Cytotoxicity induced by three commercial neonicotinoid insecticide formulations in differentiated human neuroblastoma SH-SY5Y cells.

Background: Neonicotinoid insecticides are used worldwide for crop protection. They act as agonists at postsynaptic nicotinic acetylcholine receptors (nAChRs), disrupting normal neurotransmission in target insects. Human exposure is high due to the widespread use of neonicotinoids and their residues in food. This study aimed to evaluate the in vitro neurotoxicity of three neonicotinoid commercial formulations Much 600 FS® (imidacloprid 600 g L-1), Evidence 700 WG® (imidacloprid 700 g kg-1), and Actara 250 WG® (thiamethoxam 250 g kg-1) in differentiated human neuroblastoma SH-SY5Y cell line. Methods: Cells were incubated with the pesticides for 96 h, and the cytotoxicity was evaluated through the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium·bromide (MTT) reduction and neutral red (NR) uptake assays. Toxicological pathways such as reactive oxygen (ROS) and nitrogen species (RNS) production, mitochondrial membrane potential, cell death mode, and the expression of the pro-apoptotic protein Bax were also evaluated. Results: EC50 values of 266.4, 4,175, and 653.2 mg L-1 were found for Much®, Evidence® and Actara®, respectively. Significant increases in ROS and RNS generation were observed for all pesticides, while mitochondrial membrane potential and Bax protein expression showed no significant changes. Analysis of cell death mode revealed an increase in early apoptotic cells. Conclusion: Therefore, neonicotinoid insecticides are potentially neurotoxic, reinforcing concerns about human exposure to these commercial formulations.

Sex and adrenal hormones in association with insecticide biomarkers among adolescents living in ecuadorian agricultural communities.

BACKGROUND: Organophosphate, pyrethroid, and neonicotinoid insecticides have resulted in adrenal and gonadal hormone disruption in animal and in vitro studies; limited epidemiologic evidence exists in humans. We assessed relationships of urinary insecticide metabolite concentrations with adrenal and gonadal hormones in adolescents living in Ecuadorean agricultural communities. METHODS: In 2016, we examined 522 Ecuadorian adolescents (11-17y, 50.7% female, 22% Indigenous; ESPINA study). We measured urinary insecticide metabolites, blood acetylcholinesterase activity (AChE), and salivary testosterone, dehydroepiandrosterone (DHEA), 17ß-estradiol, and cortisol. We used general linear models to assess linear (ß = % hormone difference per 50% increase of metabolite concentration) and curvilinear relationships (ß2 = hormone difference per unit increase in squared ln-metabolite) between ln-metabolite or AChE and ln-hormone concentrations, stratified by sex, adjusting for anthropometric, demographic, and awakening response variables. Bayesian Kernel Machine Regression was used to assess non-linear associations and interactions. RESULTS: The organophosphate metabolite malathion dicarboxylic acid (MDA) had positive associations with testosterone (ßboys = 5.88% [1.21%, 10.78%], ßgirls = 4.10% [-0.02%, 8.39%]), and cortisol (ßboys = 6.06 [-0.23%, 12.75%]. Para-nitrophenol (organophosphate) had negatively-trending curvilinear associations, with testosterone (ß2boys = -0.17 (-0.33, -0.003), p = 0.04) and DHEA (ß2boys = -0.49 (-0.80, -0.19), p = 0.001) in boys. The neonicotinoid summary score (ßboys = 5.60% [0.14%, 11.36%]) and the neonicotinoid acetamiprid-N-desmethyl (ßboys = 3.90% [1.28%, 6.58%]) were positively associated with 17ß-estradiol, measured in boys only. No associations between the pyrethroid 3-phenoxybenzoic acid and hormones were observed. In girls, bivariate response associations identified interactions of MDA, Para-nitrophenol, and 3,5,6-trichloro-2-pyridinol (organophosphates) with testosterone and DHEA concentrations. In boys, we observed an interaction of MDA and Para-nitrophenol with DHEA. No associations were identified for AChE. CONCLUSIONS: We observed evidence of endocrine disruption for specific organophosphate and neonicotinoid metabolite exposures in adolescents. Urinary organophosphate metabolites were associated with testosterone and DHEA concentrations, with stronger associations in boys than girls. Urinary neonicotinoids were positively associated with 17ß-estradiol. Longitudinal repeat-measures analyses would be beneficial for causal inference.

