarrier system 600 itself may have reactive sites capable of changing their biological system because it has no 601 active ingredient encapsulated. These reactive sites could interact with organic molecules of 602 the organism, inducing negative effects that indirectly decreased the mean survival time of 603 the bees (196.54 ±11.38 h; P > 0.05). By the way, further studies need to be performed in 604 order to evaluate these hypotheses. 605 Nanopesticides can be able to increase the efficiency of agrochemicals and biocides, 606 because it is possible that in the field low doses of the active ingredients can be used. 607 However, in the case of pyrethrum and SLNs this fact will be confirmed with biological 608 assays in target organisms that will be run in the future. In addition, they increase production 609 and reduce damage to the environment (PRASAD et al., 2017). However, there are still many 610 gaps in information to be filled, normative instructions to be written, and legislation to be 611 29 made before they can be extensively and safely employed in agriculture (KAH; HOFMANN, 612 2014; KOOKANA et al., 2014). According Kah et al. (2018), further studies that investigate 613 the efficacy of nanopesticides in crop farming are needed, in order to elucidate their effects 614 on biodiversity and human health, and their benefits and costs compared with conventional 615 formulations. 616 617 4. CONCLUSION 618 It is important to develop and analyze carrier systems as they have many potential 619 benefits in comparison to synthetic and natural agrochemicals, such as reducing the amount 620 of biocide in the environment and greater stability. However, nanotoxicological studies 621 should be undertaken to evaluate the effects of nanoparticles on non-target organisms. In 622 conclusion, this study demonstrates that nanoparticles loaded with pyrethrum extract at 623 sublethal dose (1 or 10 ng.µL-1) are relatively safe for honeybees, because they do not cause 624 morphological changes in digestive cells. In contrast, empty nanoparticles and 10 ng.µL-1 of 625 pyrethrum extract caused changes in digestive cells during acute exposure. The concentration 626 of 1 ng.µL-1 of pyrethrum extract could be used for pest control. These data reflect the effects 627 of a sublethal and acute exposure, and more studies are needed to check if a chronic exposure 628 to these compounds would have different effects on bees. Our results added information for 629 subsidizing future decision making, regulatory framework creation, risk assessments, and 630 legislation development, and improve food security. In addition, based on the results we are 631 planning to run biological assays in order to investigate the efficacy of the nanopesticide 632 against target organisms. 633 634 635 30 ACKNOWLEDGMENTS 636 Authors would like to thank the grant of São Paulo Research Foundation (#2017/21004-5). 637 Hellen Maria Soares-Lima and Rafaela Tadei for assisting in the toxicity bioassays, and 638 Edson Sampaio keeping the colonies of honeybees for experiments. Authors thanks Profa. 639 Dra. Leticia S. Souto from LADIVE by the availability of the microtome (FAPESP 640 2015/01424-4). 641 642 CONFLICT OF INTEREST 643 The authors declare there are no conflicts of interest in the present study. 644 645 REFERENCES 646 647 Abdelwahed, W.; Degobert, G.; Stainmesse, S.; Fessi, H. 2006. Freeze-drying of 648 nanoparticles: formulation, process and storage considerations. Adv. Drug Delivery Rev. 58 649 (15), 1688-1713. https://doi.org/10.1016/j.addr.2006.09.017 650 651 About NIFA, 2018. National Institute of Food and Agriculture (NIFA) - United States 652 Department of Agriculture (USDA). White House Office of Science and Technology 653 Policy: Alexandria, VA, 2018. https://nifa.usda.gov/about-nifa (accessed Mar 04, 2018). 654 655 Aditya, N.P.; Shim, M.; Lee, I.; Lee, Y.; Im, M.H.; Ko, S. 2013. Curcumin and gnistein 656 coloaded nanostructured lipid carriers: in vitro digestion and antiprostate cancer activity. J. 657 Agri. Food Chem. 61 (8), 1878–1883. doi: 10.1021/jf305143k. 658 659 Aljedani, D.M. 2017. Effects of Abamectin and Deltamethrin to the foragers honeybee 660 workers of Apis mellifera jemenatica (Hymenoptera: Apidae) under laboratory conditions. 661 Saudi J. Biol. Sci. 24 (5), 1007-1015. doi: http://dx.doi.org/10.1016/j.sjbs.2016.12.007. 