Neonicotinoid Pesticides and Bees Report to Syngenta Ltd January 2013

Fera produced this report for a commercial client and publication of this work shall not be regarded as an endorsement of that client. To the maximum extent permitted by law, FERA excludes all representations, warranties, and conditions relating to the report and the use of it and to the accuracy of third party data and research contained in the report. FERA, its suppliers and employees (whether or not involved in producing, maintaining or delivering the report) are not liable for any loss or damage arising from your use of, or reliance upon the information contained in the report (including without limitation and direct, indirect or consequential losses or loss of business or profits, whether or not such loss was foreseeable or Fera is advised of the possibility of such loss). You should be aware that use of the report and its content is at your own risk. Part of the data provided in this report has been collated as part of a publically tendered contract from The European Food Standards Agency (EFSA). However, this report shall not be considered as a either a scientific output or endorsement adopted by EFSA, and EFSA fully reserves its rights, view and position as regards the issues addressed and the conclusions reached in this report.

This report refers to Thompson H M (2012) Interactions Between Pesticides and Other Factors in Effects on Bees which was produced under contract to EFSA and the full version of which can be found at http://www.efsa.europa.eu/en/supporting/pub/340e.htm. It should be noted that the report was not produced by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and conclusions reached in the document, without prejudice to the rights of the authors.

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Contents 1. Executive Summary ............................................................................................................. 3 2 Introduction ............................................................................................................................. 12 3 Methodology adopted .............................................................................................................. 13 4 Results .................................................................................................................................... 14 4.1 Mode of Action and Metabolism of Neonicotinoid Insecticides .............................. 14 4.1.1 Mode of Action ..................................................................................................... 14 4.1.2 Metabolism .......................................................................................................... 15 4.1.3 Conclusions ......................................................................................................... 17 4.2 Acute Toxicity of neonicotinoids ............................................................................ 18 4.2.1 Honeybees ........................................................................................................... 18 4.2.2 Other bee species ................................................................................................ 23 4.3 Multiple exposure to pesticides (including substances used in bee medication) and potential additive and cumulative effects. ........................................................................ 35 4.3.1 Conclusions ......................................................................................................... 38 4.4 Interactions between neonicotinoids and disease ................................................. 39 4.5 Exposure of bees to pesticides ............................................................................. 41 4.6 Sublethal and chronic effects of neonicotinoids..................................................... 44 4.6.1 Honeybees ........................................................................................................... 44 4.6.2 Bumblebees ......................................................................................................... 44 5. References ....................................................................................................................... 101

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1. Executive Summary Mode of action and metabolism of neonicotinoids in bees Neonicotinoids have been shown to bind to the ligand gated ion channels at the α-bungarotoxin site on the nicotinic acetylcholine receptor (nAChR) and primarlily affect the post synaptic nAChRs. Once bound to the nAChR, neonicotinoids are not broken down by acetylcholinesterase like acetylcholine resulting in over-stimulation of the nervous system and a build up of acetylcholine in the synapse. The midgut in the honeybee is a major site of metabolism for ingested pesticides and interactions between chemicals at least in part may be influenced by effects on the detoxifying enzymes within the midgut, including microsomal oxidases, glutathione S transferases and esterases. Synergists of both Phase I and Phase II enzymes have been used to identify the metabolic pathways used in neonicotinoid detoxification and confirmed the role mixed function oxidases in the metabolism of many neonicotinoids. However P450s probably have a lesser role in the metabolism of imidacloprid which has an elimination half life of 5 hours. The main imidacloprid metabolites, urea derivative and 6-chloronicotinic acid, are found particularly in the mid gut and rectum and 4/5-hydroxy-imidacloprid and olefin are found mostly in the head, thorax and abdomen all of which are nAChR rich tissues and levels peaked 4 hours after ingestion.. Both of the latter metabolites have insecticidal properties (the olefin has toxicity similar to imidacloprid and both bind to the nAChR) and may be responsible for delayed onset of death. These profiles can be related to the two distinct phases to imidacloprid poisoning after acute exposure. There is a rapid onset of neurotoxicity in the form of hyper responsiveness, hyperactivity, and trembling; mortality is delayed until at least four hours post exposure. Acetamiprid is readily metabolised by mixed function oxidases, to seven main metabolites (none of which are insecticidal). Although metabolism is fast, the half life of acetamiprid approx 25 minutes but only 40% of the total radioactivity eliminated after 72 hours suggesting that the metabolites persist within the honeybee. The lower toxicity of acetamiprid is thought to be due to this rapid metabolism to relatively non-toxic metabolites. Acute toxicity of neonicotinoids In general, LD50 values are lower for oral exposure than for contact exposure, except for acetamiprid which is the reverse; this may be explained by low hydrophobicity and poor penetration through the cuticle of these compounds. The chloronicotinyl insecticides

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(thiamethoxam, clothianidin, imidacloprid) are more toxic than the cyano substituted (thiacloprid, acetamiprid) The oral toxicity of imidacloprid, which has been extensively reported within the literature; is highly variable with 48 hour oral LD50 values ranging from 3.7 to as high as 400 ng/bee. Data across pesticides generally suggests that the toxicity to Apis mellifera reflects that in other bee species when it is expressed on a weight basis and is supported by data for the chloronicotinyl insecticides but there are far less robust data for the cyano substituted neonicotinoids. Synergism between neonicotinoids and other pesticides (update EFSA review) Pesticides widely used both in the agricultural and urban environment (pest control and home and garden uses) as well as by beekeepers to control pests, e.g. fluvalinate, amitraz, coumaphos to control varroa, are detectable in bees and hive matrices. Exposure of honeybees to any single pesticide application may occur over the short term or, unlike many organisms, over a longer period if residues are present in pollen and/or nectar stored within the colony or due to migration of lipophilic compounds into wax. These more persistent residues are likely to be available to the colony over a period of time depending on the active ingredient and the frequency of use, e.g. multiple applications. Bees may be exposed to mixtures of products applied to plants on which they forage. Recent data indicates the extent of mixing of formulations that occur on arable, vegetable orchards and soft fruit crops in the UK and includes tank mixing of EBI fungicides with neonicotinoid insecticides. In addition to the application of products as tank mixes, the increasing use of seed treatments raises the possible scenario of nectar, pollen or guttation water containing active ingredients also being contaminated with sprays applied during the flowering period, e.g. oilseed rape. Although many reports of residues in pollen being returned to the hive by foragers are published the majority of these are based on individual pesticide residues rather than assessments of the total pesticide residue levels and the data are often not reported in sufficient detail to determine the residue levels of the individual components within multiple detections. Data reported for residues of chemicals applied by beekeepers in honeybee colonies have primarily been directed at single varroacides with some limited data after antibiotic dosing. Those that are available show that very high levels of varroacides may be present within colonies and are regularly detected in live bees The risk from most mixtures can be assessed using the additive approaches of concentration addition (or dose addition) and independent action (IA). In identifying the relevance of synergy in determining the toxicity of mixtures it is important to understand the route of metabolism of pesticides in honeybees and the effects of age, season etc on this. The role of oxidative Neonicotinoid pesticides and bees Report to Syngenta Ltd

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metabolism in detoxification of the cyano-substituted neonicotinoids in bees is highlighted by the increase in toxicity of acetamiprid and thiacloprid in combination with the EBI fungicides. The majority of synergistic effects observed in honeybees have been ascribed to inhibition rather than induction of P450s involved in pesticide metabolism. The level of exposure to the synergist also affects the scale of the synergy. The effect of exposure on the scale of synergy is important as many of the laboratory studies have been undertaken with high doses of synergists, e.g. 3-10 µg/bee and at more realistic exposure levels such high increases of toxicity have not been observed even under laboratory conditions. Contact and oral dosing with combinations of a range of EBI fungicides at more realistic exposure levels with neonicotinoid insecticides showed only low levels of synergy. There are no reports of interactions between varroacides and neonicotinoid pesticides but recent data suggest that antibiotics used in colonies may affect susceptibility to both varroacides and other pesticides Interactions between neonicotinoids and disease (update EFSA review) Honeybees are known to suffer from a wide array of bacterial, fungal and viral pathogens as well as ecto- and endo-parasite. Multiple infections are common and the impact of some pathogens can be far higher in the presence of other associated pests and diseases. Honeybees have a welldeveloped immune system for coping with bacterial and fungal infections although their immune response to viral pathogens is less well understood and there has been significant interest recently on the potential for pesticides to affect the susceptibility of bees to diseases. This has been highlighted by high pesticide residues reported in honeybee colonies in the USA and the importance of microbial communities within the hive which may be affected by pesticide residues as well as impacting on the bees directly. The dense crowding within social and eusocial bee colonies together with the relatively homeostatic nest environment with stored resources of pollen and nectar/honey results in conditions conducive to increased susceptibility to disease. This has resulted in the evolution of both individual and social immunity in the honeybee and bumble bee Factors other than pesticides can impact on the immune system in honeybees and increase susceptibility to disease, e.g. other diseases, the immunsuppressive effects of Varroa destructor, antibiotics, sulphonamides and metals and immunostimulators have been proposed. Confinement of colonies can result in immune suppression and oxidative stress in colonies and poor habitat quality may result in lowered immune response. One key factor is that although colonies show qualitatively similar immune responses the colony is a significant factor in the level of the response, i.e. there are large variations between colonies on the level of the response. There have also been suggestions that stress, e.g. isolation, weakens individual immunocompetence. This may explain Neonicotinoid pesticides and bees Report to Syngenta Ltd

