Neonicotinoids are primarily used in agriculture where they are applied as seed coatings, foliar sprays, and soil drenches or through drip irrigation. In urban areas neonicotinoids are used in home garden products and tree treatments. The maximum foraging ranges of honey bees are usually 10 – 15 km (median distances are 1 – 6 km) from the hive. Hence bee exposure to neonicotinoids is dependent upon the land use type within limited foraging distances from the hive. However, there are virtually no data showing levels of neonicotinoid use in urban areas and few studies have been done to compare urban and agricultural exposure. Several neonicotinoids have shown various toxic effects on pollinators and particularly honey bees. Honey bees have a limited arsenal of detoxification proteins to withstand neonicotinoid exposure, which makes them more sensitive and less able to develop tolerance to these insecticides compared to other insects. Sublethal exposure of honey bees to neonicotinoids can cause behavioral disturbances, orientation difficulties, impairment of social activities, and respiratory pattern changes. These behavioral changes can cause insufficient foraging behavior in honey bees due to the sublethal effect of neonicotinoids, thus putting the colony at risk of food shortage and eventually collapse. My objectives were to (1) develop a highly sensitive and selective, multi-residual analytical method for neonicotinoids in honey bee and pollen samples, (2) investigate the impacts of land use type (agriculture vs. urban) on the exposure of honey bees to neonicotinoid, (3) investigate the sublethal effect of imidacloprid on honey bees’ behavioral performance, and (4) investigate the sublethal effect of field-realistic concentrations of imidacloprid on honey bees’ metabolism at different ambient temperatures.To address my first objective (Chapter 2), I tested three sample cleanup methods (silica SPE, NH2-silica SPE, and Z-Sep SPE) based on solid phase extraction (SPE), which were investigated for determination of neonicotinoid insecticides and selected metabolites in honey bee and pollen samples by LC-MS/MS. Samples were extracted by hexane and ethyl acetate and then cleaned up with a SPE cartridge packed with silica gel, which showed a better cleanup efficiency compared to the aminopropyl silica SPE and zirconium-based sorbents method. Matrix effects of the three cleanup methods were evaluated and compared. Silica gel showed the highest analyte recoveries and method detection limit for this method were 2.0 to 9.1 μg/kg for honey bees and 2.4 to 4.7 μg/kg for pollen. Recovery studies were performed at three spiking levels (10, 60, and 120 μg/kg) and ranged from 78 to 140% with RSDs between 3 to 18% in honey bees and 83 to 124% with RSDs between 3 to 17% in pollen. The silica gel SPE cleanup method was then applied using honey bee and pollen samples that were collected from different apiaries. To address my second objective (Chapter 3), I analyzed honey bee and beebread (pollen) samples from apiaries in agricultural, developed, and undeveloped areas that were collected during two years in Virginia to assess if landscape type or county pesticide use were predictive of honey bee colony exposure to neonicotinoid insecticides. Trace concentrations of the neonicotinoid imidacloprid were detected in honey bees (3 out of 84 samples, 2.02 – 3.97 ng/g), while higher levels were detected in beebread (5 out of 84 samples, 4.68 – 11.5 ng/g) and pollen (3 out of 5 pollen trap samples, 7.86 – 12.6 ng/g). Imidacloprid was only detected in samples collected during July and August and were not detected in honey bees from hives where neonicotinoids were detected in pollen or beebread. Number of hives sampled at a site, county pesticide use, and landscape characteristics were not predictive of neonicotinoid detections in honey bees or beebread (all P>0.05). Because of the low detection rates, field surveys may underestimate honey bee exposure to field realistic levels of pesticides or the risk of exposure in different landscapes. Undetectably low levels of exposure or high levels of exposure that go undetected raise questions with regard to potential threats to honey bees and other pollinators. To address my third objective (Chapter 