662 663 Anderson, W.; Kozak, D.; Coleman, V.A.; Jämting, Å.K.; Trau, M. 2013. A comparative 664 study of submicron particle sizing platforms: Accuracy, precision and resolution analysis of 665 polydisperse particle size distributions. J. Colloid Interface Sci. 405, 322–330. 666 https://doi.org/10.1016/j.scienta.2007.11.013 667 668 Anjali, C.H.; Sudheer Khan, S.; Margulis-Goshen, K.; Magdassi, S.; Mukherjee, A.; 669 Chandrasekaran, N. 2010. Formulation of water-dispersible nanopermethrin for larvicidal 670 applications. Ecotoxicol. Environ. Saf. 73 (8), 1932-1936. doi: 671 10.1016/j.ecoenv.2010.08.039. 672 673 Bernet, D.; Schmidt, H.; Meier, W.; Burkhardt‐Holm, P.; Wahli, T. 1999. Histopathology in 674 fish: Proposal for a protocol to assess aquatic pollution. J. Fish Dis., 22 (1), 25-34. 675 https://doi.org/10.1046/j.1365-2761.1999.00134.x 676 677 31 678 Campos, E.V.R.; de Oliveira, J.L.; da Silva, C.M.; Pascoli, M.; Pasquoto, T.; Lima, R.; 679 Abhilash, P.C.; Fraceto, L.F. 2015. Polymeric and Solid Lipid Nanoparticles for Sustained 680 Release of Carbendazim and Tebuconazole in Agricultural Applications. Sci Rep. 5, 13809. 681 doi: 10.1038/srep13809 682 683 Cavalcante, V.M.; C. Cruz-Landim. 1999. Types of cells present in the midgut of the insects: 684 A review. Naturalia (Rio Claro) 24, 19-40. 685 686 Cavalcante, V.M.; Cruz-Landim, C. 2004. Padrão eletroforético de proteínas e atividade de 687 fosfatase ácida em extratos do intestino médio de Apis mellifera L. durante a 688 metamorfose. Neotrop. Entomol. 33 (2), 169-172. http://dx.doi.org/10.1590/S1519-689 566X2004000200007. 690 691 Chen, H; Chang, X.; Du, D.; Liu, W.; Liu, J.; Weng, T.; Yang, Y.; Xu, H.; Yang, X. 2006. 692 Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. J. Controlled 693 Release 110 (2), 296 – 306. doi: 10.1016/j.jconrel.2005.09.052 694 695 Chen, H., Yada, R. 2011. Nanotechnologies in agriculture: new tools for sustainable 696 development. Trends Food Sci. Technol 22, 585–594. 697 https://doi.org/10.1016/j.tifs.2011.09.004 698 699 Cruz, A.S.; da Silva-Zacarin, E.C.; Bueno, O.C.; Malaspina, O. 2010. Morphological 700 alterations induced by boric acid and fipronil in the midgut of worker honeybee (Apis 701 mellifera L.) larvae. Cell Biol. Toxicol. 26 (2), 165–176. doi: 10.1007/s10565-009-9126-x. 702 703 Cruz, L.C.; Araújo, V.A.; Dolder, H.; Araújo, A.P.; Serrão, J.E.; Neves, C.A. 2011. 704 Morphometry of the midgut of Melipona quadrifasciata anthidioides (Lepeletier) 705 (Hymenoptera: Apidae) during metamorphosis. Neotrop. Entomol. 40 (6), 677-681. 706 http://dx.doi.org/10.1590/S1519-566X2011000600007. 707 708 de Melo, N.F.S.; Araújo, D.R.; Grillo, R.; Moraes, C.M.; Matos, A.P.; Paula, E.; Rosa, A.H.; 709 Fraceto, L.F. 2012. Benzocaine-loaded polymeric nanocapsules: Study of the anesthetic 710 activities. J. Pharm. Sci. 101, 1157-1165. 711 712 de Melo, N.F.; de Macedo, C.G.; Bonfante, R.; Abdalla, H.B.; da Silva, C.M.; Pasquoto, T.; 713 de Lima, R.; Fraceto, L.F.; Clemente-Napimoga, J.T.; Napimoga, M.H. 2016. 15d-PGJ2-714 Loaded Solid Lipid Nanoparticles: Physicochemical Characterization and Evaluation of 715 Pharmacological Effects on Inflammation. PLoS One 11 (8), e0161796. 716 https://doi.org/10.1371/journal.pone.0161796 717 718 Dolezal, A.G.; Carrillo-Tripp, J.; Miller, W.A.; Bonning, B.C.; Toth, A.L. 2016. Pollen 719 Contaminated with Field-Relevant levels of Cyhalothrin affects Honey Bee Survival, 720 Nutritional Physiology, and Pollen Consumption Behavior. J. Econ, Entomol. 109 (1), 41-8. 721 https://doi.org/10.1093/jee/tov301. 722 723 32 Fader, C.M.; Colombo, M.I. 2009. Autophagy and multivesicular bodies: two closely related 724 partners. Cell Death Differ. 16 (1), p. 70–78. doi: 10.1038/cdd.2008.168. 725 726 Gamisans, F.; Lacoulonche, F.; Chauvet, A.; Espina, M.; García, M.L.; Egea, M.A. 1999. 727 Flurbiprofen-loaded nanospheres: analysis of the matrix structure by thermal methods. Int. J. 728 Pharm. 179 (1), 37-48. https://doi.org/10.1016/S0378-5173(98)00381-0 729 730 Giannini, T.C.; Cordeiro, G.D.; Freitas, B.M.; Saraiva, A.M. Imperatriz-Fonseca, V.L. 2015. 731 The dependence of crops for pollinators and the economic value of pollination in Brazil. J. 732 Econ. Entomol. 108, 1-9. doi: 10.1093/jee/tov093. 733 734 Gopal, M.; Kumar, R.; Goswami, A. 2012. Nano-pesticides - A recent approach for pest 735 control. The J. Plant Protec. Sci. 4 (2), 1-7. 736 737 Goulson, D.; Nicholls, E.; Botías, C.; Rotheray, E.L. 2015.Bee declines driven by combined 738 stress from parasites, pesticides, and lack of flowers. Science. 