Long-lasting developmental effects in rat offspring after maternal exposure to acetamiprid in the drinking water during gestation.

Neonicotinoids (NNTs) are a class of insecticides proposed to be safe for pest control in urban, suburban, and agricultural applications. However, little is known about their developmental effects after repeated low-dose exposures during gestation. Here, we tested a dose considered subthreshold for maternal toxicity in rats (6 mg/kg/day) by assessing several morphological, biochemical, and neurobehavioral features in preterm fetuses and developing pups after maternal administration of the NTT acetamiprid (ACP) dissolved in the drinking water during gestational days (GD) 2-19. The exploratory evaluation included monitoring maternal body weight gain, fetal viability, body weight and sex ratio, cephalic length, neonatal body weight and sex ratio, metabolic enzymes in the placenta, maternal blood and fetal liver, and anogenital distance and surface righting response during infancy. We also used the circling training test to study the integrity of the associative-spatial-motor response in adolescence. Results showed no consistent findings indicating maternal, reproductive or developmental toxicity. However, we found ACP effects on maternal body weight gain, placental butyrylcholinesterase activity, and neurobehavioral responses, suggestive of a mild toxic action. Thus, our study showed a trend for developmental susceptibility at a dose so far considered subtoxic. Although the ACP concentration in environmental samples of surface water and groundwater has been mostly reported to be much lower than that used in our study, our results suggest that the ACP point of departure used in current guidelines aimed to prevent developmental effects may need to be verified by complementary sensitive multiple-endpoint testing in the offspring.

Residual determination and acute toxicity of the neonicotinoid clothianidin in the neotropical stingless bee Tetragonisca angustula Latreille, 1811 (Apidae: Meliponini).

Bees play a crucial role as natural pollinators, ensuring the maintenance and stability of the world's biodiversity and agricultural crops. Native bees in neotropical regions belong to the Meliponini tribe, a larger group that differs significantly in behavior and biology from honeybees (e.g., Apis mellifera) and solitary bees (e.g., Osmia spp.). Hence, the exposure and effects of pesticides is also likely to vary among these different species. The aim of this study was to develop an analytical method to determine the presence of the neonicotinoid clothianidin in the Brazilian native stingless bee Tetragonisca angustula (local common name: Jataí). The method used for the chemical analysis involved a QuEChERS technique combined with UHPLC-MS/MS analysis. The developed method was subsequently used to analyze collected field samples. In addition, the acute toxicity of the pesticide to T. angustula was evaluated in a laboratory bioassay evaluating both lethal and sublethal endpoints. The analytical method was successfully developed with detection and quantification limits of 1.55 and 5 µg L-1, respectively, along with a linear range of 1-5 ng mL-1. Clothianidin was detected in environmental samples (9.2-32.9 ng g-1), and the exposure experiments demonstrated acute oral toxicity to adults of T. angustula, (24 h-LD50 of 0.16 ng a.i./bee), as well as no significative interference in acetylcholinesterase activity. Considering the obtained toxicity endpoints for T. angustula and those reported in the literature for other bee species, this study revealed that T. angustula is more (lethally) sensitive to clothianidin than other bee species, including those commonly used in environmental risk assessment studies. This thus also supports the call for using native test species in (regional) risk assessment evaluations.

Effect of pesticides on phosphorylation of tau protein, and its influence on Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and neurodegenerative illness which results in alterations in cognitive development. It is characterized by loss/dysfunction of cholinergic neurons, and formation of amyloid plaques, and formation of neurofibrillary tangles, among other changes, due to hyperphosphorylation of tau-protein. Exposure to pesticides in humans occurs frequently due to contact with contaminated food, water, or particles. Organochlorines, organophosphates, carbamates, pyrethroids and neonicotinoids are associated with the most diagnosed incidents of severe cognitive impairment. The aim of this study was to determine the effects of these pesticides on the phosphorylation of tau protein, and its cognitive implications in the development of AD. It was found that exposure to pesticides increased the phosphorylation of tau protein at sites Ser198, Ser199, Ser202, Thr205, Ser396 and Ser404. Contact with these chemicals altered the enzymatic activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta, and protein phosphatase-2A. Moreover, it altered the expression of the microtubule associated protein tau gene, and changed levels of intracellular calcium. These changes affected tau protein phosphorylation and neuroinflammation, and also increased oxidative stress. In addition, the exposed subjects had poor level of performance in tests that involved evaluation of novelty, as test on verbal, non-verbal, spatial memory, attention, and problem-solving skills.

