Enantioselectivity of indoxacarb during the growing, processing, and brewing of tea: Degradation, metabolites, and toxicities.

Chiral pesticides are unique hazardous materials. Here, we systematically studied the potentially harmful products of enantioselective indoxacarb degradation throughout tea growth, processing, and brewing and tested their toxicity to tea geometrid larvae and honeybees. The half-lives of S-indoxacarb and R-indoxacarb during tea growth were 2.6 d and 3.3 d, respectively. There was a trend toward the production of S-indoxacarb from R-indoxacarb. The degradation products IN-JT333, IN-MK638, IN-MF014, and IN-KG433 were also characterized in tea growth and processing and detected. IN-JT333, previously known as a direct insecticidal compound produced by the enzymatic transformation of indoxacarb in insects, was first found in plant samples. The fixation and rolling of green tea and the rolling of black tea were the most important steps that affected indoxacarb and its degradation products. The leaching rates of R-indoxacarb and S-indoxacarb were slightly higher in green tea than in black tea. The maximum leaching rates of IN-MK638 and IN-MF014 during the brewing process reached 89.9% and 94.1%, respectively. Contact toxicity tests with honeybees and tea geometrid larvae in the lab showed that the relative toxicities of the compounds could be ranked as follows: S-indoxacarb > indoxacarb (3S + 1R) ≫ R-indoxacarb. TEST toxicity predictions showed that relative toxicities were ranked IN-KG433 > indoxacarb > IN-JT333 > IN-MK638 > IN-MF014. The toxicity of the degradation product IN-KG433 is higher than that of indoxacarb itself, and its maximum leaching rate is as high as 88.2%. It therefore transfers readily from processed tea to the tea infusion during the brewing process. These findings indicate the need to pay attention to the risk of metabolites and enantiomeric differences and provide new, comprehensive insight into the risk factors for indoxacarb in tea and are relevant to the study of other chiral pesticides.

Is there a risk to honeybees from use of thiamethoxam as a sugar beet seed treatment?

The ban imposed by the European Union on the use of neonicotinoids as sugar beet seed treatments was based on the exposure of bees to residues of neonicotinoids in pollen and nectar of succeeding crops. To address this concern, residues of thiamethoxam (TMX) and clothianidin (CTD) were analyzed in soil collected from fields planted in at least the previous year with thiamethoxam-treated sugar beet seed. This soil monitoring program was conducted at 94 sites across Germany in two separate years. In addition, a succeeding crop study assessed residues in soil, guttation fluid, pollen, and nectar sampled from untreated succeeding crops planted in the season after thiamethoxam seed-treated sugar beet at eight field sites across five countries. The overall mean residues observed in soil monitoring were 8.0 ± 0.5 µg TMX + CTD/kg in the season after the use of treated sugar beet seed. Residue values decreased with increasing time interval between the latest thiamethoxam or clothianidin application before sugar beet drilling and with lower application frequency. Residues were detected in guttation fluid (2.0-37.7 µg TMX/L); however, the risk to pollinators from this route of exposure is likely to be low, based on the reported levels of consumption. Residues of thiamethoxam and clothianidin in pollen and nectar sampled from the succeeding crops were detected at or below the limit of quantification (0.5-1 µg a.i./kg) in 86.7% of pollen and 98.6% of nectar samples and, unlike guttation fluid residues, were not correlated with measured soil residues. Residues in pollen and nectar are lower than reported sublethal adverse effect concentrations in studies with honeybee and bumble bee individuals and colonies fed only thiamethoxam-treated sucrose, and are lower than those reported to result in no effects in honeybees, bumble bees, and solitary bees foraging on seed-treated crops. Integr Environ Assess Manag 2022;18:709-721. © 2021 SYNGENTA. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Concentrations of imidacloprid and thiamethoxam in pollen, nectar and leaves from seed-dressed cotton crops and their potential risk to honeybees (Apis mellifera L.).