4), I investigated the effects of sub-lethal concentrations of imidacloprid on late fall forager honey bees’ behavior by accessing their activity levels and walking performance after being fed ad libitum with six different concentrations (2 – 125 μg/kg) of imidacloprid-dosed syrup for up to 48 hours in laboratory. Honey bee activity levels and motivation to move after being released into a UV light illuminated tunnel decreased significantly as dosages of neonicotinoid in their diet increased. However, their walking speeds were not significantly affected by imidacloprid. The behavioral changes I observed in honey bees chronically exposed to neonicotinoid via diet could negatively affect individual honey bee performance of their hive duties and consequently, colony survival during late fall and winter. To address my fourth objective (Chapter 5), I measured honey bee (Apis mellifera) foragers’ CO2 production rates at different temperatures (25, 30, or 35°C) after they consumed syrup dosed with a field realistic (5 μg/L) or high (20 μg/L) concentration of a neonicotinoid insecticide (i.e. imidacloprid) for 48h. We found that imidacloprid exposure significantly disrupted honey bees’ non-flight metabolic rates and there was a significant interaction between imidacloprid dosage and ambient temperature. Honey bee foragers dosed with 5 μg/L imidacloprid displayed higher average metabolic rates and those dosed with 20 μg/L imidacloprid displayed similar average metabolic rates compared to the corresponding control group across all temperatures. Exposure to field realistic concentrations of neonicotinoid may have a higher energetic cost for honey bees at 25℃ than at higher ambient temperatures. Disrupted energy costs in honey bees fed imidacloprid might be due to the thermoregulation, nerve excitation, or detoxification processes. Metabolic rate changes caused by pesticide exposure could result in less available energy for honey bees to perform hive duties and forage, which could negatively affect colony health.

The whitefly Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) is a serious pest of protected vegetable and ornamental crops in most temperate regions of the world. Neonicotinoids, pymetrozine (a feeding blocker), spiromesifen (a tetronic acid derivative), bifenthrin (a pyrethroid), and pyriproxyfen (a juvenile hormone mimic) are among the most important insecticides used to control this species. Bioassays were used to quantify responses of recently-collected strains of T. vaporariorum to three neonicotinoids (imidacloprid, thiamethoxam, and acetamiprid), pymetrozine, spiromesifen, bifenthrin, and pyriproxyfen. 454 pyrosequencing was exploited to generate the first transcriptome for this species. PCR-sequencing was used to identify mutations in the target proteins of spiromesifen and bifenthrin potentially associated with resistance to these compounds. Microarray sequencing technology was employed to investigate differences in gene expression associated with pyriproxyfen resistance. Resistance to neonicotinoids was age-specific in expression and consistently associated with resistance to pymetrozine, supporting a hypothesis of metabolic resistance analogous to that in the tobacco whitefly, Bemisia tabaci. Bioassays also showed moderate to high level resistance to spiromesifen, bifenthrin and pyriproxyfen in some strains. Analysis of the transcriptome identified genes encoding enzymes involved in the detoxification of xenobiotics (cytochrome P450s, carboxyl/cholinesterases, and glutathione-s transferases) and ones encoding insecticide targets: acetyl-coA carboxylase (ACCase), the target of spiromesifen and the voltage-gated sodium channel protein targeted by pyrethroids. PCR-sequencing revealed a single nucleotide polymorphism in the ACCase gene, which was consistently associated with spiromesifen resistance. Three amino-acid substitutions in the sodium channel of pyrethroid-resistant T. vaporariorum were found in positions previously implicated in pyrethroid resistance in B. tabaci. Microarray sequencing disclosed that a cytochrome P450 gene (CYP4G61) was overexpressed in a strain selected for increased pyriproxyfen resistance. The implications of these results and opportunities for further work are discussed.