Behavioral and physiological response of the passerine bird Agelaioides badius to seeds coated with imidacloprid.

Neonicotinoids are globally used insecticides, and there are increasing evidence on their negative effects on birds. This study is aimed at characterizing the behavioral and physiological effects of the neonicotinoid imidacloprid (IMI) in a songbird. Adults of Agelaioides badius were exposed for 7 days to non-treated peeled millet and to peeled millet treated with nominal concentrations of 75 (IMI1) and 450 (IMI2) mg IMI/kg seed. On days 2 and 6 of the trial, the behavior of each bird was evaluated for 9 min by measuring the time spent on the floor, the perch, or the feeder. Daily millet consumption, initial and final body weight, and physiological, hematological, genotoxic, and biochemical parameters at the end of exposure were also measured. Activity was greatest on the floor, followed by the perch and the feeder. On the second day, birds exposed to IMI1and IMI2 remained mostly on the perch and the feeder, respectively. On the sixth day, a transition occurred to sectors of greater activity, consistent with the disappearance of the intoxication signs: birds from IMI1 and IMI2 increased their time on the floor and the perch, respectively. Control birds always remained most of the time on the floor. IMI2 birds significantly decreased their feed intake by 31% the first 3 days, compared to the other groups, and significantly decreased their body weight at the end of the exposure. From the set of hematological, genotoxic, and biochemical parameters, treated birds exhibited an alteration of glutathione-S-transferase activity (GST) in breast muscle; the minimal effects observed are probably related to the IMI administration regime. These results highlight that the consumption of less than 10% of the bird daily diet as IMI-treated seeds trigger effects at multiple levels that can impair bird survival.

Effect of Systemic Insecticides Applied via Drench on the Mortality of Diaphorina citri on Curry Leaf.

Huanglongbing (HLB), the most serious disease in citriculture, is caused by the bacteria Candidatus Liberibacter spp., which is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. HLB is mainly controlled with insecticides, necessitating the development of alternative methods, e.g., the use of trap plants such as curry leaf Bergera koenigii, which is highly attractive to the ACP. We evaluated the effects of the main systemic insecticides used by citrus growers, applied via drench to adults of D. citri on the curry leaf tree. We tested the persistence of three pesticides: thiamethoxam, thiamethoxam + chlorantraniliprole, and imidacloprid in protected cultivation and the field condition at 7, 14, 28, 42, 56, 70, 98, and 154 days after the application. Different concentrations of insecticides containing the active ingredient thiamethoxam were tested on adults to determine the LC10 and LC50. Finally, we assessed the sublethal effects on the oviposition and development of D. citri. The insecticides controlled the adults for long periods. However, in the field experiment, from 42 days after application there was a decrease in mortality caused by pesticides applied via drench, while in the protected cultivation, mortality did not decline until the last day of evaluation. The median lethal concentration (LC50) for thiamethoxam was 0.031 g of active ingredient per plant, and for thiamethoxam in a mixture, the LC50 was 0.028 g a.i. per plant. In the experiment with sublethal doses, D. citri did not oviposit on the treated plants. Our findings suggest that the attract-and-kill system using the curry leaf tree and systemic insecticides is effective for the control of D. citri and contributes to the integrated management of HLB.

Behavioral, biochemical, and endocrine responses of zebrafish to 30-min exposure with environmentally relevant concentrations of imidacloprid-based insecticide.