Neonicotinoid insecticides (NIs) have recently been recognized as co-factors in the decline of honeybee colonies because most neonicotinoids are systemic and can transfer into the pollen and nectar of many pollinated crops. In this study, we collected pollen, nectar and leaves from a cotton crop treated with imidacloprid and thiamethoxam to measure the residue levels of these two NIs at different application doses during the flowering period. Then, the residual data were used to assess the risk posed by the systemic insecticides to honeybees following mandated methods published by the European Food Safety Authority (EFSA), and a highly toxic risk to honeybees was highlighted. Imidacloprid was found in both pollen and nectar samples, whereas thiamethoxam was found in 90% of pollen samples and over 60% of nectar samples. Analysis of the pollen and nectar revealed residual amounts of imidacloprid ranging from 1.61 to 64.58 ng g-1 in the pollen and from not detected (ND) to 1.769 ng g-1 in the nectar. By comparison, the thiamethoxam concentrations in pollen and nectar ranged from ND to 14.521 ng g-1 and from ND to 4.285 ng g-1, respectively. The results of this study provide information on the transfer of two NIs from seed treatment to areas of the plant and provides an understanding of the potential exposure of the bee and other pollinators to systemic insecticides.

Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee.

Apis mellifera perform important pollination roles in agroecosystems. However, there is often intensive use of systemic pesticides in crops, which can be carried to the colony by forage bees through the collection of contaminated pollen and nectar. Inside the colony, pollen loads are stored by bees that add honey and several enzymes to this pollen. Nevertheless, intra-colonial chronic exposure could induce sublethal effects in young bees exposed to a wide range of pesticides present in these pollen loads. This study was aimed to both determine the survival rate and evaluate the sublethal effects on the hepato-nephrocitic system in response to continuous oral exposure to lower concentrations of neonicotinoid thiamethoxam (TXT) and picoxystrobin fungicide (PXT). Exposure to a single chemical and co-exposure to both pesticides were performed in newly emerged honeybee workers. A significant decrease in the bee survival rates was observed following exposure to TXT (0.001 ng a.i./µL) and PXT (0.018 ng a.i./µL), as well as following co-exposure to TXT+PXT/2. After five days of continuous exposure, TXT induced sub-lethal effects in the organs involved in the detoxification of xenobiotics, such as the fat body and pericardial cells, and it also induced a significant increase in the hemocyte number. Thus, the hepato-nephrocitic system (HNS) reached the greatest level of activity of pericardial cells as an attempt to eliminate this toxic compound from hemolymph. The HNS was activated at low levels by PXT without an increase in the hemocyte number; however, the mobilization of neutral glycoconjugates from the trophocytes of the fat body was prominent only in this group. TXT and PXT co-exposure induced intermediary morphological effects in trophocytes and pericardial cells, but oenocytes from the fat body presented with atypical cytoplasm granulation only in this group. These data showed that the realistic concentrations of these pesticides are harmful to newly emerged Africanized honeybees, indicating that intra-colonial chronic exposure drastically reduces the longevity of bees exposed to neonicotinoid insecticide (TXT) and the fungicide strobilurin (PXT) as in single and co-exposure. Additionally, the sublethal effects observed in the organs constituting the HNS suggest that the activation of this system, even during exposure to low concentrations of theses pesticides, is an attempt to maintain homeostasis of the bees. These data together are alarming because these pesticides can affect the performance of the entire colony.

Application of the combination index (CI)-isobologram equation to research the toxicological interactions of clothianidin, thiamethoxam, and dinotefuran in honeybee, Apis mellifera.

Due to complex pest control scenarios and the needs of agricultural production, different neonicotinoids may be used in certain agricultural applications. Consequently, honeybees may be exposed to these substances through distribution throughout plant tissues via the vascular system through several pathways, such as surface water, the exudates excreted from plants, and air pollution via drift of dust as well as contaminated pollen and nectar. In the current study, the single and combined toxicity of clothianidin, dinotefuran, and thiamethoxam to honeybees was examined after 48 h exposure by the acute oral method and combination index (CI)-isobologram equation. At the 48 h interval, our results showed that 1) the order of toxicities for the single insecticides was ranked as clothianidin > thiamethoxam > dinotefuran and that 2) all binary and ternary combinations showed synergism or additive effect at the effect (fa) 0.5. Therefore, our results not only provided meaningful guidelines in evaluating the safety risk of the mixtures of the three neonicotinoids towards honeybees but also suggested that there is a significant interest in the study of mixture toxicities of neonicotinoids against honeybees because risk assessment of neonicotinoids against honeybees conducted only in individual insecticides may underestimate the realistic toxicity.