347 (6229), 1255957. Doi: 739 10.1126/science.1255957 740 741 Grillo, R.; Abhilash, P.C.; Fraceto, L.F. 2016. Nanotechnology applied to Bio-encapsulation 742 of pesticides. J. Nanosci. Nanotechnol. 16, 1231-1234. 743 https://doi.org/10.1166/jnn.2016.12332 744 745 Guirguis, O.W.; Moselhey, M.T.H. 2012. Thermal and structural studies of poly(vinyl 746 alcohol) and hydroxypropyl cellulose blends. Natural Sci. 4 (1), 57-67. 747 http://dx.doi.org/10.4236/ns.2012.41009 748 749 Jacques, M.T.; Oliveira, J.L.; Campos, E.V.; Fraceto, L.F.; Ávila, D.S. 2017. Safety 750 assessment of nanopesticides using the roundworm Caenorhabditis elegans. Ecotoxicol. 751 Environ. Saf. 139, 245–253. doi: 10.1016/j.ecoenv.2017.01.045 752 753 Johnson, R.M.; Ellis, M. D.; Mullin, C. A.; Frazier, M. 2010. Pesticides and honey bee 754 toxicity – USA. Apidologie 41, 312–331. doi: 10.1051/apido/2010018 755 756 Kah, M.; Machinski, P.; Koerner, P.; Tiede, K.; Grillo, R.; Fraceto, L.F. 2014. Hofmann 757 T.Analysing the fate of nanopesticides in soil and the applicability of regulatory protocols 758 using a polymer-based nanoformulation of atrazine. Environ. Sci. Pollut. Res. 21 (20), 11699-759 707. doi: 10.1007/s11356-014-2523-6. 760 761 Kah, M.; Hofmann, T. 2014. Nanopesticide research: Current trends and future priorities. 762 Environ. Int. 63, 224-235. doi: 10.1016/j.envint.2013.11.015. 763 764 Kah, M.; Kookana, R.S.; Gogos, A.; Bucheli, T.D. 2018. A critical evaluation of 765 nanopesticides and nanofertilizers against their conventional analogues. Nat. Nanotechnol. 766 13, 677–684. doi: 10.1038/s41565-018-0131-1. 767 768 33 Kakamand, F.A.K.; Mahmoud, T. T.; Amin, A.M. 2008. The role of three insecticides in 769 disturbance the midgut tissue in honey bee Apis mellifera L. Workers. J. Dohuk Univ. 11 (1), 770 144-151. 771 772 Kearns, C.A.; Inouye, D. W. 1997. Pollinators, Flowering Plants, and Conservation Biology. 773 BioScience 47 (5), 297-307. 774 775 Kilic, A. C.; Capan, Y.; Vural, I.; Gursoy, R.N.; Dalkara, T.; Cuine, A.; Hincal, A.A. 2005. 776 Preparation and characterization of PLGA nanospheres for the targeted delivery of NR2B-777 specific antisense oligonucleotides to the NMDA receptors in the brain. J. 778 Microencapsulation 22, 633–641. 779 780 Kim, D.Y.; Kadam, A.; Shinde, S.; Saratale, R.G.; Patra, J.; Ghodake, G. 2018. Recent 781 developments in nanotechnology transforming the agricultural sector: a transition replete 782 with opportunities. J. Sci. Food Agric. 98 (3), 849-864. doi: 10.1002/jsfa.8749. 783 784 Kookana, R.S.; Boxall, A.B.; Reeves, P.T.; Ashauer, R.; Beulke, S.; Chaudhry, Q.; Cornelis, 785 G.; Fernandes, T.F.; Gan, J.; Kah, M.; Lynch, I.; Ranville, J.; Sinclair, C.; Spurgeon, D.; 786 Tiede, K.; Van den Brink, P.J. 2014. Nanopesticides: guiding principles for regulatory 787 evaluation of environmental risks. J. Agric. Food. Chem. 62 (19), 4227-4240. 788 doi: 10.1021/jf500232f 789 790 Liao, C.H.; He, X.J.; Wang, Z.L.; Barron, A.B.; Zhang, B.; Zeng, Z.J.; Wu, X.B. 2018. Short-791 Term Exposure to Lambda-Cyhalothrin Negatively Affects the Survival and Memory-792 Related Characteristics of Worker Bees Apis mellifera. Arch. Environ. Contam. Toxicol. 75 793 (1), 59-65. doi: 10.1007/s00244-018-0514-1. 794 795 Liu, Y.; Tong, Z.; Prud'homme, R.K. 2008. Stabilized polymeric nanoparticles for controlled 796 and efficient release of bifenthrin. Pest. Manag. Sci. 64 (8), 808-812. doi: 10.1002/ps.1566. 797 798 Lourenço, C.; Teixeira, M.; Simões, S.; Gaspar, R. 1996. Steric stabilization of nanoparticles: 799 Size and surface properties. Int. J. Pharm. 138 (1), 1-12. https://doi.org/10.1016/0378-800 5173(96)04486-9 801 802 Malagoli, D.; Abdalla, F.C.; Cao, Y.; Feng, Q.; Fujisaki, K.; Gregorc, A.; Matsuo, T.; Nezis, 803 I.P.; Papassideri, I.S.; Sass, M.; Silva-Zacarin, E.C.; Tettamanti, G.; Umemiya-Shirafuji, R. 804 2010. Autophagy and its physiological relevance in arthropods: current knowledge and 805 perspectives. Autophagy. 6 (5), 575-88. doi: 10.4161/auto.6.5.11962. 806 807 Martins, G.F.; Neves, C.A.; Campos, L.A.; Serrão, J.E. 2006. The regenerative cells during 808 the metamorphosis in the midgut of bees. Micron 37 (2), 161-168. 