The potential harm caused to bees and other pollinators by the widespread use of neonicotinoids has the capacity to pose a real and immediate threat to both the environment and humans. The benefits that bees and other pollinators provide, combined with the potential of harm they may face, are important enough to warrant a more comprehensive testing apparatus by which to evaluate threats to their population. Environmentally, bees and other pollinators are an important piece of ecosystemic balance--from pest management to pollination of plants that are a part of many species' diet. Anthropologically speaking, the way of life humans have been accustomed to and even need in order to survive is also largely dependent on a healthy population of bees and other pollinators; up to 70% of plants and vegetables we eat are directly a result of pollinators, and one third of every mouthful humans consume is attributed to pollinators. Without a healthy population of pollinators, the agricultural variety and nutritional availability would drastically decrease. Moreover, these agricultural products pollinators are responsible for also affect billions of dollars on both a national and global level. In many ways, the economic stability of the United States is at an equal risk as the pollinators. For example, an inability to produce many of our own agricultural staples would leave local and regional livelihoods disrupted and change the United States' import/export position. Moreover, this is not just a national problem. Pollinators are responsible for over 150 billion dollars globally in agriculture. Many of the nutrients humans need to be healthy would be in short supply. While scientists continue to study the possible effects of neonicotinoids on pollinators, how should policy makers respond? In this thesis, I argue that the various and drastic ways in which pollinators impact our environment and every day life, combined with the potential of the harsh threats their collapse would entail, warrant a more stringent approach to the evaluation of potential harms like neonicotinoids. An ethical risk assessment, as I define one, would be an appropriate tool to apply to this situation to guide policy makers in drafting regulations even in the absence of scientific certainty. Ethical risk assessments are a tool by which to evaluate the moral and ethical responsibilities in a whole host of different scenarios, one of which is neonics and pollinators. In other words, this ethical risk assessment will be used as an instrument by which to determine whether or not there is a sufficient risk to the population of pollinators, thus determining whether regulation is appropriate. Through application of this risk assessment, I will show that in this particular case regulation is appropriate due to the risks neonics pose to pollinators in light of the evidence that we do have. I develop a set of criteria for an ethical risk assessment. The criteria are a result of a combination of existing literature and some novel connections I draw here. This list, I argue, is what constitutes an ethical risk assessment. Ethical risk assessment, grounded in Utility Theory, is appropriate here because of its calculative apparatus and sociopolitical applicability.

Neonicotinoid insecticides are widespread in surface waters across the agriculturally-intensive Midwestern US. We report for the first time the presence of three neonicotinoids in finished drinking water and demonstrate their general persistence during conventional water treatment. Periodic tap water grab samples were collected at the University of Iowa over seven weeks in 2016 (May-July) after maize/soy planting. Clothianidin, imidacloprid, and thiamethoxam were ubiquitously detected in finished water samples and ranged from 0.24-57.3 ng/L. Samples collected along the University of Iowa treatment train indicate no apparent removal of clothianidin and imidacloprid, with modest thiamethoxam removal (~50%). In contrast, the concentrations of all neonicotinoids were substantially lower in the Iowa City treatment facility finished water using granular activated carbon (GAC) filtration. Batch experiments investigated potential losses. Thiamethoxam losses are due to base-catalyzed hydrolysis at high pH conditions during lime softening. GAC rapidly and nearly completely removed all three neonicotinoids. Clothianidin, hydrolysis products of thiamethoxam and known metabolites of imidacloprid are susceptible to reaction with free chlorine and may undergo transformation during chemical disinfection via chlorination or during distribution with chlorine residual. We identify several transformation products resulting from these oxidation and hydrolysis reactions, and discuss implications for human health. Our work provides new insights into the persistence of neonicotinoids and their potential for transformation during water treatment and distribution, while also identifying GAC as a potentially effective management tool to lower neonicotinoid concentrations in finished drinking water.