The combined effects of a monotonous diet and exposure to thiamethoxam on the performance of bumblebee micro-colonies.

There is a pressing need to better understand the factors contributing to declines of wild pollinators such as bumblebees. Many different contributors have been postulated including: loss of flower-rich habitats and nesting sites; monotonous diets; impacts of invasive pathogens; exposure to pesticides such as neonicotinoids. Past research has tended to investigate the impacts of these stressors in isolation, despite the increasing recognition that bees are simultaneously exposed to a combination of stressors, with potentially additive or synergistic effects. No studies to date have investigated the combined effects of a monotonous diet and exposure to pesticides. Using queenless micro-colonies of Bombus terrestris audax, we examined this interaction by providing bees with monofloral or polyfloral pollen that was either contaminated with field-realistic levels of thiamethoxam, a commonly used neonicotinoid, or not contaminated. Both treatments were found to have a significant effect on various parameters relating to micro-colony performance. Specifically, both pesticide-treated micro-colonies and those fed monofloral pollen grew more slowly than those given polyfloral pollen or pollen without pesticides. The two factors appeared to act additively. Micro-colonies given monofloral pollens also exhibited lower reproductive efforts and produced smaller drones. Although further research is needed to examine whether similar effects are found in whole colonies, these findings increase our understanding of the likely effects of multiple stressors associated with agricultural intensification on bee declines.

Review of field and monitoring studies investigating the role of nitro-substituted neonicotinoid insecticides in the reported losses of honey bee colonies (Apis mellifera).

The nitro-substituted neonicotinoid insecticides, which include imidacloprid, thiamethoxam and clothianidin, are widely used to control a range of important agricultural pests both by foliar applications and also as seed dressings and by soil application. Since they exhibit systemic properties, exposure of bees may occur as a result of residues present in the nectar and/or pollen of seed- or soil-treated crop plants and so they have been the subject of much debate about whether they cause adverse effects in pollinating insects under field conditions. Due to these perceived concerns, the use of the three neonicotinoids imidacloprid, clothianidin and thiamethoxam has been temporarily suspended in the European Union for seed treatment, soil application and foliar treatment in crops attractive to bees. Monitoring data from a number of countries are available to assess the presence of neonicotinoid residues in honey bee samples and possible impacts at the colony level and these are reviewed here together with a number of field studies which have looked at the impact of clothiandin on honey bees in relation to specific crop use and in particular with oilseed rape. Currently there is considerable uncertainty with regards to the regulatory testing requirements for field studies. Accordingly, a testing protocol was developed to address any acute and chronic risks from oilseed rape seeds containing a coating with 10 g clothianidin and 2 g beta-cyfluthrin per kg seeds (Elado®) for managed honey bee (Apis mellifera) colonies, commercially bred bumble bee (Bombus terrestris) colonies and red mason bees (Osmia bicornis) as a representative solitary bee species. This is described here together with a summary of the results obtained as an introduction to the study details given in the following papers in this issue.

Effect of dehydrogenase, phosphatase and urease activity in cotton soil after applying thiamethoxam as seed treatment.

Soil enzymes are indicators of microbial activities in soil and are often considered as an indicator of soil health and fertility. They are very sensitive to the agricultural practices, pH of the soil, nutrients, inhibitors and weather conditions. To understand the effect of an insecticide, thiamethoxam, on different soil enzyme activities, the experiments were conducted at cotton experimental fields of Punjab Agricultural University, Ludhiana. The results here were presented to understand the impact of thiamethoxam on soil enzyme activities. Thiamethoxam was applied as seed treatment to control the pest. Soil from three localities, i.e. soil in which seed was treated with recommended dose at 2.1 g a.i. kg(-1), soil in which seed was treated with four times recommended dose at 8.4 g a.i. kg(-1) and from the control field, were tested for different enzyme activities. Phosphatase and dehydrogenase activities were high in control soil in comparison to control soil while no effect of this insecticide on urease activity. Thiamethoxam had inhibitory effects on dehydrogenase and phosphatase activities. Therefore, it can be attributed that agricultural practices, weather conditions and use of thiamethoxam might be responsible for the different level of enzyme activities in soil.

A field study examining the effects of exposure to neonicotinoid seed-treated corn on commercial bumble bee colonies.