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why some immune competence effects are evident in under laboratory conditions but not in colonies. Pollen is the primary source of protein in honeybees is well established to affect longevity, development of the hypopharyngeal glands and ovaries and the susceptibility to pathogens. Pollen quantity does not affect individual or social immunity, however, diet diversity (polyfloral pollen) increases glucose oxidase activity which has a key role in social immunity and protein quality has been reported to affect melanisation, e.g. of the gut wall. This parallels observations of immune function in other insects where antibacterial activity was higher for individuals fed high-quality diets. Pollen is also important for haemocyte function in honeybees and has been shown to be a key parameter in bumble bees The major fungal disease of honeybees is the microsporidian Nosema. It appears that N. ceranae spore count, unlike N. apis, is not a useful measure of the state of a colony’s health and in-hive bees are unsuitable as indicators of the degree of infection of the colony N. ceranae infection but not N apis infection appears to significantly suppresses the honey bee immune response. Such immune suppression would also increase susceptibility to other bee pathogens. After infection with N. apis the honey bee immune system quickly activates defence mechanisms, which includes the increase in the expression of genes encoding antimicrobial peptides and other immunity-related enzymes. Whereas N. ceranae infection seems to suppress the immune response by reducing the transcription of some of these genes. There have been conflicting reports over the interactions between imidacloprid and Nosema which are confounded by the effects of Nosema on the energetic of individuals and results in significantl;y increased food intake. A similar issue was identified in an increase in mortality was observed when bees (five days after emergence) were infected with 125,000 spores of N ceranae and after a further 10 days were exposed to sublethal (LD50/100) doses of thiacloprid or fipronil for 10 days. The bees clearly showed energetic stress after Nosema infection by their increased consumption of sucrose with infected bees consuming approximately twice the amount of sucrose compared with uninfected bees. Although there was no apparent difference in overall daily intake when exposed to the insecticides, there were large difference in intake on day 1 of the pesticide exposure period There was one report identified which suggests that N ceranae may increase the susceptibility of bees to fipronil but there were inconsistencies in the reported results. The data do show the variability of mortality caused by N ceranae: N ceranae mortality ranged between 22 and 39% when dosed on day 0 and between 22 and 37% when dosed on day 7 and fipronil resulted in 2931% mortality whether dosed from day 0 or day 7. Neonicotinoid pesticides and bees Report to Syngenta Ltd

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It is also vital to understand the disease status of individuals used in pesticide assessments since both the honeybee (Apis mellifera) and the bumble-bee (Bombus terrestris) perform poorly in proboscis extension reflex (PER) memory tests when their immune systems were challenged by lipopolysaccharide. Honeybees are the target of a large number of viruses with a total of 18 identified to date. Often virus vectoring by Varroa, which is a significant stressor in honeybee colonies by feeding on the haemolymph causes a variety of physical and physiological effects on the colony, and results in infections from viruses which are otherwise present as covert infections resulting in severe disease and mortality within the colony. In addition viruses can be transmitted within the colony by trophallaxis, contact, faeces and salivary gland secretions. However to date there are no reports of interactions between neonicotinoids and viruses Exposure (update EFSA review) including available data from incident monitoring schemes Bees are exposed to pesticides via a number of routes and the relative importance of each depends on the life stage of the insect and the mode of application of the pesticide. Adults may be exposed directly to pesticides through direct overspray or flying through spray drift, by consumption of pollen and nectar (which may contain directly over-sprayed or systemic residues), by contact with treated surfaces (such as resting on recently treated leaves or flowers), by contact with dusts generated during drilling of treated seeds, or by exposure to guttation fluid potentially as a source of water or as dried residues on the surface of leaves. The exposure of larvae is primarily via processed pollen and nectar in brood food. Data available in the literature includes residues in pollen, wax and nectar within colonies, pollen and nectar residues from plants, in pollen loads on bees returning to the hives and in adult workers. Such data also includes the residues of veterinary medicines detected and the distribution of chemicals around the hive. The routes of exposure of bees to pesticides has been assessed and recently reviewed in an EFSA Scientific Opinion particularly in relation to quantifying uptake and extended to include other non-Apis species where data were available. The exposure of bumble bees to pesticides has also been reviewed and showed there are key times in the year when exposure of queens may be particularly important in determining the fate of a colony. A review of residues in bees after pesticide applications in the EFSA review (2012) provides evidence of the exposure of bees to applications aggregated through all routes of exposure, i.e. through direct overspray, foraging on treated crops and consumption of treated food and water as samples were collected over time after exposure. This showed peak residues in the first sample after application with declines for spray applications over the following week. No data from systemic seed or soil application field studies were available but residues of imidacloprid and its Neonicotinoid pesticides and bees Report to Syngenta Ltd

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metabolite 6-chloronicotinic acid and fipronil and its metabolites were detected at low levels in monitoring studies Studies on residues deposited as dust containing neonicotinoids are summarised obviously this does not take into account recent EU requirements to limit dust emissions through the use of professionally treated seeds and deflectors. Drift during agricultural treatment determines the deposition of pesticides within a small distance from the field edge. What is less obvious is how to calculate the drift onto flowering weeds in the field margin as dust drift is unlikely to behave in the same way as spray drift due to the wide variations in particle size. Unfortunately the deposition data generated to date for grasses and flowering weeds in flower margins have limitations in terms of the reporting of sowing rates and seed treatment rates. Contact of bees with treated surfaces may occur through resting on treated leaves or during foraging on treated flowers. The major issue is that bees do not come into contact with the treated plant over the entire surface of their body but primarily through their feet; however during resting and cleaning they may transfer residues from their feet to other parts of their bodies. The exposure of bees to pesticides in pollen depends on both the residues present and the amounts of pollen collected by the bees. The amount of pollen collected by a colony per day is highly variable and depends on pollen availability, crop species and the needs of the colony. On oilseed rape the amount of pollen collected varied with the stage of flowering with most collected in the latter stage. Bee bread is pollen processed from the pollen loads by bees for storage by combining with nectar or honey and addition of antimicrobial agents. This results in higher residues in bee bread than in pollen which may relate to differences in availability for residue analysis following processing of the pollen by bees. Flower morphology is an important factor in the pesticide content of nectar: flowers in which the nectar is deeper, such as clover, were less contaminated than shallower flowers such as cabbage and nectar yield/flower was less important in determining pesticide content. To date, there are no reports of pesticide residues in aphid honeydew after spray application but the intake by bees may be expected to be similar to that of nectar sources. Residues in honey formed from contaminated nectar and stored within the hive will depend on the concentration of nectar through evaporation of water to produce honey and degradation of residues through biological and chemical factors in honey. Both factors are slow and counter each other to some extent and there are differences between honey contained in open and sealed cells. The residues of neonicotinoids pesticides detected in stored nectar and honey in field studies and available monitoring data for samples taken directly from colonies are summarised. Monitoring data for processed honey has been excluded as honey is combined from a large number of Neonicotinoid pesticides and bees Page 8 of 133 Report to Syngenta Ltd

colonies and therefore residues may be diluted. For pesticides (not acaracides) the residues detected in the monitoring studies are lower than those reported in field studies. Water is collected by honeybees to dilute thickened honey, to produce brood food from stored pollen, to maintain humidity within the hive and to maintain temperature within the brood area. Water is not stored in combs by temperate bee colonies. The amount of water required depends on the outside air temperature and humidity, the strength of the colony and the amount of brood present. The production of water by evaporation of nectar to form honey may address at least some of this need. Water consumption by honeybee colonies has been assessed using confined of colonies provided with a source of water within the hive. To date there have been no published studies that demonstrate significant exposure of bees to guttating crops as a source of water in the field. Guttation fluid is unlikely to be identified by honeybees as a source of sugar due to the low levels present. Bees are less subject to dessication than most terrestrial insects due to their nectar diet and high metabolic water production Beeswax is produced by worker bees within the colony to house stores of nectar and pollen and for brood production. Production begins when the worker is slightly less than one week old, peaking at around two weeks and then reducing. It takes between 24 and 48 hours for any particular honeybee worker to produce a moderate-sized wax scale. If unchanged by a beekeeper wax within the colony may accumulate lipophilic residues over time both from contaminated pollen and nectar brought into the hive and from chemicals used within the hive, e.g. varroacides. There are no reports of neonicotinoids in beeswax from colonies Propolis is collected by bees as resin from trees, e.g. buds, primarily poplars and pine trees and is used within the hives to block small gaps and as a defense at the hive entrance against ants etc. and also as an anti-bacterial antifungal agent within the hive. The main propolis plants in Europe are poplar, birch, oak, alder, willow and hazel.