809 https://doi.org/10.1016/j.micron.2005.07.003 810 811 Maruyama, C.R.; Guilger, M.1.; Pascoli, M.; Bileshy-José, N.; Abhilash, P.C.; Fraceto, L.F.; 812 de Lima, R. 2016. Nanoparticles Based on Chitosan as Carriers for the Combined Herbicides 813 Imazapic and Imazapyr. Sci. Rep. 6 (19768). Doi: 10.1038/srep19768 814 815 34 Masarudin, M.J.; Cutts, S.M.; Evison, B.J.; Phillips, D.R.; Pigram, P. J. 2015. Factors 816 determining the stability, size distribution, and cellular accumulation of small, monodisperse 817 chitosan nanoparticles as candidate vectors for anticancer drug delivery: application to the 818 passive encapsulation of [14C]-doxorubicin. Nanotechnol. Sci. Appl. 8, 67–80. 819 http://doi.org/10.2147/NSA.S91785 820 821 Mishra, S.; Keswani, C.; Abhilash, P.C.; Fraceto, L.F.; Singh, H.B. 2017. Integrated 822 Approach of Agri-nanotechnology: Challenges and Future Trends. Front. Plant. Sci. 8, 1-12. 823 824 Montanha, F.P.; Pimpão, C.T. 2012. Efeitos toxicológicos de piretróides (cipermetrina e 825 deltametrina) em peixes. Rev. Cient. Elet. Med. Vet. 9 (18), 1-58. 826 827 Mukhopadhyay, S.S. 2014. Nanotechnology in agriculture: prospects and constraints. 828 Nanotechnol., Sci. Appl. 7, 63–71. doi: 10.2147/NSA.S39409 829 830 Muller, R. H.; Rühl, D.; Runge, S.A. 1996. RungeBiodegradation of solid lipid nanoparticles 831 as a function of lipase incubation time. Int. J. Pharm. 144 (1), 115-121. 832 833 Muller, H.R.; Mäder, K.; Gohla, S. 2000. Solid lipid nanoparticles (SLN) for controlled drug 834 delivery: a review of the state of the art. Eur. J. Pharm. Biopharm. 50 (1), 161-177. 835 https://doi.org/10.1016/S0939-6411(00)00087-4 836 837 Muller, H.; Shegokar, R.; Keck, C.M. 2011. 20 Years of Lipid Nanoparticles (SLN & NLC): 838 Present State of Development & Industrial Applications. Curr. Drug. Discov. Technol. 8, 839 207-227. https://doi.org/10.2174/157016311796799062 840 841 Nafee, N.; Schneider, M.; Schaefer, U.F.; Lehr, C.M. 2009. Relevance of the colloidal 842 stability of chitosan/PLGA nanoparticles on their cytotoxicity profile. Int. J. Pharm. 381 843 (2, 3), 130-139. https://doi.org/10.1016/j.ijpharm.2009.04.049 844 845 Naseri, N.; Valizadeh, H.; Zakeri-Milani, P. 2015. Solid Lipid Nanoparticles and 846 Nanostructured Lipid Carriers: Structure, Preparation and Application. Adv. Pharm. Bull. 5 847 (3), 305–313. http://doi.org/10.15171/apb.2015.043 848 849 Nasseri, M.; Golmohammadzadeh, S.; Arouiee, H.; Jaafari, M.R.; Neamati, H. 2016. 850 Antifungal activity of Zataria multiflora essential oil-loaded solid lipid nanoparticles in-vitro 851 condition. Iran. J. Basic Med. Sci. 19 (11), 1231-1237. Doi: 10.22038/ijbms.2016.7824 852 853 Neves, C.A.; Bhering, L.L.; Serrão, J.E.; Gitirana, L.B. 2002. FMRFamide-like midgut 854 endocrine cells during the metamorphosis in Melipona quadrifasciata anthidioides 855 (Hymenoptera Apidae). Micron 33 (5), 453-460. https://doi.org/10.1016/S0968-856 4328(01)00043-9 857 858 Oliveira, J.L.; Campos, E.V.; Gonçalves da Silva, C.M.; Pasquoto, T.; Lima, R.; Fraceto, 859 L.F. 2015. Solid Lipid Nanoparticles Co-loaded with Simazine and Atrazine: Preparation, 860 Characterization, and Evaluation of Herbicidal Activity. J. Agric. Food Chem. 63, 422-432. 861 Doi: 10.1021/jf5059045 862 35 863 Oliveira, J.L.; Campos, E.V.R.; Pereira, A.E.S.; Pasquoto, T.; Lima, R.; Grillo, R.; Andrade, 864 D.J.; Santos, F.A.D.; Fraceto, L.F. 2018. Zein Nanoparticles as Eco-Friendly Carrier Systems 865 for Botanical Repellents Aiming Sustainable Agriculture. J. Agric. Food Chem. 66, 1330-866 1340. doi: 10.1021/acs.jafc.7b05552. 867 868 Oliveira, R.A.; Roat, T.C.; Carvalho, S.M.; Malaspina, O. 2014. Side‐effects of 869 thiamethoxam on the brain and midgut of the africanized honeybee Apis 870 mellifera (Hymenopptera: Apidae). Environ. Toxicol. 29 (10), 1122-33. doi: 871 10.1002/tox.21842. 872 873 Oliveira, R.L.; Passos, F.B. 2013. Estudo da oxidação parcial do etanol em catalisadores de 874 Rh por DRIFTS. Quim. Nova 36 (3), 375-381. 875 876 Palmquist, K.; Salatas, J.; Fairbrother, A. 2012. Pyrethroid insecticides: use, environmental 877 fate, and ecotoxicology, in: Perveen, F. (Ed.), Insecticides: Advances in Integrated Pest 878 Management, InTech, Rijeka: Croatia, cap. 11, pp. 251-278. 879 880 Pasquoto-Stigliani, T.; Campos, E.V.R.; Oliveira, J.L.; Silva, C.M.G.; Bilesky-José, N.; 881 Guilger, M.; Troost, J.; Oliveira, H.C.; Stolf-Moreira, R.; Fraceto, L.F.; de Lima, R. 2017. 882 Nanocapsules Containing Neem (Azadirachta Indica) Oil: Development, Characterization, 883 And Toxicity Evaluation. Sci. Rep. 7 (5929). doi:10.1038/s41598-017-06092-4 884 885 Peay, S.; Hiley, P.D.; Collen, P.; Martin, I. 2006. Biocide treatment of ponds in Scotland to 886 eradicate signal crayfish. Bull. Fr. Pêche Piscic. 