Neonicotinoid insecticides have been studied as possible contributors to bumble bee declines in North America and Europe. This has potential significance in corn agro-ecosystems since this crop is frequently treated with neonicotinoids and dominates much of the agricultural landscape in North America and Europe where bumble bees and other pollinators are commonplace. We conducted an experiment where commercial bumble bee (Bombus impatiens) hives were placed during pollen shed next to corn (Zea mays) fields that were grown from "conventional" seed that was treated with neonicotinoids, or "organic" seed that was not treated with pesticides. Samples of pollen were collected from corn plants for neonicotinoid residue analysis, pollen types carried by worker bees returning to hives were determined, and in autumn hives were dissected to measure various endpoints that serve as markers of colony vigor. Clothianidin was detected (0.1-0.8 ng/g) in pollen collected from all conventional fields, but was not detected in pollen from organic fields. Corn pollen was only rarely collected from bumble bee foragers and the vast majority of pollen was from wild plants around the corn fields. All hives appeared healthy and neonicotinoid seed treatments had no effect on any hive endpoints measured, except the number of workers, where significantly fewer workers were recovered from hives placed next to conventional fields (96 ± 15 workers per hive) compared to organic fields (127 ± 17 workers per hive). The results suggest that exposure during pollen shed to corn grown from neonicotinoid-treated shed poses low risk to B. impatiens.

A four-year field program investigating long-term effects of repeated exposure of honey bee colonies to flowering crops treated with thiamethoxam.

Neonicotinoid residues in nectar and pollen from crop plants have been implicated as one of the potential factors causing the declines of honey bee populations. Median residues of thiamethoxam in pollen collected from honey bees after foraging on flowering seed treated maize were found to be between 1 and 7 µg/kg, median residues of the metabolite CGA322704 (clothianidin) in the pollen were between 1 and 4 µg/kg. In oilseed rape, median residues of thiamethoxam found in pollen collected from bees were between <1 and 3.5 µg/kg and in nectar from foraging bees were between 0.65 and 2.4 µg/kg. Median residues of CGA322704 in pollen and nectar in the oilseed rape trials were all below the limit of quantification (1 µg/kg). Residues in the hive were even lower in both the maize and oilseed rape trials, being at or below the level of detection of 1 µg/kg for bee bread in the hive and at or below the level of detection of 0.5 µg/kg for hive nectar, honey and royal jelly samples. The long-term risk to honey bee colonies in the field was also investigated, including the sensitive overwintering stage, from four years consecutive single treatment crop exposures to flowering maize and oilseed rape grown from thiamethoxam treated seeds at rates recommended for insect control. Throughout the study, mortality, foraging behavior, colony strength, colony weight, brood development and food storage levels were similar between treatment and control colonies. Detailed examination of brood development throughout the year demonstrated that colonies exposed to the treated crop were able to successfully overwinter and had a similar health status to the control colonies in the following spring. We conclude that these data demonstrate there is a low risk to honey bees from systemic residues in nectar and pollen following the use of thiamethoxam as a seed treatment on oilseed rape and maize.

Movement of soil-applied imidacloprid and thiamethoxam into nectar and pollen of squash (Cucurbita pepo).

There has been recent interest in the threat to bees posed by the use of systemic insecticides. One concern is that systemic insecticides may translocate from the soil into pollen and nectar of plants, where they would be ingested by pollinators. This paper reports on the movement of two such systemic neonicotinoid insecticides, imidacloprid and thiamethoxam, into the pollen and nectar of flowers of squash (Cucurbita pepo cultivars "Multipik," "Sunray" and "Bush Delicata") when applied to soil by two methods: (1) sprayed into soil before seeding, or (2) applied through drip irrigation in a single treatment after transplant. All insecticide treatments were within labeled rates for these compounds. Pollen and nectar samples were analyzed using a standard extraction method widely used for pesticides (QuEChERS) and liquid chromatography mass spectrometric analysis. The concentrations found in nectar, 10 ± 3 ppb (mean ± s.d) for imidacloprid and 11 ± 6 ppb for thiamethoxam, are higher than concentrations of neonicotinoid insecticides in nectar of canola and sunflower grown from treated seed, and similar to those found in a recent study of neonicotinoids applied to pumpkins at transplant and through drip irrigation. The concentrations in pollen, 14 ± 8 ppb for imidacloprid and 12 ± 9 ppb for thiamethoxam, are higher than those found for seed treatments in most studies, but at the low end of the range found in the pumpkin study. Our concentrations fall into the range being investigated for sublethal effects on honey bees and bumble bees.

anticonvulsant and toxicity evaluation of newer 4H-benzo[1,4]oxazin-3-ones: The effect of two hydrogen bonding domains.