Foragers collect the resin in their pollen baskets to

return it to the hive and can carry approximately 10 mg. The chemical composition of propolis varies between sources but is a mixture of resins, terpenes and volatiles. Due to the range of sources of propolis and storage within the hive it can contain a range of contaminants but only a small number of reports exist of trace residues of pesticides present in propolis collected from colonies and propolis tinctures prepared from this and no reports of neonicotinoid pesticides There are three possible sources of inhalation exposure of bees to pesticides. During applications of pesticides (is a similar manner to flying through spray), through vapour generated from residues on the crop after application and from stored pollen and nectar within the hive (and potentially water evaporated within the hive). There are no reports of exposure associated with inhalation of neonicotinoid pesticide residues. Neonicotinoid pesticides and bees Report to Syngenta Ltd

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Nectar collected by foragers from plants is transferred to in-hive bees at the colony entrance which then to further bees for transport to storage or brood combs. During spring and summer large quantities of nectar are stored for use in periods of shortage, e.g. during breaks in nectar flow, periods of poor weather, or for over-wintering. Nectar is placed both in storage combs and also in brood combs close to larvae so it is readily available for brood rearing. The majority of published studies relate to in-hive treatments with varroacides and antibiotics and solely measured residues in honey intended for human consumption. However, there are a small number of studies which specifically address the distribution of incoming contaminated nectar within hives, including that releasing just six foragers fed with radiolabelled sμgar into a colony resulted in about 20% of the workers in the brood area receiving some labelled food within 3.5 hours and this included nurse bees which demonstrated the potential exposure of brood. The nectar delivered to brood comb is used rapidly by nurse bees to feed larvae. For spray applications the residue per unit dose (RUD) can be calculated and used to determine the relative amounts of a pesticide available through each routes of exposure. The data for all routes of exposure is currently limited and would benefit feom a larger dataset. For seed treatments and soil applications the data available for calculation of an RUD approach is far more limited and there are a number of issues which require additional research, e.g. crop dependence, concentration dependence and active ingredient dependency of the RUD. Overspray can be related to the surface area of the bee which suggests the RUD for honeybees should be increased but although the surface area of bumble bees is likely to increase significantly due to their greater size they are also far more variable in size making any predictions unreliable. For bumble bees intake data are far more limited than for honeybees but some data are available for adults from queenless microcolonies under laboratory conditions. For larvae intake of sucrose is unclear but an approximation is available. However, the intake of foragers is not reported and therefore the data only relate to intake for metabolic requirements. There data can be used to identify possible RUDs but limited confidence can be held in these. There was insufficient data available to assess the exposure of solitary bee species. Sublethal and chronic effects of neonicotinoids, A large number of studies have been undertaken on the sublethal and chronic effects of neonicotinoid pesticides on bees using a number of different exposure scenarios and endpoints and by far the majority of the reported literature relates to imidacloprid that a large number of dosing studies have been conducted at dose rates and concentrations in excess of the reported maximum concentrations for imidacloprid and thiamethoxam in nectar following use as seed Neonicotinoid pesticides and bees Report to Syngenta Ltd

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treatments. Where effects were observed at or below rates of imidaclorpid close to the maximum reported in nectar only one appears to be a non-biomarker effect at the colony level. There were far fewer studies with thiamethoxam and none reported effects at or below the maximum field nectar residue reported following seed treatment. The vast majority of studies with non-Apis species have been undertaken with bumble bees and again in the majority imidacloprid was used at concentrations in excess of the maximum rate reported in nectar although 2 studies report effects following continuous dosing at field realistic rates. Only a small number of studies have been undertaken in bumble bees with clothianidin and thiamethoxam and they have not been reported to show effects at field realistic rates.

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2. Introduction This literature review encompasses specific aspects of Neonicotinoid pesticide impacts on bees: 1. Mode of action and metabolism of neonicotinoids in bees 2. Acute toxicity of neonicotinoids 3. Synergism between neonicotinoids and other pesticides (update EFSA review) 4. Interactions between neonicotinoids and disease (update EFSA review) 5. Exposure (update EFSA review) including available data from incident monitoring schemes key differences from honeybees including available data from incident monitoring schemes 6. Sublethal and chronic effects of neonicotinoids,

Sections 3-5 draw heavily on the EFSA review (2012) http://www.efsa.europa.eu/en/supporting/pub/340e.htm and are not reproduced here The key considerations in compiling the data were:  To primarily concentrate on European data  Where already covered in recent EFSA review (Thompson 2012) provide any additional information, e.g. more recent papers  How much can be toxicity and exposure be extrapolated from honeybees to other bees- what are the key differences – can these be quantified?  Can the exposure data in the sublethal/chronic studies be compared to estimated field exposure?

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3. Methodology adopted The set of search terms selected and databases searched for the study are shown in Appendix 1. All search results are fully documented in Appendix 1. The database was searched for duplicates which were removed and the cleaned database transferred to EndNote. The literature was evaluated systematically and the criteria for including or excluding the references stated (see Appendix 1). Any reports of studies that were identified as useful were evaluated to assess their reliability. Reliability covers the inherent quality of the test relating to the test methodology and the way the performance and results of the test are described. The criteria used for assessing reliability of the identified literature were based on that identified in “Submission of scientific peer-reviewed open literature for the approval of pesticide active substances under Regulation (EC) No 1107/2009” which provides a definition of scientific peer-reviewed open literature and instructions on how to minimise bias in the identification, selection and inclusion of peer-reviewed open literature in dossiers, according to the principles of systematic review (i.e. methodological rigour, transparency, reproducibility). For each identified data source the reason for inclusion or exclusion is clearly stated in the main report for those included and in Appendix 1 for those excluded. The information from previous reports and the review of ‘new’ literature was combined to provide an overview of the interactions between pesticides and other factors in effects on bees considering:

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4. Results 2.1 Mode of Action and Metabolism of Neonicotinoid Insecticides In depth studies have been undertaken to fully understand the mechanisms involved in both neonicotinoid mode of action and target sites in addition to the detoxification mechanisms. Most of the bee studies have been directed at the neonicotinoid imidacloprid, although one paper has investigated the metabolism of acetamiprid. Clothianidin is a metabolite of thiamethoxam in honeybees. 2.1.1 Mode of Action In insects, including the honeybee, acetylcholine is the main neurotransmitter; it binds to nicotinic acetylcholine receptors (nAChR) mediating fast cholinergic synaptic transmission (Barbara et al., 2008; Thany et al., 2010). Honeybee sensory and motor functions are dependent on central and cholinergic pathways (Tomizawa and Yamamoto, 1992; Barbara et al., 2008). Although widely distributed throughout the body, key areas of cholinergic transmission in the honeybee are the antennal lobes and mushroom bodies (thought to be involved in some aspects of olfactory conditioning, e.g. odour disrimination) located in the head (Barbara et al., 2008). Neonicotinoids have been shown to bind to the ligand gated ion channels (LGIC) at the αbungarotoxin site on nAChR, along with other ligands including acetylcholine (Lind et al., 1999; Liu and Casida, 1993; Matsuo et al., 1998; Nakayama and Sukekawa, 1998; Schmuck et al., 2003; Tomizawa et al., 1995; Tomizawa and Yamamoto, 1992; Tomizawa and Yamamoto, 1993). Although nAChRs are located on both the pre and post-synaptic membrane, neonicotinoids are more likely to affect post synaptic nAChRs (Buckingham et al., 1997). Once bound to the nAChR, neonicotinoids are not broken down by acetylcholinesterase like acetylcholine. This leads to over-stimulation of the nervous system and a build up of acetylcholine in the synapse. Studies have confirmed that neonicotinoids bind to a single binding site in honeybees (Tomizawa et al., 1995). Studies evaluating the effects of neonicotinoids on continued nerve transmission across the synapses, have identified different binding properties. Imidacloprid is a partial agonist of the nAChR that binds to the acetylcholine (ACh) recognition site (Barbara et al., 2008; Barbara et al., 2005; Benzidane et al., 2011; Deglise et al., 2002). Clothianidin was found to be a full agonist in cockroaches (Periplaneta americana) although this has not been confirmed in honeybees (Benzidane et al., 2011). Nitenpyram, despite having a neonicotinoid structure, behaves more like nicotine in that it was found to bind to both the ACh site and an allosteric site within the nAChR (Tomizawa and Yamamoto, 1993). Other insect species, such as the peach potato aphid (Myzus persicae) appear to have an allosteric binding interaction between at least two nicotinic binding sites (Lind et al., 1999). The LGIC are pentameric molecules composed of five identical subunits (homomeric receptors) or different subunits (heteromeric receptors) arranged round a central pore, which is selective to Na+, K+ and Ca2+ cations (Millar and Lansdell, 2012; Thany et al., 2007). Genome studies have identified 11 nAChR subunit genes in the honeybee (Jones et al., 2007; Jones et al., 2006), compared with 10 each in the fruit fly (Drosophila melanogaster) Neonicotinoid pesticides and bees Report to Syngenta Ltd