The imidacloprid-based insecticides (IBIs) are among the most used insecticides worldwide, and chronic and acute toxic effects (days exposure protocols) have been reported in several species in studies of IBIs at lethal concentrations. However, there is little information on shorter time exposures and environmentally relevant concentrations. In this study, we investigated the effect of a 30-min exposure to environmentally relevant concentrations of IBI on the behavior, redox status, and cortisol levels of zebrafish. We showed that the IBI decreased fish locomotion and social and aggressive behaviors and induced an anxiolytic-like behavior. Furthermore, IBI increased cortisol levels and protein carbonylation and decreased nitric oxide levels. These changes were mostly observed at 0.013 and 0.0013 µg·L-1 of IBI. In an environmental context, these behavioral and physiological disbalances, which were immediately triggered by IBI, can impair the ability of fish to evade predators and, consequently, affect their survival.

Neurocytotoxicity of imidacloprid- and acetamiprid-based comercial insecticides over the differentiation of SH-SY5Y neuroblastoma cells.

Neonicotinoids are effective insecticides with specificity for invertebrate nicotinic acetylcholine receptors. Neonicotinoids are chemically stable and tend to remain in the environment for long so concerns about their neurotoxicity in humans do nothing but increase. Herein, we evaluated the chronic toxic effects of acetamiprid- and imidacloprid-based insecticides over the differentiation of human neuroblastoma SH-SY5Y cells, which were exposed to these insecticides at a concentration range similar to that applied to crop fields (0.01-0.5 mM). Both insecticides did not have acute cytotoxic effects in both non-differentiated and in staurosporine-differentiated SH-SY5Y cells cytotoxicity as measured by the MTT and vital-dye exclusion tests. However, after a chronic (7-day) treatment, only imidacloprid dose-dependently decreased the viability of SH-SY5Y cells (F(4,39) = 43.05, P < 0.001), largely when administered-during cell differentiation (F(4,39) = 51.86, P < 0.001). A well-defined dose-response curve was constructed for imidacloprid on day 4 (R2 = 0.945, EC50 = 0.14 mM). During differentiation, either imidacloprid or acetamiprid dose-dependently caused neurite branch retraction on day 3, likely because of oxidative stress, to the extent that cells turned into spheres without neurites after 7-day treatment. Despite their apparent safety, the neurodevelopmental vulnerability of SH-SY5Y neurons to the chronic exposure to imidacloprid and to a lesser extent to acetamiprid points to a neurotoxic risk for humans.

Sex-specific alterations in adaptive responses of Chironomus columbiensis triggered by imidacloprid chronic and acute sublethal exposures.

The use of imidacloprid is a common pest control practice in the Neotropical region. However, the imidacloprid unintended sublethal effects on Neotropical aquatic non-target arthropods and undesirable consequences for aquatic environments remain unclear. Here, we assessed the susceptibility of Chironomus columbiensis (Diptera: Chironomidae) larvae to the neonicotinoid imidacloprid and evaluated whether sublethal exposure types would trigger sex-dependent adaptive responses (e.g., emergence, body mass, reproduction, wing morphology). We conducted a concentration-mortality curve (96 h of exposure) and established chronic and acute sublethal exposure bioassays. While chronic sublethal exposures consisted of exposing individuals during their entire larval and pupal stages, the acute sublethal exposures represented a single short duration (24 h) exposure episode during either the first or fourth larval instar. Our results revealed that chronic sublethal exposure reduced the body mass of males, while acute sublethal exposures during the first instar resulted in heavier males than those that were not exposed to imidacloprid. Chronic exposure also reduced the reproduction of males and females, while the acute sublethal exposure only affected the reproduction of individuals that were imidacloprid-exposed on their later larval instar. Chronic and acute sublethal exposures did differentially affect the wing properties of C. columbiensis males (e.g., increased size when chronically exposed and highly asymmetric wings when acutely exposed in early larval phase) and females (e.g., highly asymmetric wings when chronically and acutely exposed). Collectively, our findings demonstrated that imidacloprid can cause unintended sublethal effects on C. columbiensis, and those effects are dependent on sex, exposure type, and developmental stage.

Thirty-minute test to detect insecticide resistance under field conditions: the case of the Asian citrus psyllid.