380–381, 1363–1379. 887 https://doi.org/10.1051/kmae:2006041 888 889 Perez-de-Luque, A.; Rubiales, D. 2009. Nanotechnology for parasitic plant control. Pest 890 Manage. Sci. 65 (5), 540-545. https://doi.org/10.1002/ps.1732 891 892 Polleto, F.S.; Jäger, E.; Ré, M.I.; Guterres, S.S.; Pohlmann, A.R. 2007. Rate modulating 893 PHBHV/PCL microparticles containing weak acid model drugs. Int. J. Pharm. 345, 70-80. 894 https://doi.org/10.1016/j.ijpharm.2007.05.040 895 896 Potts, S.G.; Biesmeijer, J.C.; Kremen, C.; Neumann, P.; Schweiger, O.; Kunin, W.E. 2010. 897 Global pollinator declines: trends, impacts and drivers. Trends Ecol. Evol. 25 (6), 345-353. 898 https://doi.org/10.1016/j.tree.2010.01.007 899 900 Prasad, R., Bhattacharyya, A.; Nguyen, Q.D. 2017. Nanotechnology in Sustainable 901 Agriculture: Recent Developments, Challenges, and Perspectives. Front. Microbiol. 20; 902 8:1014. https://doi.org/10.3389/fmicb.2017.01014 903 904 Rossi, C.A., Roat, T.C.; Tavares, D.A.; Cintra-Socolowski, P.; Malaspina, O. 2011. Effects 905 of sublethal doses of imidacloprid in malpighian tubules of africanized Apis 906 mellifera (Hymenoptera, Apidae). Microsc. Res. Tech. 76 (5), 552-558. 907 https://doi.org/10.1002/jemt.22199 908 909 36 Santos, M.A.T.; Areas, M.A.; Reyes, F.G. 2007. Piretróides – uma visão geral. Alim. Nutr. 910 18 (3), 339-349. 911 912 Sarangi, M.J.; Padhi, S. Solid lipid nanoparticles – A Review. J. Crit. Rev. 2016, 3 (3), 5-12. 913 914 Sarlak, N.; Taherifar, A.; Salehi, F. 2014. Synthesis of Nanopesticides by Encapsulating 915 Pesticide Nanoparticles Using Functionalized Carbon Nanotubes and Application of New 916 Nanocomposite for Plant Disease Treatment. J. Agric. Food Chem 62 (21), 4833–483. 917 doi.10.1021/jf404720d 918 919 Schaffazick, S.R.; Guterres, S.S.; Freitas, L.L.; Pohlmann, A.R. 2003. Caracterização e 920 estabilidade físico-química de sistemas poliméricos nanoparticulados para administração de 921 fármacos. Quím. Nova 26 (5), 726-737. http://dx.doi.org/10.1590/S0100-922 40422003000500017. 923 924 Schleier, J.J.; Peterson, R.K.D. 2011. Pyrethrins and Pyrethroid Insecticides, in: López, O.; 925 Fernández-Bolaños, J.G. (Eds.), Green Trend. Insect Cont, J. Publishing. London: Burlington 926 House, cap. 3, pp. 94-131. 927 928 Schwarz, C.; Mehnert, W.; Lucks, J.S.; Müller, R.H. 1994. Solid lipid nanoparticles (SLN) 929 for controlled drug delivery. I. Production, characterization and sterilization. J. Controlled 930 Release. 30 (1), 83-96. https://doi.org/10.1016/0168-3659(94)90047-7 931 932 Serrão, J. E., C. Cruz-Landim. 1996. Ultrastructure of digestive cells in stingless bees of 933 various ages (Hymenoptera, Apidae, Meliponinae). Cytobios 88, 161-171. 934 935 Scott, N.; Chen, H. 2012. Nanoscale Science and Engineering for Agriculture and Food 936 Systems. Ind. Biotechnol. 9 (1), 17-18. https://doi.org/10.1089/ind.2013.1555 937 938 Sekhon, B.S. 2014. Nanotechnology in agri-food production: an overview. Nanotechnol., Sci. 939 Appl. 7, 31–53. http://doi.org/10.2147/NSA.S39406 940 941 Silva-Zacarin, E.C.M.; Chauzat, M.P.; Zeggane, S.; Drajnudel, P.; Schurr, F.; Faucon, J.P.; 942 Malaspina, O.; Engler, J.A. 2012. Protocol for optimization of histological, histochemical 943 and immunohistochemical analyses of larval tissues: application in histopathology of honey 944 bee. In: Méndez-Vilas, A. (Ed.), Current microscopy contributions to advances in science 945 and technology, Formatex Research Center: Badajoz, 5. ed., v. 1, pp. 696-703. 946 947 Silva, M. dos S. Cocenza, D. S. Grillo, R. de Melo, N. F. S. Tonello, P. S. Oliveira, L. C. de 948 Cassimiro, D. L. Rosa, A. H. Fraceto, L. F. 2011. Paraquat-loaded alginate/chitosan 949 nanoparticles: preparation, characterization and soil sorption studies. J. Hazard. Mater. 190 950 (1-3), 366-374. 951 952 Soares, H.M. Avaliação dos efeitos do inseticida imidacloprido para abelhas sem ferrão 953 Scaptotrigona postica Latreille, 1807 (Hymenoptera, Apidae, Meliponini). Dissertação de 954 Mestrado, Universidade Estadual Paulista - campus Rio Claro/SP, 2012. 955 956 37 Soares-Lima, H.M.; Silva-Zacarin, E.C.M.; Camargo, I; Nocelli, R.C.F.; Malaspina, O. 957 Histopathological alterations on honeybees midgut infected by Nosema ceranae and exposed 958 to imidacloprid. In: Society of Environmental Toxicology and Chemistry Asia-Pacific 959 Conference, Daegu, Korea, Sep 16-19, 2018; SETAC AP, Eds.; SETAC: Korea, 2018. 