abstract: Six high-production-volume neonicotinoids were traced through a municipal wastewater treatment plant (WWTP) and engineered wetland located downstream, in a study motivated by reports on these insecticides posing threats to non-target invertebrate species and potentially playing a role in the global honeybee colony collapse disorder. An array of automated samplers was deployed in a five-day monitoring campaign and resultant flow-weighted samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using the isotope dilution method. Concentrations in WWTP influent and effluent were 54.7 ± 2.9 and 48.6 ± 2.7 ng/L for imidacloprid, respectively, and 3.7 ± 0.3 and 1.8 ± 0.1 ng/L for acetamiprid, respectively. A mass balance over the WWTP showed no (p=0.09, CI = 95%) removal of imidacloprid, and 56 ± 6% aqueous removal of acetamiprid. In the constructed wetland downstream, a lack of removal was noted for both imidacloprid (from 54.4 ± 3.4 ng/L to 49.9 ± 14.6 ng/L) and acetamiprid (from 2.00 ± 0.03 ng/L to 2.30 ± 0.21 ng/L). Clothianidin was detected only inconsistently in the WWTP and wetland (>2 to 288 ng/L; 60% detection frequency), whereas thiamethoxam (<10 ng/L), thiacloprid (<2 ng/L), and dinotefuran (<180 ng/L) were not detected at all. Thus, imidacloprid and acetamiprid were identified as recalcitrant sewage constituents (estimated U.S. WWTP discharge of 1920- 4780 kg/y) that persist during conventional wastewater treatment to enter U.S. surface waters at potentially harmful concentrations. / Dissertation/Thesis / Masters Thesis Civil and Environmental Engineering 2015

Dans cette étude, des aspects importants du rapport entre pesticides et abeilles domestiques (Apis mellifera L.) ont été traités. Dans la première partie, les effets de l’exposition des abeilles aux poussières contaminées avec trois neonicotinoides et le fipronil ont été étudiés. En fait, des quantités considérables de ces pesticides, utilisés pour l’enrobage des semences, sont dispersées pendant le semis du mais, et peuvent donc représenter une voie d’exposition des abeilles.En particulier, une voie spécifique d’exposition, le contact indirect, a été pris en compte. Les effets létaux et sub-létaux (mortalité aigue, butinage, développement des colonies, capacité d’orientation) des poussières ont été évalués en laboratoire, en tunnel et en plein champ. La dispersion réelle des poussières pendant le semis avait été évalué précédemment.Les résultats ont montré un effet significatif, sur la mortalité, de l’exposition aux poussières contaminées avec neonicotinoides et fipronil, en laboratoire et en tunnel. Par contre, la capacité d’orientation des abeilles n’a été pas influencée par l’exposition aux concentrations testées.Dans la deuxième partie de la recherche, on a étudié l’impact de différentes pesticides (chimiques et biologiques) sur les changements biochimiques et physiologiques des abeilles exposées. Ces paramètres on été évalué pour différentes façons et durées d’exposition. En particulier, trois expérimentations ont été effectuées en combinant les spores de Bacillus thuringiensis avec la deltamethine, les spores de Bt avec le fipronil et le traitement avec deltamethrine et le fongicide difenoconazole. Certains enzymes impliqués dans la détoxification, le stress oxydant et le métabolisme énergétique (GST, ALP, SOD, CAT, G6PDH, GAPDH) ont été sélectionnés pour évaluer les variations de leur activité suite à l’exposition aux pesticides. L’analyse des différents indicateurs biochimiques, comme le GST et le ALP, a mis en évidence des variations physiologiques qui peuvent être liés à l’exposition aux pesticides. Cette méthodologie pourrait donc représenter un nouvel aspect de l’évaluation des effets sub-létaux des pesticides chez l’abeille. / In this study, some important aspects of the relationship between honey bees (Apis mellifera L.) and pesticides have been investigated. In the first part of the research, the effects of the exposure of honey bees to neonicotinoids and fipronil contaminated dusts were analyzed. In fact, considerable amounts of these pesticides, employed for maize seed dressing treatments, may be dispersed during the sowing operations, thus representing a way of intoxication for honey bees. In particular, a specific way of exposure to this pesticides formulation, the indirect contact, was taken into account. To this aim, we conducted different experimentations, in laboratory, in semi-field and in open field conditions in order to assess the effects on mortality, foraging behaviour, colony development and capacity of orientation. The real dispersal of contaminated dusts was previously assessed in specific filed trials. The results showed a significant effect on mortality of neonicotinoids and fipronil contaminated dusts, both in laboratory and in semi-field trials. However, no effects were evidenced in honey bees orientation capacity.In the second part, the impact of various pesticides (chemical and biological) on honey bee biochemical-physiological changes, was evaluated. Different ways and durations of exposure to the tested products were also employed. Three experimentations were performed, combining Bt spores and deltamethrin, Bt spores and fipronil, difenoconazole and deltamethrin. Several important enzymes (GST, ALP, SOD, CAT, G6PDH, GAPDH) were selected in order to test the pesticides induced variations in their activity. In particular, these enzymes are involved in different pathways of detoxification, oxidative stress defence and energetic metabolism. The analysis of different biochemical indicators highlighted some interesting physiological variations that can be linked to the pesticide exposure. We therefore stress the attention on the possibility of using such a methodology as a novel toxicity endpoint in environmental risk assessment.