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and mosquito (Anopheles mellifera) (Jones et al., 2007). Although studies by Le Novere et al. (2002) and Millar (2003) cited in Millar and Lansdell (2012) identified seventeen subunits in vertebrates (α1 – α10, β1- β4, γ, δ, ε), only α and β subunits have been found in the honeybee (Dupuis et al., 2011). The combination of subunits determines the functional and pharmacological properties of the receptor (Jones et al., 2007) however, it is thought that the α subunits of two adjacent cysteine residues in Loop C are important in ACh binding (Kao et.al (1984) cited in Jones et al., (2007)). There are core nAChR subunits that are conserved between different insect species with over 60% homology in their amino acid sequences (Jones et al., 2007; Sattelle, 2009). However, the fruit fly, mosquito and honeybee have at least one divergent subunit with less than 20% homology (Sattelle, 2009). The subunits found in the honeybee are α1, α2, α3, α4, α5, α6, α8, α7, α9, β1, β2 (Dupuis et al., 2011) of which only α5, α8 and α9 have not been sequenced (Rocher and Marchand-Geneste, 2008). Different combinations of subunits within the LIGC are present in different receptors throughout the body and may explain the susceptibility of some areas of the honeybee compared with others. For example, α2, α8 and β1 subunits were expressed in adult honeybee Kenyon Cells, where as an additional subunit α7 found in the Antennal Lobes (Dupuis et al., 2011). Through structure activity relationship (SAR) it has been possible to identify the 3pyridylmethylamine moiety as essential for providing the insecticidal properties of neonicotinoids (Tomizawa and Yamamoto, 1992; Tomizawa and Yamamoto, 1992; Tomizawa and Yamamoto, 1993; Yamamoto et al., 1995). Furthermore the difference in binding affinity at the nAChR may explain differences in toxicity between species (Matsuo et al., 1998; Rocher and Marchand-Geneste, 2008; Tomizawa and Yamamoto, 1993; Yamamoto et al., 1995). 2.1.2 Metabolism 2.1.2.1 Detoxifying enzymes in honeybees The honeybee genome has substantially fewer protein coding genes than Drosophila melanogaster and Anopheles gambiae with some of the most marked differences occurring in three superfamilies encoding xenobiotic detoxifying enzymes (Claudianos et al., 2006). This variation makes extrapolation of responses to both individual pesticides and pesticide mixtures between species less reliable as there are only about half as many of the three major xenobiotic metabolising enzymes glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. The glutathione-S-transferase group of enzymes catalyse the metabolism of pesticides by conjugation of reduced glutathione — via a sulfhydryl group — to electrophilic centers on a wide variety of substrates. The P450s catalyse a range of reactions including oxidation and demethylation which may result in decrease in activity or produce active metabolites, e.g. the conversion of the neonicotinoid thiamethoxam to clothianidin. The midgut in the honeybee is a major site of metabolism for ingested pesticides and interactions between chemicals at least in part may be influenced by effects on the detoxifying enzymes within the midgut, including microsomal oxidases, glutathione S transferases and esterases. Microsomal oxidase assay required intact midgut because an inhibitor of P450 is released when midguts are dissected and midgut microsomal preparations contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain the low microsomal oxidase activity in microsomes (Johnson et al 2009). The microsomal oxidase activities include aldrin epoxidase activity which is inhibited by malathion and permethrin, N-demethylase activity which is induced by diazinon and EPN Neonicotinoid pesticides and bees Report to Syngenta Ltd

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and O-demethlase activity which is induced by diazinon. Of the glutathione S-transferases, aryltransferase activity is significantly induced by diazinon and moderately induced by permethrin. Carboxylesterase activity is moderately inhibited by malathion and permethrin (Suh and Shim, 1988; Yu et al., 1984). The P450s are thought to play a central role in insects in the metabolism of phytochemicals (Li et al., 2007). Examples of such phytochemicals relevant to honeybees are the flavonoids (flavonoles e.g.quercetin, kaempherol, galangin, fisetin, flavanones e.g. pinocembrin, naringin, hesperidin and flavones e.g. apigenin, acacetin, chrysin, luteolin) which occur as glycosides in nectar and are hydrolysed to aglycones during the formation of honey and are also present in propolis and pollen (Viuda-Martos et al., 2008). However, when compared with other insects there are a significantly lower number of CYP3 clans (which include the CYP6s and CYP9s) associated with xenobiotic metabolism encoded in the honeybee genome (Johnson et al., 2010). Although this may be related to the reduced exposure to chemically-defended plant tissues there is some suggestion that others e.g. the CYP6AS subfamily, have undergone an expansion relative to other insects (Mao et al., 2011). CYP6 enzymes are recognised as being involved in the metabolism of dietary constituents in herbivorous insects (Liu et al., 2006). Therefore this expansion may be due to the presence of specific phytochemicals in the diet, e.g. in pollen and nectar, which may be concentrated in honey and bee bread (Adler, 2000). The link to phytochemical exposure in honeybees is supported by the upregulation of three of the CYP6AS genes in response to consumption of honey (Johnson, 2008). Detailed studies by Suchail et al. (2004a; 2004b) using radiolabelled [C14] imidacloprid have identified the distribution and metabolic pathways of imidacloprid in honeybees. Imidacloprid is readily metabolised (possibly by mixed function oxidases (Phase I metabolism)) to more water soluble products with an elimination half life of 5 hours; there is no evidence of conjugation prior to elimination. Six and 24 hours after ingestion of imidacloprid at 20 and 50 µg/kg-1 bee, no imidacloprid was detected in the honeybee (Suchail et al., 2004b). There are 5 metabolic products; 6-chloronicotinic acid is formed by the oxidative cleavage of the imidacloprid methylene bridge, urea derivative is formed by the reduction of the nitro group, hydroxylation of the imidazolidine ring to form 4,5-dihydroxy-imidacloprid and then 4/5hydroxy-imidacloprid, and the dehydration of the 4/5-hydroxy-imidacloprid and/or desaturation of the imidazolidine moiety of imidacloprid to for olefin. The main metabolites are the urea derivative and 6-chloronicotinic acid. Unlike mammals, there was no guanidine derivative detected (Suchail, et al. 2004). Suchail et al., (2004a) also reported the distribution of imidacloprid and its metabolites throughout the body of the honeybee. The rapid appearance of metabolites throughout the body, combined with variations in the kinetic profile suggests that metabolism also occurs outside of the main focus in the midgut. The main metabolites, urea derivative and 6chloronicotinic acid, were found particularly in the mid gut and rectum. In addition, 4/5hydroxy-imidacloprid and olefin were found mostly in the head, thorax and abdomen, all of which are nAChR rich tissues. Furthermore, concentrations of these two metabolites peaked 4 hours after ingestion of 100 µg kg-1 bee. These profiles can be related to the two distinct phases to imidacloprid poisoning after acute exposure. There is a rapid onset of neurotoxicity in the form of hyper responsiveness, hyperactivity, and trembling; mortality is delayed until at least four hours post exposure (Suchail et al., 2004a; Suchail et al., 2004b; Suchail et al., 2001). Insecticidal properties were found with olefin, and 4/5-hydroxy-imidacloprid all of which retain nitroguanidine Neonicotinoid pesticides and bees Report to Syngenta Ltd