BACKGROUND: Diaphorina citri Kuwayama is one of the most destructive citrus pests worldwide. It is controlled mainly through applications of conventional insecticides. Methodologies used to estimate resistance to insecticides do not correlate with field efficacy, and do not provide timely and reliable information to make decisions at a site where spraying is needed. The use of diagnostic doses with 30-min exposure is proposed for estimating the resistance of D. citri to imidacloprid, spinosad, malathion and chlorpyrifos at the orchard level. RESULTS: Under laboratory conditions, we estimated the lowest doses that caused 100% mortality within 30 min of exposure (diagnostic dose) in a susceptible D. citri colony. The diagnostic doses for imidacloprid, spinosad, malathion and chlorpyrifos were 7.4, 4.2, 1.0 and 5.5 mg a.i. L-1 , respectively. Under field conditions, we applied the diagnostic doses to D. citri feeding on Citrus aurantifolia Swingle at five localities in Michoacan state, Mexico (Nueva Italia, Santo Domingo, El Varal, Gambara and El Ceñidor). Additionally, the field efficacy of these insecticides against these populations was evaluated. A significant correlation between field efficacy and mortality was observed with the diagnostic doses for imidacloprid, malathion and chlorpyrifos (R2 ≥ 0.93). The correlation for spinosad could not be estimated because the mortality caused by the diagnostic dose and its field efficacy at all study sites was consistently >98%. CONCLUSIONS: Field efficacy and resistance were estimated based on the field diagnostic doses with 30-min exposure for all tested insecticides. Consequently, growers and pest management technicians can estimate the performance of the evaluated insecticides at the orchard level and before insecticide application. © 2023 Society of Chemical Industry.

Individual and combined toxicity of imidacloprid and two seed dressing insecticides on collembolans Folsomia candida.

The aim of this study was to examine the chronic toxicity of imidacloprid (IMI), clothianidin (CLO) and fipronil (FIP) as a single exposure, as well as binary mixtures of IMI with CLO or FIP toward collembolans Folsomia candida, which are fauna present in the soil. Chronic toxicity assays were performed following an ISO guideline in a Tropical Artificial Soil (TAS), and the influence on the number and growth of the juveniles produced were determined. The range of nominal concentrations used in the tests with the individual compounds was 0.08-1.28 mg/kg (IMI), 0.079-1.264 mg/kg (FIP) and 0.007-0.112 mg/kg (CLO), whereas the mixture assays were performed with half the value used in the tests with individual compounds. Based upon single exposures, IMI produced a similar impact of reducing reproduction by 50% (EC50 ranging from 0.74 to 0.85 mg/kg) compared to FIP (EC50 = 0.78 mg/kg), whereas CLO was the most toxic to F. candida (EC50 = 0.08 mg/kg). Their mixtures generally resulted in a diminished effect on reproduction, as evidenced by the higher EC50 values. In contrast, in the case of the IMI+FIP combination at high concentrations at the EC50 level, a synergistic effect on toxicity was observed. The single exposure to the three insecticides and the mixture of IMI-FIP also decreased the size of generated juveniles, which was evidenced by the reduction in the proportion of large juveniles and increased proportion of small juveniles. However, both binary mixtures (IMI-FIP and IMI-CLO) presented antagonistic effects as evidenced by less than expected reductions in growth. Data on the toxic effects of IMI in a mixture with other seed dressing insecticides to collembolans provides useful information to environmental risk assessors by diminishing the uncertainties on the ecological risk of exposure to pesticides, enabling soil management degradation by utilizing multiple insecticides.

Influence of temperature and soil moisture on the toxic potential of clothianidin to collembolan Folsomia candida in a tropical field soil.