<p>The western corn rootworm (WCR) <i>Diabrotica virgifera virgifera</i> Leconte is a major pest of maize in the United States. Currently, it is principally managed using Bt maize hybrids and neonicotinoid seed treatments (NSTs), which were concurrently introduced in the early 2000s. This simultaneous release, and subsequent rapid adoption, created a situation in which Bt maize hybrids were never assessed in the absence of NSTs, and vice versa. Consequently, neonicotinoids’ influence on refuge function, primarily whether these insecticides aid or hinder the production of a sufficient population of susceptible beetles to delay resistance, has not been assessed. Moreover, a mounting suite of detrimental environmental effects of NSTs have been documented, lending some urgency to questions about their necessity.</p><p>To determine the influence of NSTs on refuge function, untreated and treated Bt maize fields were planted with 5% untreated refuge marked with ¹⁵N. Throughout the field seasons of 2019 and 2020, adults were collected from these fields and analyzed to determine their natal host plant. Results documented that the numbers of refuge beetles produced by the 5% seed blend are likely insufficient to result in rates of mating to delay resistance development.</p><p>To determine if the effect of using NSTs in combination with Bt maize hybrids is additive, synergistic, or neutral at managing secondary soil pests in the Midwest, four 16 block fields were planted in 2018, 2019 and 2020 comparing four treatments (1. Untreated, Bt seed; 2. NST, Bt seed; 3. Untreated, non-Bt seed; 4. NST, non-Bt seed). Compact method sampling, root rating, and yield were used to document the presence of secondary soil pests. All three sampling years documented low abundance of white grubs and wireworms. There was minimal influence of NSTs on maize yield (2018: p = 0.07; 2019: p = 0.62; 2020: p = 0.056) and root damage (0-3 scale) (2018: M = 0.0092; 2019: M = 0.0091; 2020: M = 0.0361). These same fields were used to document the presence of NSTs in soil, as well as residues in nearby waterways. Results documented greatest soil and water clothianidin levels earlier in the season and declined as the season progressed.</p><p><br></p>

Amphibians have been facing global declines over the last decades due to direct and indirect effects of anthropogenic activities. One of the leading causes is environmental contamination, particularly that of waterbodies which are used by many amphibian species for reproduction, development, and adult life. An important source of contamination comes from agricultural runoffs of pesticides such as neonicotinoids, which are known to alter anuran survival, behaviour, predation stress response, and development. However, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors which could alter the strength and direction of observed neonicotinoid effects. This study investigated how a concentration of imidacloprid (a neonicotinoid) measured in surface waters interacted with high population density, an important environmental stressor, to influence behaviour and development across metamorphosis in wood frogs (Lithobates sylvaticus) known to breed in agricultural landscapes. I reared tadpoles in a fully crossed design experiment, between two densities (0.33 and 1 tadpole/L) and clean vs contaminated water (10 µg/L imidacloprid). Behaviours were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. I found that imidacloprid did not interact with population density or independently affect behaviours in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural behavioural response (i.e., “freezing”) of tadpoles to predation cues. Both treatments also slightly accelerated metamorphosis while only density altered final mass at metamorphosis. Finally, I found that distance travelled was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. This study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioural assays and stressors in studies of amphibian ecotoxicology.