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(CH4N4O2). LD50 values show that the olefin (48 hour oral LD50 >36 ng/bee), in particular, is of similar toxicity to imidacloprid (48 hour oral LD50 41 ng/bee) to bees (Nauen et al., 2001). Furthermore, both metabolites were found to bind to the nAChR. The delayed onset of death is thought to be as a result of the appearance of these toxic metabolites (Nauen et al., 2001; Schmuck et al., 2003). A similar metabolic fate study was conducted by (Brunet et al., 2005) using radiolabelled [14C]-acetamiprid. Acetamiprid is readily metabolised by mixed function oxidases. Seven metabolites were detected, with the main ones being 6-choronicotinic acid (IC 0) and U1. Although metabolism is fast, with 50% of acetamiprid metabolised 30 minutes after oral dosing with 100µg kg-1 bee, only 40% of the total radioactivity was eliminated after 72 hours . This suggests that both the metabolites and parent compound persist within the honeybee. However, none of the breakdown products have insecticidal qualities (Iwasa et al., 2004). The low toxicity of acetamiprid to honeybees is believed to be because of this rapid metabolism (half life 25 min). Brunet et al., (2005) also evaluated the distribution of acetamiprid and its metabolites in different compartments of the honeybee. Acetamiprid was rapidly distributed in all compartments and metabolised. Initially, acetamiprid was mainly detected in nAChR tissues of the abdomen, head and thorax with a distribution profile similar to that of imidacloprid (Suchail et al., 2004a). However unlike imidacloprid, significant levels of metabolites were detected at 72 hours throughout the honeybee compartments. The metabolism of neonicotinoids was further explored by Iwasa et al., (2004), who used synergists of both Phase I and Phase II enzymes to identify the metabolic pathways used in neonicotinoid detoxification. This study confirmed the role mixed function oxidases in the metabolism of many neonicotinoids. However, the toxicity of imidacloprid was not increased by the presence of potent cytochrome P450 inhibitors, such as piperonyl butoxide and Ergosterol Biosynthesis Inhibitors (EBI) suggesting P450s have a lesser role. 2.1.3 Conclusions Neonicotinoids have been shown to bind to the ligand gated ion channels at the αbungarotoxin site on the nicotinic acetylcholine receptor (nAChR) and primarlily affect the post synaptic nAChRs. Once bound to the nAChR, neonicotinoids are not broken down by acetylcholinesterase like acetylcholine resulting in over-stimulation of the nervous system and a build up of acetylcholine in the synapse. The midgut in the honeybee is a major site of metabolism for ingested pesticides and interactions between chemicals at least in part may be influenced by effects on the detoxifying enzymes within the midgut, including microsomal oxidases, glutathione S transferases and esterases. Synergists of both Phase I and Phase II enzymes have been used to identify the metabolic pathways used in neonicotinoid detoxification and confirmed the role mixed function oxidases in the metabolism of many neonicotinoids. However P450s probably have a lesser role in the metabolism of imidacloprid which has an elimination half life of 5 hours. The main imidacloprid metabolites, urea derivative and 6-chloronicotinic acid, are found particularly in the mid gut and rectum and 4/5-hydroxy-imidacloprid and olefin are found mostly in the head, thorax and abdomen all of which are nAChR rich tissues and levels peaked 4 hours after ingestion.. Both of the latter metabolites have insecticidal properties (the olefin has Neonicotinoid pesticides and bees Report to Syngenta Ltd

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toxicity similar to imidacloprid and both bind to the nAChR) and may be responsible for delayed onset of death. These profiles can be related to the two distinct phases to imidacloprid poisoning after acute exposure. There is a rapid onset of neurotoxicity in the form of hyper responsiveness, hyperactivity, and trembling; mortality is delayed until at least four hours post exposure. Acetamiprid is readily metabolised by mixed function oxidases, to seven main metabolites (none of which are insecticidal). Although metabolism is fast, the half life of acetamiprid approx 25 minutes but only 40% of the total radioactivity eliminated after 72 hours suggesting that the metabolites persist within the honeybee. The lower toxicity of acetamiprid is thought to be due to this rapid metabolism to relatively non-toxic metabolites.

2.2 Acute Toxicity of neonicotinoids 2.2.1 Honeybees Toxicity data from the ANSES Agritox databse are shown in

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Table 1. Most publications have concentrated on oral and contact LD50 imidacloprid, but other neonicotinoids are represented along with imidacloprid metabolites (Table 8); Contact exposure can be from either of two methods; directly where the dose is applied to the body of the bee, or indirectly where bees are exposed through contact with a contaminated surface. All the studies have reported at the effects of neonicotinoids on worker bees and have concentrated on Apis mellifera and its subspecies; however one study has looked at toxicity in the Indian Honeybee (Apis cerana indica) (Jeyalakshmi et al., 2011). In general, LD50 values are lower for oral exposure than for contact exposure, except for acetamiprid which is the reverse. For imidacloprid this may be explained its low hydrophobicity and poor penetration through the cuticle (Yamamoto et al., 1995). Residue data undertaken for the UK Wildlife Incident Investigation Scheme suggests that there is only slow penetration of the neonicotinoid active ingredients through the cuticle (Figure 1)

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Table 1: Oral and contact 48 LD50 values for neonicotinoid pesticides in honeybees from 1 ANSES Agritox database (http://www.dive.afssa.fr/agritox/php/fiches.php) or EPA factsheets (United States Environmental Protection Agency)

Insecticide Acetamiprid

Clothianidin

Dinotefuran

Imidacloprid

Thiacloprid

Thiamethoxam

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Exposure Route

48 LD50

Oral

14.5 µg/bee

Contact

8.1 µg/bee

Oral

3.79 ng/bee

Contact

44.3 ng/bee

Oral

23 ng/bee

Contact

47 ng/bee

Oral

3.7 ng/bee

Contact

81 ng/bee

Oral

17.32 µg/bee

Contact

38.83 µg/bee

Oral

5 ng/bee

Contact

24 ng/bee

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Figure 1: Relationship between dose applied and residue after 4 hours in bees dosed by contact and oral exposure with imidacloprid, acetamiprid and thiacloprid (from Defra report PS2548 and PS2549).

There is a greater degree of variability within the published data than is evident from Neonicotinoid pesticides and bees Report to Syngenta Ltd

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Table 1. It is particularly true for imidacloprid which has been extensively reported within the literature; Table 2 shows 48 hour oral LD50 values ranging from 3.7 to over 109 ng/bee with values as high as 400 ng/bee identified at Fera (Defra project PS2368). Table 2: Mean, maximum and minimum LD50 values for imidacloprid in honeybees (Apis sp), compiled from published data that is expressed as ng/bee (Table 1).

Exposure route Exposure time (hours) Mean LD50 (ng/bee) n

Max – Min (ng/bee)

Oral

191.0 – 3.2

Contact

24

112.7

5

48

44.3

14 109.6 – 3.7

72

64.0

5

97.4 – 31

96

43.5

2

50 - 37

24

15.9

4

23.8 – 6.7

48

80.6

11 242.6 – 6.7

72

104.0

1

n/a

Variability could be due to a number of factors, such as different test conditions, honeybee species, and/or timing in the season. Inter-laboratory variation was highlighted by Nauen et al., (2001) and Schmuck et al., (2003) who presented data on 48 hour acute oral toxicity for imidacloprid provided by laboratories in Germany and the United Kingdom following internationally adopted guidelines (EPPO 170, 1992). LD50 values ranged 2 fold (Table 3, Table 8). Hashimoto et al., (2003) found that oral LC50 values for thiamethoxam were 0.047 ng/mL for newly emerged worker bees increasing to 0.101 ng/mL for 21 day old worker bees. A similar pattern was seen with contact LC50 ranging from 3.21 mg/mL in newly emerged worker bees to 4.51 mg/mL in 14 day old worker bees however the experimental details do not allow the study to be fully reconstructed. Table 3: Acute contact toxicity (48 hours) of imidacloprid to honeybees derived by different laboratories using honeybees from different apiaries. Based on data presented by (Nauen et al., 2001; Schmuck et al., 2003)

Contact LD50 ng/bee

95 % confidence limits

Test Period

Origin of Honeybees

61.0

26.0 – 90.0

May 2000

Germany II

50

9.1 – 71.0

May 2000

United Kingdom

42.0

20.0 – 59.0

May 2000

Germany III

42.9

34.6 – 53.2

May 2000

Germany IV

74.9

61.8 – 90.9

July 2000

Germany V

Inter-hive variablity was studied by Laurino et al., (2010) who looked at acute oral toxicity of clothianidin, imidacloprid and thiamethoxam on three different strains of the Italian honeybee (Apis mellifera ligustica) by using bees from three different hives. Other factors such as methodology and timing were standardised. Variation between the three hives was small (Table 4), and the toxicity of the three neonicotinoids tested is in line with other published Neonicotinoid pesticides and bees Page 22 of 133 Report to Syngenta Ltd

data. The LD50 for imidacloprid is higher than those of Nauen et al., (2001) and Schmuck et al., (2003) however, there is some overlap between the ranges of the two sets of data. The variation observed for clothianidin and thiamethoxam is less. Table 4: Mean oral LD50 values for three neonicotinoid insecticides. Data taken from (Laurino et al., 2010)