A series of (Z)-2-(substituted aryl)-N-(3-oxo-4-(substituted carbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl) hydrazine carboxamides (6a-r) was synthesized using 2-amino-5-nitrophenol as a starting material. All the synthesized compounds possessed two hydrogen-bonding domains and their effect on the activity was studied thereof. The anticonvulsant activity was assessed by the maximal electroshock test (MES), subcutaneous pentylenetetrazole test (scPTZ) and intraperitoneal thiosemicarbazide test (ipTSC). Compounds (6b, 6h, 6i, and 6p) were found to be the most potent of the series as they showed 83-100% protection in the MES test. They also displayed considerable activity in the chemically induced seizure tests. Most of the tested compounds were devoid of the neurotoxic and hepatotoxic effects.

Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), two naturally occurring benzoxazinones contained in sprouts of Gramineae are potent aneugens in human-derived liver cells (HepG2).

Benzoxazinoids (BAs) are toxic constituents of sprouts of Gramineae such as wheat, maize and rye and are part of the plant defence system against pests. In the last years, sprouts have been increasingly consumed as health foods and are also used for the production of dietary supplements. In the present study we investigated the mutagenic activities of the two most abundant BAs, namely 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) in the Salmonella/microsome assay and additionally, in micronucleus (MN) assay and single cell gel electrophoresis (SCGE) assay in a human-derived liver cell line (HepG2). DIBOA caused significant induction of his(+) revertants in all three strains in the range between 0.02 and 0.50 mg/plate; the highest activity was observed in TA100 (fivefold increase over the background at the highest dose level). The effect in YG1024 (a derivative of TA98 with increased acetyltransferase activity) was only slightly higher than the effect in the parental strain indicating that acetylation plays no crucial role in the activation of this BA. DIMBOA was in general less active and a positive result was only seen in the base substitution strain (TA100). Addition of rat liver homogenate (S9-mix) led to a significant (ca. twofold) increase of the mutagenic activities of both BAs. In SCGE assays with HepG2 cells consistently negative results were obtained with both compounds whereas in MN assays significant dose dependent effects were observed under similar experimental conditions. DIMBOA caused significant effects already at concentrations > or =1 microM; at the highest dose (20 microM) the MN frequency was sevenfold higher than the background level. DIBOA caused weaker effects and was positive at doses > or =2.5 microM, the maximal induction (twofold over background) was observed with 20 microM. Overall, DIMBOA was ca. 30-fold more active as DIBOA. Subsequent experiments with pancentromeric probes showed that >80% of the MN induced at the highest doses gave a centromere positive signal indicating that both BAs are aneugenic. This is an interesting observation as it is assumed that aneuploidy is a key event in cancer induction and at present no other aneugenic plant-derived substances of dietary relevance are known.

Male reproductive assessment of the rubber accelerator N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide in rats.

The rubber accelerator N-oxydiethylene thiocarbamyl-N-oxydiethylene sulfenamide (OTOS) was evaluated to determine its potential to cause reproductive effects. No evidence of a compound-related effect on mating, fertility, gestation length, number of implants or live births, pup growth, and survival was observed using Sprague-Dawley rats. Furthermore, light and electron microscopy of the testes from the high-dose males failed to reveal any morphological changes compared to the controls. Groups of 12 male Sprague-Dawley rats were continuously administered diets containing 0, 60, 200, or 600 ppm OTOS for 12 weeks. Following 56 days of exposure, the males were subsequently cohoused nightly with two females for a maximum of 21 days. During this mating period, males continued to receive control and OTOS-containing diets; however, feeders were removed for nightly cohabitation. Although a 4 to 8% reduction in body weight was observed in the 600 ppm animals, statistical significance was reached only at the end of the first week of treatment. In the 60 and 200 ppm males body weights generally were slightly elevated compared to the control, with the 60 ppm body weights showing statistically significant differences during Weeks 5 to 7 of exposure.