Under 2013 förbjöds behandling av blommande grödor med de systematiska neonikotinoiderna imidakloprid, klotianidin och tiametoxam i EU. Ett flertal studier och rapporter hade visat att pollinerande biarter kan uppleva subletala effekter av att konsumera neonikotinoid. Oro rådde om bin blir förgiftade av neonikotinoider funna i pollen och nektar hos dessa blommade grödor. Därför har jag undersökt på en kommunal nivå om neonikotinoidbetade rapsareal generellt har medfört en negativ effekt på förekomst av rapspollinerande vildbin. Studien använde observationsdata från medborgarforskning angående 46 vildbiarter som är vanliga i och omkring åkrar, varav 19 är vanliga i rapsfält. Jag sammanställde data över pollinatörer och areal åker med andel raps mellan 2002 och 2020 för 202 kommuner i södra Sverige. Med hjälp av ”Generalized linear mixed model” undersökte jag sambandet mellan oddset att observera rapspollinerande arter bland pollinatörerna och rapsareal. Resultatet visade ingen effekt av ökad mängd raps på rapsbesökare och ingen lägre mängd pollinatörer mellan 2008–2013 då neonikotinoidbetad raps förekom som mest. Frånvaro av negativ effekt på vildbin kan vara på grund av låga koncentrationer av neonikotinoider i nektar och pollen över skalan för denna studie. En eventuell negativ effekt av neonikotinoider från mer raps kan möjligtvis ha vägts upp av den positiv effekt i form av extra resurser som ökar tillväxten hos lokala populationer. Framtida studier uppmanas att vidare undersöka variationen i exponering av neonikotinoider från raps beroende på beteendemässiga skillnader som tillexempel flygtid och socialstruktur. / In 2013, treatment of flowering crops with the systematic neonicotinoids imidacloprid, clothianidin and thiamethoxam were prohibited by the EU. Several studies and reports had shown that pollinating bees can experience sublethal effects by consuming neonicotinoids. Concerns regarded whether bees would be poisoned by neonicotinoids found in pollen and nectar of these flowering crops. Therefore, I have examined on a municipal level, whether area of rapeseed treated with neonicotinoids have generally caused a negative effect on the abundance of wild bees which pollinate on rapeseed. The study used citizen-based observational data of 46 wild bee-species that are common in and around agricultural fields, 19 of which are common in rapeseed-fields. I compiled data regarding pollinators and area of agricultural field with proportions of rapeseed-fields between 2002 and 2020 for 202 municipalities in South Sweden. Using Generalized linear mixed model, I examined the connection between odds of observing rapeseed-pollinating species among pollinators and the area of rapeseed. The result showed no effect from increased amount of rapeseed on rapeseedpollinators and no reduced number of pollinators between 2008-2013, the periodwhenneonicotinoid-treated rapeseed was most abundant. The absence of a negative effect on wild bees could be caused by low concentrations of neonicotinoids in nectar and pollen. Also, possible negative effects of neonicotinoids from rapeseed could possibly be outweighed by a positive effect from additional resources that increases local population growth. Future studies are encouraged to further examine varied exposure of neonicotinoids from rapeseed based of behavioural differences like flight-time and social structure of wild bees.

Premise of The Study: Studies on the effects of heterospecific pollen (HP) transfer have been focused mainly on insect-pollinated species, despite evidence of insect visitation to wind-pollinated species and transfer of their pollen onto stigmas of insect-pollinated plants. Thus, the potential consequences of HP transfer from wind-pollinated species remain largely unknown. Furthermore, accumulation of pesticide residues in pollen of wind-pollinated crops has been documented, but its potential effects on wild plant species via HP transfer have not been tested. Methods: We evaluated the effect of wind-dispersed Zea mays pollen on pollen tube growth of the insect-pollinated Mimulus nudatus via hand pollinations. We further evaluated whether pesticide-contaminated Z. mays pollen has larger effects on M. nudatus pollen success than non-contaminated Z. mays pollen. Key Results: We found a significant negative effect of Z. mays pollen on M. nudatus pollen tube growth even when deposited in small amounts. However, we did not observe any difference in the magnitude of this effect between pesticide-laden Z. mays pollen and non-contaminated Z. mays pollen. Conclusions: Our results suggest that wind-pollinated species can have negative effects as HP donors on insect-pollinated recipients. Thus, their role in shaping co-flowering interactions for wind- and insect-pollinated species deserves more attention. Although we did not find evidence that pesticide contamination increased HP effects, we cannot fully rule out the existence of such an effect, because pollen load and thus the pesticide dose applied to stigmas was low. This result should be confirmed using other HP donors and across a range of HP loads, pesticide types, and concentrations.