Duration (hours) Clothianidin Mean 95% CI

Imidacloprid

Thiamethoxam

Mean

Mean 95% CI

95% CI

24

4.48

3.96 – 4.90 183.78 174.5 -190.7

3.55

2.82 – 4.43

48

4.32

3.86 – 4.65 104.12 99.53 – 108.99 3.35

2.68 – 4.25

72

4.21

3.81 – 4.50 72.94

2.59 – 3.13

49.55 – 95.15

2.88

Suchail et al., (2000) identified differences in LD50 response to imidacloprid between two subspecies of honeybees Apis mellifera mellifera (the dark European Honeybee) and Apis mellifera caucasica (the Caucasian Honeybee). Similar values were obtained for oral exposure of 5.4 and 6.6 ng/bee LD50 (24 hour) and 4.8 and 6.5 ng/bee LD50 (48 hour) A. m. mellifera and A. m. caucasica respectively. However, there were marked differences in responses between the subspecies after contact exposure. At low doses (between 1 and 7 ng/bee) A. m. mellifera mortality rates apparently increased, between 7 to 15 ng/bee mortality rates decreased, and at concentrations above 15ng/bee mortality rates increased in a dose dependent manner (Error! Reference source not found.A). As a result, two 24 hour LD50 values, 6.7ng/bee and 23.8 ng/bee, were calculated for A. m. mellifera based on the two ascending parts of the dose response curve. A similar drop in mortality was seen with A. m. caucasica (Error! Reference source not found.B), but this was less marked, and a single LD50 value of 15.1 ng/bee was calculated. A similar increase in mortality was seen in A. m. mellifera over 48 hours at low doses; however this was not followed by a fall below that expected. Notwithstanding this, 48 hour LD50 values were again calculated from the two ascending parts of the dose response curve with values of 6.7 and 24.3 ng/bee for A. m. mellifera; 48 hour contact LD50 for A. m. caucasica was 12.8 ng/bee. Interestingly, the deviation between replicates is small, and experiments were repeated at least three times. Suchail et al., (2000) suggested this was a result of biphasic mortality at low concentrations, particularly via contact exposure. Howeever the result is predicated primarily on the mortality observed at one dose level around 7 ng/bee. Unfortunately none of the other reported studies tested imidacloprid at doses this low; the minimum dose in the reported literature was 40 ng/bee (Nauen et al., 2001; Schmuck et al., 2003) and at Fera the lowest dose tested in Defra study PS2368 was 12.5 ng/bee. No other studies have reported to replicate this phenomenon. Studies by Nauen et al., (2001), Schmuck et al., (2003), and Suchail et al., (2001a) have looked at the acute toxicity of imidacloprid and the five main metabolites to A. mellifera. Two metabolites were found to have insecticidal properties; these are olefin and 5-OH imidacloprid with 48 hour oral LD50 values of >39 and 159 ng/bee respectively (Nauen et al., 2001; Schmuck et al., 2003) and 28 and 258 ng/bee (Suchail et al., 2001a). Nauen et al., (2001) obtained an LD50 for di-hydroxy-imidacloprid of >49 ng/bee; its insecticidal significance was identified as low due to its weak receptor affinity and lack of receptor activation despite retaining the nitro guanidine pharmocophore, (Schmuck et al., 2003). Neonicotinoid pesticides and bees Report to Syngenta Ltd

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Incidentally, (Suchail et al., 2001a) reported oral LD50 values of >1000 ng/bee for di hydroxy-imidacloprid for exposures between 48 and 96 hours and thus concluded it had no insecticidal qualities. The major plant metabolites of acetamiprid (N-demethyl acetamiprid, 6chloro-3-pyridylmethanol and 6-chloro-nictinic acid) were also tested for insecticidal properties (Iwasa et al., 2004), however no mortality was detected when applied topically at 50 µg/bee. 2.2.2 Other bee species Table 5 summarises the toxicity data for a range of bee species, including bumble bees, other Apis species and non-Apis species. Data across pesticides generally suggests that the toxicity to Apis mellifera reflects that in other bee species when it is expressed on a weight basis (Figure 2). However Table 6 and Table 7 suggest that although this is true for the chloronicotinyl insecticides there are far less robust data for the cyano substituted neonicotinoids acetamiprid and thiacloprid. 10000 B. Terrestris 210 mg 1000

B lucorum 210 mg ug/g bee

100 B agrorum (pascuorum) 120 mg

10

M rotundata 86.6mg

1 0.01

1

100

10000

O lignaria 90mg

0.1 Nomia melanderi 30.8mg 0.01 Apis mellifera ug/ g bee

Figure 2 Comparison of the toxicity of a range of pesticides in Apis and non-Apis bee species expressed on a weight organism basis

The toxicity of imidacloprid to B terrestris was reported by Marletto et al (2003); unfortunately the weights of the bees were not reported so the toxicity in µg/g bee can only be estimated (based on 210mg/bee). They reported the acute contact LD50 was 0.04 µg/ bee (0.19 µg/ g bee) at 24 hours and 0.02 µg/bee (0.095 µg/ g bee) at 72 hours. Following oral exposure the 24 hour LD50 was not reported but the 72 hour LD50 was 0.02 µg/bee (0.095 µg/g bee). Mayer and Lunden (1997) assessed the toxicity of field weathered imidacloprid residues (after application of a 240FS formulation at 0.168 Kg ai/ha) to a range of bees species,. Exposure for 24 hours to cranberry or alfalfa leaves aged for 2 hours resulted in 56% mortality in exposed B occidentalis individuals; 28% mortality in exposed alkali bees (N. melanderi) and 66% mortality in exposed leafcutter bees (M rotundata) compared with 14% morality in exposed A. mellifera. Bortolotti et al (2001) assessed the toxicity of the imidacloprid formulation Confidor (17.8%) to B terrestris; again the bodyweight is not reported so is estimated). They reported the Neonicotinoid pesticides and bees Report to Syngenta Ltd

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contact LD50 as 445 ng/bee (2119 ng ai/ g bee) at 24 hours, 14.2 ng/bee (67.6 ng ai/ g bee) at 48 hours and 5.3 ng/bee (25.2 ng ai/ g bee ) at 72 hours. The oral LD50 was 7.1 ng/bee (33.9 ng ai/ g bee) at 24 hours, 5.3 ng/bee (25.2 ng ai/ g bee ) at 48 hours and 4.6 ng/bee (22.0 ng ai/ g bee ) at 72 hours. There is no readily available LD50 data available for acetamiprid but Horgan (2007) compared the residual toxicity of a 20% formulation of acetamiprid at 100ppm to that of 50ppm imidacloprid and showed that the residue of acetamiprid was safe to B terrestris after 1 days whereas the imidacloprid formulation was still toxic after 3 days. The toxicity of imidacloprid to the stingless bee N perilampoides (average weight 8.2 mg/bee) showed the contact LD50 after 24 hours was 0.0011 µg/bee (0.0008-0.0015) (0.13 µg/g bee) that of thiamethoxam was 0.004 µg/bee (0.003-0.006) (0.49 µg/ g bee), whereas thiacloprid was 0.007 µg/bee (0.004-0.01) (0.85 µg/g bee) suggesting that the relative safety of thiacloprid to honeybees was not reflected by stingless bees (Valdovinos-Nunez et al 2009). Kumar and Regupathy (2005) reported the toxicity of thiamethoxam and imidacloprid to A mellifera, A. indica (the Indian honeybee), A. florea (the little bee) and Trigona irridipenis (the dammer bee) and reported these were similar or less toxic than in A mellifera in the same laboratory (thiamethoxam 0.0666 µg ai/g bee and imidacloprid 0.0281 µg ai/g bee at 24 hours). Scott-Dupree et al (2009) assesed the toxicity of imidacloprid and clothianidin to B. impatiens, M rotundata and Osmia lignaria using treated surfaces. Unfortunately this prevents assessment of the dose per bee as the LC50 was expressed as a treatment rate (% solution wt/vol) on the surface of the arena. Based on this they assessed the toxicity (LC50) of imidacloprid to B impatiens as 3.22 x 10-3%, to M rotundata as 0.17 x 10-3%, and to O lignaria as 0.07 x 10-3%. The LC50 of clothianidin was B impatiens 0.39 x 10-3%, to M rotundata as 0.08 x 10-3%, and to O lignaria as 0.10 x 10-3%. The toxicity of fipronil to A. mellifera, the alfalfa leafcutter bee M. rotundata and the alkali bee Nomia melanderi was reported by Mayer and Lunden (1999). The LD50 was highest in N melanderi (1.13 µg/bee; 13.2 µg/g bee), intermediate in A mellifera (0.013 µg/bee; 0.103 µg/g bee ) and lowest in M. rotundata (0.004 µg/bee; 0.132 µg/g bee).