Climate change can alter the toxic effects of pesticides on soil invertebrates. However, the nature and magnitude of the influence of climatic factors on clothianidin impacts in tropical soils are still unknown. The influence of increasing atmospheric temperature and the reduction in soil moisture on the toxicity and risk of clothianidin (seed dressing formulation Inside FS®) were assessed through chronic toxicity tests with collembolans Folsomia candida in a tropical field soil (Entisol). The risk of clothianidin for collembolans was estimated using the Toxicity-Exposure Ratio (TER) approach. Organisms were exposed to increasing clothianidin concentrations at 20, 25 and 27 °C in combination with two soil moisture conditions (30 and 60% of the maximum water holding capacity-WHC). The effect of temperature and soil moisture content on clothianidin toxicity was verified through the number of F. candida juveniles generated after 28 days of exposure to the spiked soil. The toxicities estimated at 25 °C (EC50_30%WHC = 0.014 mg kg-1; EC50_60%WHC = 0.010 mg kg-1) and 27 °C (EC50_30%WHC = 0.006 mg kg-1; EC50_60%WHC = 0.007 mg kg-1) were 2.9-3.0-fold (25 °C) and 4.3-6.7-fold (27 °C) higher than those found at 20 °C (EC50_30%WHC = 0.040 mg kg-1; EC50_60%WHC = 0.030 mg kg-1), indicating that clothianidin toxicity increases with temperature. No clear influence of soil moisture content on clothianidin toxicity could be observed once the EC50 values estimated at 30% and 60% WHC, within the same temperature, did not significantly differ. A significant risk was detected in all temperatures and soil moisture scenarios studied, and the TER values indicate that the risk can increase with increasing temperatures. Our results revealed that temperature could overlap with soil moisture in regulating clothianidin toxicity and reinforce the importance of including climatic factors in the prospective risk assessment of pesticides.

Toxicity of the neonicotinoid insecticides thiamethoxam and imidacloprid to tadpoles of three species of South American amphibians and effects of thiamethoxam on the metamorphosis of Rhinella arenarum.

The present study examined the acute and chronic toxicity of the neonicotinoid insecticides imidacloprid (IMI) and thiamethoxam (TIA) on the neotropical amphibian species Rhinella arenarum, Rhinella fernandezae and Scinax granulatus. The median lethal concentration after 96 hr exposure (96 hr-LC50) ranged between 11.28 and >71.2 mg/L amongst all species and development stages tested, indicating that these pesticides are not likely to produce acute toxicity in the wild. The subchronic toxicity was also low, with 21 day-LC50 values ranging between 27.15 and >71.2 mg/L. However, tadpoles of Rhinella arenarum exposed to thiamethoxam from stage 27 until completion of metamorphosis presented a significantly lower metamorphic success rate together with a smaller size at metamorphosis, starting from the lowest concentration tested. Although a number of studies previously examined the effects of neonicotinoids on amphibian tadpoles, these investigations focused on the time to metamorphosis and reported a variety of results including retardation, acceleration or lack of effect. Here, data demonstrated that thiamethoxam predominantly impacts metamorphosis through reduction of the transformation success and body weight, rather than by affecting the timings of metamorphosis. By closely monitoring progression of tadpoles through the different stages, impairment of metamorphosis was demonstrated to occur during the transition from stage 39 to 42, suggesting an effect on the thyroid system. An asymmetry in the length of the arms was also observed in metamorphs treated with thiamethoxam. Overall, these results indicate that thiamethoxam, and conceivably other neonicotinoids, have the potential to significantly impair metamorphosis of amphibians and diminish their performance and survival in the wild.

The multigenerational effects of clothianidin on Chironomus xanthus: Larvae exposed to this acetylcholine super agonist show no clear resistance.

Clothianidin (CLO) is an insecticide belonging to the second-generation class of neonicotinoids. In this study, we evaluated how CLO affects the survival and the complete life cycle of the tropical insect Chironomus xanthus, a non-target species, considering the Parental (P) and Filial (F1) generations. We found a 48 h-lethal concentration (LC50) of CLO of 3.78 µg/L. The lowest observed effect concentrations (LOECs) were: i) for body growth and head capsule width in P generation = 47.3 ng/L CLO; ii) for body growth and head capsule width in F1 generation larvae = 80 and 36.4 ng/L CLO, respectively; iii) for cumulative emergence it was 80 ng/L CLO in the P generation, while there was no significant difference in the F1 generation; iv) for total developmental time for males and females = 61.53 ng/L in P generation; v) in the F1 generation, the LOEC was determined to be 36.4 ng/L for males and 80 ng/L for females; vi) The number of total hatched eggs and total hatched eggs/female had LOECs of 36.4 ng/L CLO for both generations. Our study reveals that environmentally relevant concentrations of the CLO-based insecticide are highly toxic to C. xanthus. It also shows that the F1 generation, resulting from parents exposed to CLO was not clearly resistant to the insecticide. This fact might be explained by the different effects observed for males and females of F1 generation. Understanding the sub-types of acetylcholine receptors present on target and non-target insect species and toxicological effects of neonicotinoids seems to be desirable for the insecticide industry to deal with insect pests and the environmental protection of non-target organisms.