960 961 Terra, W.R.; Ferreira, C. 2012. Biochemistry and molecular biology of digestion. In, Gilbert, 962 L.I. (Ed.), Insect Molecular Biology and Biochemistry, Academic Press: San Diego, pp. 365–963 418. https://doi.org/10.1016/C2009-0-62118-8 964 965 U.S. Department of Agriculture, National Institute of Food and Agriculture (USDA/NIFA) 966 2018; National Nanotechnology Initiative (NANO). White House Office of Science and 967 Technology Policy: Alexandria, VA, 2018. https://www.nano.gov/node/137 (accessed Mar 968 04, 2018). 969 970 Data Evaluation Record - Pyrethrum extract 1991; U.S. Environmental Protection Agency 971 (USEPA). White House Office of Science and Technology Policy: Philadelphia, PA, 1991. 972 https://www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-069001_3-Sep-973 91_a.pdf (accessed Mar 14, 2018). 974 975 Reregistration Eligibility Decision for Pyrethrins 2006; U.S. Environmental Protection 976 Agency (USEPA). White House Office of Science and Technology Policy: Philadelphia, PA, 977 2006. http://www.epa.gov/oppsrrd1/REDs/pyrethrins_red.pdf (accessed Mar 14, 2018). 978 979 Venkatraman, S. S. 2005. Micelle-like nanoparticles of PLA–PEG–PLA triblock copolymer 980 as chemotherapeutic carrier. Int. J. Pharm. 298, 219–232. 981 https://doi.org/10.1016/j.ijpharm.2005.03.023 982 983 Vitorino, C.; Carvalho, F.A.; Almeida, A.J.; Sousa, J.J.; Pais, A.A. 2011. The size of solid 984 lipid nanoparticles: An interpretation from experimental design. Colloids Surf., B. 84 (1), 985 117-130. https://doi.org/10.1016/j.colsurfb.2010.12.024 986 987 Walker, G.W.; Kookana, R.S.; Smith, N.E.; Kah, M.; Doolette, C.L.; Reeves, P.T.; Lovell, 988 W.; Anderson, D.J.; Turney, T.W.; Navarro, D.A. 2017. Ecological risk assessment of nano-989 enabled pesticides: A perspective on problem formulation. J. Agric. Food Chem. 66 (26), 990 6480-6486. https://doi: 10.1021/acs.jafc.7b02373 991 992 Wang, Q.; Guan, Y.X.; Yao, S.J.; Zhu, Z.Q. 2010. Microparticle formation of sodium 993 cellulose sulfate using supercritical fluid assisted atomization introduced by hydrodynamic 994 cavitation mixer. Chem. Eng. J. 159(1-3), 220-229. https://doi.org/10.1016/j.cej.2010.02.004 995 996 Zain, N.A.M.; Suhaimi, M.S.; Idris, A. 2011. Development and modification of PVA–997 alginate as a suitable immobilization matrix. Process Biochem. 46 (11), 2122-2129. 998 https://doi.org/10.1016/j.procbio.2011.08.010 999 1000 Zhao, D.; Jiao, X.; Zhang, M.; Ye, K.; Shi, X.; Xihua, Lu. Qiu, G.; Sheac, K.J. 2016. 1001 Preparation of high encapsulation efficiency fragrance microcapsules and their application 1002 in textiles. RSC Adv. 84, 80924-80933. doi: 10.1039/C6RA16030A 1003 38 1004 Zhou, T.; Zhou, W.; Wanga, Q.; Dai, P.L.; Feng, L.; Zhang, Y.L; Sun, J.H. 2011. Effects of 1005 pyrethroids on neuronal excitability of adult honeybees Apis mellifera. Pestic. Biochem. 1006 Physiol. 100 (1), 35-40. https://doi.org/10.1016/j.pestbp.2011.02.001 1007 1008 HIGHLIGHTS Nanoparticles showed good properties to be used as pyrethrum carrier system Pyrethrum extract in nanocarrier and sublethal concentrations is safer for honeybees Pyrethrum and nanotechnology showed promising results aiming agriculture applications *Highlights (3 to 5 bullet points (maximum 85 characters including spaces per bullet point) Nanopesticide based on botanical insecticide pyrethrum and its 1 potential effects on honeybees 2 Cristiane R. Oliveiraa,b; Caio E. C. Dominguesc; Nathalie F. S. de Melod; Thaisa C. Roatc; Osmar 3 Malaspinac; Monica Jones-Costab; Elaine C. M. Silva-Zacarinb and Leonardo F. Fracetoa* 4 5 a Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho”, Instituto de Ciência e Tecnologia de 6 Sorocaba, Laboratório de Nanotecnologia Ambiental, Av. Três de Março, 511, Alto da Boa Vista, 18087-180, 7 Sorocaba, SP, Brazil. 8 b Universidade Federal de São Carlos (UFSCar), Campus Sorocaba, Departamento de Biologia (CCHB), 9 Laboratório de Fisiologia da Conservação e Laboratório de Ecotoxicologia e Biomarcadores em Animais, 10 Rodovia João Leme dos Santos km 110, Itinga, 18052-780, Sorocaba, SP, Brazil. 11 c Universidade Estadual Paulista (UNESP) – “Júlio de Mesquita Filho”, Campus Rio Claro, Departamento de 12 Biologia, Centro de Estudos de Insetos Sociais (CEIS), Av. 24 A, 1515, Jardim Bela Vista, 13506-900, Rio 13 Claro, SP, Brazil. 14 d Faculdade de Medicina São Leopoldo Mandic, Campus Araras. Av. Dona Renata, 71, Santa Cândida, 13600-15 001, Araras, SP, Brazil. 