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Table 5:Summary of toxicity of neonicotinoids in other bee species

Pesticide

Species

Acetamiprid Mosplian (3% ai) Acetamiprid Mosplian (3% ai) Acetamiprid Mosplian (3% ai) Imidacloprid (formulation) Imidacloprid (formulation) Imidacloprid Confidor (17.8% ai)

B ignites 48 hr oral B hypocrite 48 hr oral B patagiatus 48 hr oral B terrestris 72 hour contact B terrestris 72 hour oral B terrestris 24 hour contact

Imidacloprid Confidor (17.8% ai) Imidacloprid Confidor (17.8% ai) Imidacloprid Confidor (17.8% ai) Imidacloprid Confidor (17.8% ai) Imidacloprid Confidor (17.8% ai) Imidacloprid

B terrestris 48 hour contact B terrestris 72 hour contact B terrestris 24 hour oral B terrestris 48 hour oral B terrestris 72 hour oral A indica 24 hr contact A florae 24 hr contact T irridipenis 24 hr contact N perilampoides 24 hr contact B terrestris 24 hour contact N perilampoides 24 hr contact A indica 24 hr contact A florae 24 hr contact T irridipenis 24 hr contact N perilampoides

Imidacloprid Imidacloprid Imidacloprid Imidacloprid (formulation) Thiacloprid Thiamethoxam Thiamethoxam Thiamethoxam Thiamethoxam

Neonicotinoid pesticides and bees Report to Syngenta Ltd

µg a.i./ bee (95%CL) 0.0023 (0.00210.0024) 0.0028 (0.00180.0031) 0.0021 (0.00200.0023) 0.02

µg ai/g bee

Ref

0.02

0.095

0.445

2.119

0.0142

0.0676

Bortolotti et al (2001)

0.0053

0.0252

Bortolotti et al (2001)

0.0071

0.0339

Bortolotti et al (2001)

0.0053

0.0252

Bortolotti et al (2001)

0.0046

0.0220

0.0025

0.0362

0.0022

0.0767

0.0020

0.5275

0.0011

0.13

0.04

0.19

0.007

0.85

0.0056

0.0819

0.0056

0.1905

0.0051

1.3381

0.004

0.49

Bortolotti et al (2001) Kumar and Regupathy (2005) Kumar and Regupathy (2005) Kumar and Regupathy (2005) Valdovinos-Nunez et al 2009 Marletto et al (2003) Valdovinos-Nunez et al 2009 Kumar and Regupathy (2005) Kumar and Regupathy (2005) Kumar and Regupathy (2005) Valdovinos-Nunez

Wu et al (2010) Wu et al (2010) Wu et al (2010) 0.095

Marletto et al (2003) Marletto et al (2003) Bortolotti et al (2001)

Page 26 of 133

24 hr contact

et al 2009

. The lowest LD50 reported for each study are shown in Table 6 for contact toxicity and Table 7 for oral toxicity. These data suggest that the contact toxicity is generally within an order of magnitude of the A. mellifera for imidacloprid and thiamethoxam whereas N perilampoides is several orders of magnitude more sensitive following contact exposure to thiacloprid. Table 7 shows the toxicity of oral exposure to acetamiprid but this should be interpreted with care as the exposure profile differed significantly with the Bombus species exposed ad libitum to treated sucrose for 48 hrs and Apis only for 2-4 hrs. . Table 6: Summary data using lowest contact LD50 for each reported study (µg/g bee) A B A T N mellifera terrestris indica A florea irridipenis perilampoides (India) 0.025imidacloprid 0.19 0.036 0.077 0.53 0.13 0.028 thiacloprid

A mellifera 0.81

0.85

thiamethoxam

0.082

0.191

1.34

0.49

388 0.066

0.24

Table 7: Summary data using lowest oral LD50 for each reported study (µg/g bee) 4 hrs access to treated 48hrs access to treated sucrose sucrose B ignitus

B hypocrite

B patagiatus

Bterrestris

A mellifera

0.01

0.013

0.01

0.0220.095

145

acetamiprid

Conclusions In general, LD50 values are lower for oral exposure than for contact exposure, except for acetamiprid which is the reverse; this may be explained by low hydrophobicity and poor penetration through the cuticle of these compounds. The chloronicotinyl insecticides (thiamethoxam, clothianidin, imidacloprid) are more toxic than the cyano substituted (thiacloprid, acetamiprid) The oral toxicity of imidacloprid, which has been extensively reported within the literature; is highly variable with 48 hour oral LD50 values ranging from 3.7 to as high as 400 ng/bee. including bumble bees, other Apis species and non-Apis species. Data across pesticides generally suggests that the toxicity to Apis mellifera reflects that in other bee species when it is expressed on a weight basis and is supported by data for the chloronicotinyl insecticides but there are far less robust data for the cyano substituted neonicotinoids.

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 27 of 133

Table 8: Showing published acute toxicity data for Apis mellifera (Compounds in italics are metabolites)

Insecticide Concentration LD50 – 24 hours Oral Imidacloprid 5.4 ng/bee

Range

Bee Type

Pre-treatment

Reference

5.2-1.6 (95% CI)

Starvation 2 hr

Imidacloprid

6.6 ng/bee

5.1-8.1 (95% CI)

Imidacloprid (0.15 – 150 ppm) Clothianidin

183.78 ng/bee

Clothianidin

4.48 ng/bee

174.5 – 190.7 (95% CI) 1.733-4.045 (95% CI) 4.0 – 4.9 (95% CI)

Worker Bees (A. m. mellifera) Worker Bees (A. m. caucasica) Apis mellifera ligustica Forager Bee

Not stated

Thiamethoxam

4.679 ng/bee

Apis mellifera ligustica Forager Bee

Thiamethoxam

3.54 ng/bee

Apis mellifera ligustica

Not stated

(Suchail et al., 2000) (Suchail et al., 2000) (Laurino et al., 2010) (Laurino et al., 2011) (Laurino et al., 2010) (Laurino et al., 2011) (Laurino et al., 2010)

Worker Bees

CO2 anaesthesia

2.844 ng/bee

3.862-5.552 (95% CI) 2.8 – 4.4 (95% CI)

LD50 – 24 hours – Contact (applied directly to the bee) Acetamiprid 7.07 µg/bee 4.57-11.2 (95% CI) IM-2-1 >50 µg/bee (Acetamiprid metabolite) IC-0 (Acetamiprid >50 µg/bee metabolite) IM-O (Acetamiprid >50 µg/bee metabolite) Imidacloprid 17.9 ng/bee 9.2-31.5 (95% CI) (dose range to give 8 – 100% mortality) Neonicotinoid pesticides and bees Report to Syngenta Ltd

Starvation 2 hr Not stated Not stated

Not stated

(Iwasa et al., 2004) (Iwasa et al., 2004)

(Iwasa et al., 2004)

Worker Bees

CO2 anaesthesia

Page 28 of 133

(Iwasa et al., 2004)

Insecticide Imidacloprid

Concentration 6.7 ng/bee

Range 5.2-8.2 (95% CI)

Imidacloprid

23.8 ng/bee

22.3-25.3 (95% CI)

Imidacloprid

15.1 ng/bee

11.9-18.3 (95% CI)

Imidacloprid 17.8% SL (5 – 52 ng/bee) Nitenpyram

27 ng/bee

Thiacloprid Dinotefuran Clothianidin Clothianidin 50% WDG Thiamethoxam Thiamethoxam

14.6 µg/bee 75.0 ng/bee 21.8 ng/bee 14 ng/bee

0.138 µg/bee

29.9 ng/bee 26 ng/bee

Bee Type Worker Bees (A. m. mellifera) Worker Bees (A. m. mellifera) Worker Bees (A. m. caucasica) Apis cerana indica

Pre-treatment CO2 anaesthesia

0.0717-0.259 (95% CI) 9.53-25.4 (95% CI) 62.8-89.6 (95% CI) 10.2-46.5 (95% CI)

Worker Bees

CO2 anaesthesia

(Iwasa et al., 2004)

Worker Bees Worker Bees Worker Bees Apis cerana indica

CO2 anaesthesia CO2 anaesthesia CO2 anaesthesia CO2 anaesthesia

20.8-42.9 (95% CI)