A systematic review of research conducted by pioneer groups in ecotoxicological studies with bees in Brazil: advances and perspectives.

Brazil presents the most threatened endemic or rare species among neotropical regions, with the Hymenoptera order, to which bees belong, classified as a high-risk category. In Brazil, the main cause of bee death is the indiscriminate use of pesticides. In this context, groups such as Bee Ecotoxicology and Conservation Laboratory (LECA in Portuguese) and Bees and Environmental Services (ASAs in Portuguese) have become a reference in studies evaluating the impacts of pesticides on bees since 1976. Thus, the objective of this review was to conduct a quantitative and qualitative review of the studies conducted by these groups to evaluate and compile the advances made over the years, identify potential knowledge gaps for future studies, and support the sensitivities of stingless bees when compared to the species Apis mellifera. The quantitative analyses showed that most studies were carried out in the genus Apis, under laboratory conditions. However, more recently (since 2003), studies have also focused on stingless bees and the neonicotinoid class of insecticides. The most relevant gaps identified were the lack of studies under field conditions and on bee biology. The qualitative analyses indicated that Brazilian stingless bees are more susceptible to pesticides than A. mellifera and require a much lower average dose, concentration, or lethal time to display morphological and behavioral damage or decreased lifespan. Thus, future studies should work towards establishing more representative protocols for stingless bees. Furthermore, public policies must be created for the protection and conservation of bees native to Brazil.

Detection and Concentration of Neonicotinoids and Other Pesticides in Honey from Honey Bee Colonies Located in Regions That Differ in Agricultural Practices: Implications for Human and Bee Health.

This is a preliminary study conducted to analyze the presence and concentration of pesticides in honey obtained from honey bee colonies located in two regions with managed ecosystems that differ in the intensity and technification of agricultural practices. Fourteen pesticides at variable concentrations were detected in 63% of the samples analyzed. The pesticides most frequently found at higher concentrations were insecticides (neonicotinoids, followed by organophosphates), herbicides, and fungicides. The number, frequency, and concentration of pesticides were higher in samples collected from hives located where intensive and highly-technified agriculture is practiced. Forty-three percent of the samples from that zone had residues of imidacloprid, compared with only 13% of the samples from the less-technified zone. Furthermore, 87.5% of those samples had imidacloprid concentrations that were above sublethal doses for honey bees (>0.25 ng/g) but that are not considered hazardous to human health by the European Commission. The results of this study suggest that honey can be used as a bioindicator of environmental contamination by pesticides, which highlights the need to continue monitoring contaminants in this product to determine the risks of pesticide impacts on pollinator health, on ecosystems, and on their potential implications to human health and other non-target organisms.

Evaluation of the Bioefficacy and Insecticide Compatibility of Entomopathogens for Management of Whitefly (Hemiptera: Aleyrodidae) on Upland Cotton Under Laboratory and Polyhouse Conditions.