16 17 ABSTRACT 18 Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and 19 nanopesticides can control agricultural pests and increase farm productivity with little 20 environmental impact. However, it is important to evaluate their toxicity on non-target 21 organisms, such as honeybees (Apis mellifera) that forage on crops. The aims of this study 22 were to develop a nanopesticide that was based on solid lipid nanoparticles (SLNs) loaded 23 with pyrethrum extract (PYR) and evaluate its physicochemical properties and short-term 24 toxicity on a non-target organism (honeybee). SLN+PYR was physicochemically stable after 25 120 days. SLN+PYR had a final diameter of 260.8 ± 3.7 nm and a polydispersion index of 26 0.15 ± 0.02 nm, in comparison with SLN alone that had a diameter of 406.7 ± 6.7 nm and a 27 polydispersion index of 0.39 ± 0.12 nm. SLN+PYR had an encapsulation efficiency of 99%. 28 The survival analysis of honeybees indicated that PYR10ng presented shorter longevity than 29 those in the control group (P ≤ 0.01). Empty nanoparticles and PYR10ng caused morphological 30 alterations in the bees’ midguts, whereas pyrethrum-loaded nanoparticles had no significant 31 effect on digestive cells, so are considered safer, at least in the short term, for honeybees. 32 These results are important in understanding the effects of nanopesticides on beneficial 33 insects and may decrease the environmental impacts of pesticides. 34 35 KEYWORD: Nanopesticide; Biocide; Sustainable agriculture, Solid lipid nanoparticles; 36 Bees. 37 38 Corresponding Authors 39 * Elaine C. M. Silva Zacarin - Universidade Federal de São Carlos (UFSCar), Campus Sorocaba, Departamento 40 de Biologia (Dbio, CCHB), Laboratório de Fisiologia da Conservação e Laboratório de Ecotoxicologia e 41 Biomarcadores em Animais, Rodovia João Leme dos Santos km 110, Itinga, 18052-780, Sorocaba, SP, Brazil. 42 Email: elaine@ufscar.br 43 *Leonardo Fernandes Fraceto – Universidade Estadual Paulista (UNESP), Instituto de Ciência e Tecnologia de 44 Sorocaba, Av. Três de Março, 511, Alto da Boa Vista, 18087-180, Sorocaba, SP, Brazil. Email – 45 leonardo.fraceto@unesp.br 46 47 *Revised manuscript with no changes marked Click here to view linked References mailto:elaine@ufscar.br mailto:leonardo.fraceto@unesp.br http://ees.elsevier.com/chem/viewRCResults.aspx?pdf=1&docID=89035&rev=2&fileID=2095523&msid={ADFEA4FE-0F1F-4AD4-8FA4-102F5488AE22} 2 1. INTRODUCTION 48 Agri-food production and population growth are amongst the greatest challenges 49 facing humanity. Agriculture is one of the primary drivers of the economy by providing food 50 to the population and benefiting producing countries, but increased population growth has 51 significantly increased humanity’s global ecological footprint, surpassing the biocapacity of 52 the Earth (SEKHON, 2014). Human populations increase exponentially over time, whereas 53 food production increases in a linear manner. Conventional agricultural practices generally 54 have negative impacts on the environment and biodiversity, as they require many resources 55 such as energy, water, and soil, and large amounts of agrochemicals and fertilizers are used 56 to improve productivity. 57 The U.S. Department of Agriculture’s (USDA) National Institute of Food and 58 Agriculture (NIFA, 2018) aims to find innovative solutions to issues related to agriculture, 59 food, the environment, and communities. NIFA’s priorities include global food security and 60 hunger, food safety, plant health and production, and animal health and production (NANO, 61 2018). Many of these issues may be resolved using nanotechnology, which has demonstrated 62 great potential in providing novel solutions to agricultural problems (SCOTT and CHEN, 63 2012; MUKHOPADHYAY, 2014). In the last few decades, nanoscience and nanotechnology 64 have been at the forefront of the development of several nanomaterials for different medical 65 and industrial purposes. Nanoparticles have been developed for a wide variety of applications 66 in the biomedical and electronic fields, while research on nanoparticles as carriers of 67 pesticides has only been conducted in the last decade, and there are still many variables to be 68 investigated before their use on crops (LIU et al., 2008; ANJALI et al., 2010; GOPAL et al., 69 2012; KAH et al., 2014; SARLAK et al., 2014; MISHRA et al., 2017; KIM et al., 2018). 