Entomopathogens (EPFs) are potential alternatives to chemical insecticides for managing Bemisia tabaci (Genn.), an invasive pest of the cotton crop. EPFs alone may not always provide enough insect pest control, but combining EPFs with pesticides, provided both components are compatible, can make an integrated pest management program considerably more effective. Hence, the bioefficacy of EPFs against whitefly, their compatibility with pesticides, and the factors responsible for determining compatibility were studied. The highest nymphal mortality was recorded with the Beauveria bassiana strains Bb-4511 (95.1%) and Bb-4565 (89.9%), and Metarhizium anisopliae Ma-1299 (86.7%) at 1 × 106 conidia ml-1. Lower LC50 values were observed for Cordyceps javanica Cj-089 and Bb-4511, 0.2 × 104 and 0.5 × 104 conidia ml-1, respectively. The toxicity index values in insecticide sensitivity assays ranged from 19.4 to 119.6% among all the EPFs. Comparatively, all the EPFs except Bb-4543 and Bb-4565 showed compatible to moderately toxic reactions to neonicotinoids and spinosyns. Organophosphates (ethion) and pyrethrins (bifenthrin) were toxic to very toxic to all the EPFs except Bb-4511, Fv-083, and Ma-1299. Cj-102 and Cj-089 were compatible with 50% of the average recommended dose of bifenthrin and ethion, and the average recommended dose for the field application of neonicotinoids and spinosyns. Principal component analysis showed that spore production and toxicity index values correlate with each other and are responsible for determining the EPF compatibility with insecticides. The EPF spore production and toxicity index are important factors for determining chemical compatibility. Compatible EPFs can be used individually or in combination as promising and compatible biological alternatives to insecticides in the management of whitefly in cotton.

The effects of thiamethoxam on coffee seedling morphophysiology and Neotropical leaf miner (Leucoptera coffeella) infestations.

BACKGROUND: Coffee (Coffea arabica L.) is one of the main commodities produced in Brazil. Insecticides like the (systemic) neonicotinoid thiamethoxam are widely used to suppress pest populations during coffee production, in particular the Neotropical leaf miner (Leucoptera coffeella Guérin-Mèneville & Perrottet, 1842) (Lepidoptera: Lyonetiidae). In addition to its efficacy against this pest species, thiamethoxam is also thought to be a bioactivator of plant metabolism, but has not yet been tested for such activity. Thus, the objectives of the present study were (1) to assess the concentration-response effects of thiamethoxam on the vegetative vigor of coffee seedlings (C. arabica 'Catuaí 144' cultivar) at different concentrations [2, 20, 40, 80 and 200 mg active ingredient (a.i.) kg-1 ] applied via soil drenching and (2) to evaluate if the plant response interferes with the effectiveness of thiamethoxam in controlling leaf miner populations. The morphophysiological traits of the coffee seedlings were evaluated 20, 40, 60 and 80 days after application, and leaf miner infestations were recorded starting 20 days after the insecticide application with the releasing of adults, and every 20 days afterwards. RESULTS: The results indicated that thiamethoxam has a deleterious effect on the morphophysiological traits of the plants compromising their development with increase in concentrations. However, leaf area exhibited a different pattern with a peak at 50 mg a.i. kg-1 consistent with thiamethoxam-induced hormesis (i.e. biphasic response with stimulatory effect at sublethal range of a toxic substance at the higher concentration). Nonetheless, such bioactivator effect did not affect thiamethoxan effectiveness against the leaf miner even at the lowest concentration tested. CONCLUSION: Thiamethoxan exhibited bioactivation effect on leaf at low concentration, but without compromising efficacy against leaf miner populations. Therefore, its proposed metabolism-boosting properties may encourage the unnecessary use of this insecticide, potentially leading to higher selection for insecticide resistance and an eventual decline in its effectiveness against the Neotropical leaf miner. © 2022 Society of Chemical Industry.

Neonicotinoids exposure assessment in Africanized honey bees (Apis mellifera L.) by using an environmentally-friendly sample preparation technique followed by UPLC-MS/MS.

A miniaturized QuEChERS extraction method followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to analyze thiamethoxam and imidacloprid in 0.3 g of lyophilized Africanized honey bees (Apis mellifera L.). The work aimed to estimate honey bees' exposure to pesticides at the nanogram/gram (ng g-1) levels, using small sample and reagent quantities. Low amounts of solvents and salts were employed - 15× less than used in traditional methods. Average recoveries ranged from 64.5% to 99.7%, with repeatability below 20% for samples spiked at 3 and 167 ng g-1. LOD and LOQ were 0.7, and 3 ng g-1 for both pesticides. Applying the proposed approach, honey bee samples from different apiaries from the State of São Paulo (Brazil) were analyzed. The pesticides were detected in concentrations between 7.0 and 27.0 ng g-1. Thus, the proposed method can be used as a greener alternative to analyze the two neonicotinoids at trace levels in small quantities of bees, consequently saving chemicals and waste.