70 3 Nanotechnology can deliver agricultural substances such as nanopesticides and 71 nanofertilizers that increase farm productivity, decrease the environmental impact and the 72 amount of resources used, improve pest control, and support sustainable agriculture, 73 particularly in developing countries. Furthermore, nanocarriers of pesticides and fertilizers 74 have economic advantages for agriculture, because their stability and controlled-release 75 mechanism increase efficiency and reduce the amount of chemicals required on crops 76 (PEREZ-DE-LUQUE and RUBIALES; 2009; CHEN and YADA, 2011; GRILLO et al., 77 2016; PRASAD et al., 2017; WALKER et al., 2017). 78 However, the effects of nanoparticles should be fully evaluated before they are 79 incorporated into sustainable agriculture. The U.S. National Science Foundation (NSF) and 80 Environmental Protection Agency (EPA) encourage the investigation of various aspects of 81 nanomaterials, such as their toxicity to non-target organisms, their destination, transportation, 82 and safety in the environment, and their status in terms of food legislation, and support the 83 creation of a nanomaterial database and the maintenance of food regulations (SCOTT and 84 CHEN, 2012). 85 Pyrethrum extract is a natural botanical insecticide that is extracted from 86 chrysanthemum (Chrysanthemum cinerariaefolium and Chrysanthemum cineum) flowers, is 87 composed of pyrethrin types I and II and jasmolin, and can be used on crops to control pest 88 insects (PEAY et al., 2006). Natural pyrethrum (a.i.) is highly lipophilic, photodegradable, 89 has low water solubility (<10 mg.L-1), does not exhibit biomagnification (SCHLEIER and 90 PETERSON, 2011), and leaves no toxic residues in plants. However, it is more expensive 91 than synthetic pyrethroids (PEAY et al., 2006) and is highly toxic to insects, aquatic 92 invertebrates, and fish (USEPA, 2006). Pyrethroids are insecticides that were developed to 93 improve the photodegradation of natural pyrethrin, and thus be used as an insecticide in the 94 4 field (SANTOS et al., 2007), and have great stability and target selectivity. Examples of 95 pyrethroids include deltamethrin, permethrin, and cypermethrin (MONTANHA and 96 PIMPÃO, 2012). 97 However, for the use of pyrethrum extract in the field it is necessary, at first, to load 98 it into solid lipid nanoparticles (SLNs) to prevent its fast degradation, improving its stability 99 and efficiency to allow its application on crops. Many benefits can be obtained by using 100 SLNs, such as lower large-scale production costs, greater physicochemical stability, the 101 possibility of hydrophilic and hydrophobic drug encapsulation, and the use of natural 102 products in the formulation preparation (MULLER et al., 2000; MULLER et al., 2011; 103 NASERI et al., 2015; SARANGI and PADHI et al., 2016). 104 Interactions between biological systems and nanomaterials are complex, so it is 105 important to evaluate their toxicity to non-target organisms (JACQUES et al., 2017), 106 particularly to beneficial insects such as honeybees (Apis mellifera), which play an important 107 role in pollinating agricultural crops (GIANNINI et al., 2015). Honeybee populations are 108 declining worldwide, and although multiple factors contribute to this decline (GOULSON et 109 al., 2015), it is mainly caused by agrochemicals sprayed on crops visited by bees (POTTS et 110 al., 2010). In this context, the physicochemical characterization of nanopesticides can enable 111 their future use in organic farming and contribute to sustainable agriculture, because these 112 carriers may have little effect on the environment and biodiversity (GRILLO et al., 2016; 113 PRASAD et al., 2017). However, this carrier system must have low toxicity to honeybees 114 and other beneficial insects. 115 The objectives of this study were to develop a nanopesticide that was based on SLNs 116 loaded with pyrethrum extract biocide (nanobiocide), characterize its physicochemical 117 properties, and evaluate its toxicity to honeybees (Africanized A. mellifera). We evaluated 118 5 sublethal effects on the histopathology of the bee midgut, an organ that plays a central role 119 in food digestion and nutrient absorption. It is important to emphasize the fact that there are 120 gaps of information in the literature regarding the toxicity of nanopesticides to non-target 121 organisms, such as pollinator insects including honeybees. Our results can b