Worker Bees Apis cerana indica

CO2 anaesthesia CO2 anaesthesia

(Iwasa et al., 2004) (Iwasa et al., 2004) (Iwasa et al., 2004) (Jeyalakshmi et al., 2011) (Iwasa et al., 2004) (Jeyalakshmi et al., 2011)

LD50 – 24 hours – Indirect Contact (contact with contaminated surface) Clothianidin 4.485 ng/µl 3.820-5.167 (95% Forager Bee CI) Imidacloprid 0.10 ppm 0.015-0.63 (Fiducial Worker bees (dose range to Limits) give 10-90% mortality after 24 hrs) Thiamethoxam 5.200 ng/µl 4.302-6.227 (95% Forager Bee CI) Thiamethoxam 15.16 ppm 2.84-97.00 (Fiducial Worker bees Neonicotinoid pesticides and bees Report to Syngenta Ltd

CO2 anaesthesia CO2 anaesthesia CO2 anaesthesia

Not stated Starvation 2 hours

Not stated Starvation 2 hours Page 29 of 133

Reference (Suchail et al., 2000) (Suchail et al., 2000) (Suchail et al., 2000) (Jeyalakshmi et al., 2011)

(Laurino et al., 2011) (Singh and Karnatak, 2005)

(Laurino et al., 2011) (Singh and

Insecticide

Concentration

Range Limits) LC50 - 24 hours - Contact (applied directly to the bee) Imidacloprid 2.2 (%w/v x10-3) 1.5-2.6 (Fiducial (0.00008 – 1.0% limits x10-3) n sol ) LD50 – 48 hours – Oral Imidacloprid 41 ng/bee (0.1 to 81 ng/bee)

Bee Type

Pre-treatment

Reference Karnatak, 2005)

Forager Bees

CO2 anaesthesia

(Bailey et al., 2005)

Worker Bees

Starvation up to 2 hr

Imidacloprid

4.8 ng/bee

4.5-5.1 (95% CI)

Starvation 2 hr

Imidacloprid

6.5 ng/bee

4.7-8.3 (95% CI)

Imidacloprid (1 - 1000 ng/bee) Imidacloprid (1 – 1000 ng/bee) Imidacloprid (0.2 – 3.2 mg/L-1) Imidacloprid

70 ng/bee

Worker Bees (A. m. mellifera) Worker Bees (A. m. caucasica) Worker Bees

Starvation 2 hours

Imidacloprid

>21.0 ng/bee

Worker Bees

Imidacloprid

40.9 ng/bee

Worker Bees

Imidacloprid (as formulation WG70) Imidacloprid (as formulation

11.6 ng/bee

7.3-18.3 (95% CI)

Worker Bees

(Nauen et al., 2001; Schmuck et al., 2003) (Suchail et al., 2000) (Suchail et al., 2000) (Suchail et al., 2001b) (Suchail et al., 2001a) (Decourtye et al., 2003) (Schmuck et al., 2001) (Schmuck et al., 2001) (Schmuck et al., 2001) (Schmuck et al., 2001)

21.2 ng/bee

15.0-29.6 (95% CI)

Worker Bees

57 ng/bee

± 28 (SD)

Worker Bees

30.6 ng/bee

26.7-36.3 (95% CI)

Worker Bees

3.7 ng/bee

2.6-5.3 (95% CI)

Worker Bees

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Starvation 2 hr Not stated

(Schmuck et al., 2001) Page 30 of 133

Insecticide SC200) Imidacloprid (0.15 – 150 ppm) Olefin

Concentration

Range

Bee Type

Pre-treatment

Reference

104.12 ng/bee

99.5 – 109.0 (95% CI) ± 19 (SD)

Apis mellifera ligustica Worker Bees

Not stated

Olefine

>36 ng/bee

Worker Bees

Starvation up to 2 hr

5-OH-Imidacloprid

159 ng/bee

Worker Bees

Starvation up to 2 hr

5-OH-Imidacloprid

258 ng/bee

± 7 (SD)

Worker Bees

Starvation 2 hours

5-OH-imidacloprid

153.5

125.9-196.9 (95% CI)

Worker Bees

Di-OHImidacloprid

>49 ng/bee

Worker Bees

Starvation up to 2 hr

4.5 Di-OHImidacloprid Urea metabolite

>1000 ng/bee

Worker Bees

Starvation 2 hours

>99500 ng/bee

Worker Bees

Starvation up to 2 hr

Urea metabolite

>1000 ng/bee

Worker Bees

Starvation 2 hours

6-Chloronicotinic acid

>121500 ng/bee

Worker Bees

Starvation up to 2 hr

6-Chloronicotinic acid

>1000 ng/bee

Worker Bees

Starvation 2 hours

(Laurino et al., 2010) (Suchail et al., 2001a) (Nauen et al., 2001; Schmuck et al., 2003) (Nauen et al., 2001; Schmuck et al., 2003) (Suchail et al., 2001a) (Decourtye et al., 2003) (Nauen et al., 2001; Schmuck et al., 2003) (Suchail et al., 2001a) (Nauen et al., 2001; Schmuck et al., 2003) (Suchail et al., 2001a) (Nauen et al., 2001; Schmuck et al., 2003) (Suchail et al., 2001a)

28 ng/bee

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Starvation 2 hours

Page 31 of 133

Insecticide Clothianidin

Concentration 2.689 ng/bee

Clothianidin

4.32 ng/bee

Thiamethoxam

4.411 ng/bee

Thiamethoxam

3.34 ng/bee

Range 1.749-3.679 (95% CI) 3.86 – 4.65 (95% CI) 3.612-5.252 (95% CI) 2.7 – 4.2 (95% CI)

LD50 48 hours – Contact (applied directly to the bee) Imidacloprid 61.0 ng/bee 26.0-90.0 (95% CI) (40-154 ng/bee)

Bee Type Forager Bee

Pre-treatment Not stated

Apis mellifera ligustica Forager Bee

Not stated

Apis mellifera ligustica

Not stated

Worker Bees

CO2 anaesthesia

Not stated

Reference (Laurino et al., 2011) (Laurino et al., 2010) (Laurino et al., 2011) (Laurino et al., 2010)

Imidacloprid (40-154 ng/bee)

50.0 ng/bee

9.1-71.0 (95% CI)

Worker Bees

CO2 anaesthesia

Imidacloprid (40-154 ng/bee)

42.0 ng/bee

20.0–59.0 (95% CI)

Worker Bees

CO2 anaesthesia

Imidacloprid (40-154 ng/bee)

42.9 ng/bee

34.6-53.2 (95% CI)

Worker Bees

CO2 anaesthesia

Imidacloprid (40-154 ng/bee)

74.9 ng/bee

61.8-90.9 (95% CI)

Worker Bees

CO2 anaesthesia

Imidacloprid

6.7 ng/bee

4.4-9.0 (95% CI)

CO2 anaesthesia

Imidacloprid

24.3 ng/bee

22.0-26.6 (95% CI)

CO2 anaesthesia

(Suchail et al., 2000

Imidacloprid

12.8 ng/bee

9.7-15.9 (95% CI)

Worker Bees (A. m. mellifera) Worker Bees (A. m. mellifera) Worker Bees (A. m. caucasica)

(Nauen et al., 2001; Schmuck et al., 2003 (Nauen et al., 2001; Schmuck et al., 2003 (Nauen et al., 2001; Schmuck et al., 2003 (Nauen et al., 2001; Schmuck et al., 2003 (Nauen et al., 2001; Schmuck et al., 2003 (Suchail et al., 2000

CO2 anaesthesia

(Suchail et al., 2000

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 32 of 133

Insecticide Imidacloprid

Concentration 81.0 ng/bee

Imidacloprid

230.3 ng/bee

Range 55.0-119.0 (95% CI)

Bee Type Worker Bees Worker Bees

Imidacloprid (as 242.6 ng/bee 173.3-353.4 (95% Worker Bees formulation CI) WG70) Imidacloprid (as 59.7 ng/bee 39.1-92.7 (95% CI) Worker Bees formulation (SC200) LD50 – 48 hours Indirect Contact (contact with contaminated surface) Clothianidin 2.967 ng/µl 2.398-3.467 (95% Forager Bee CI) Thiamethoxam4 3.313 ng/µl 2.786-3.806 (95% Forager Bee CI) LD50 – 72 hours – Oral Imidacloprid 37 ng/bee ± 10 (SD) Worker Bees (1 - 1000 ng/bee) Imidacloprid 70 ng/bee Worker Bees (1 - 1000 ng/bee) Imidacloprid 72.94 ng/bee 49.5 – 95.1 (95% CI) Apis mellifera (0.15 – 150 ppm) ligustica 5-OH-Imidacloprid 206 ng/bee ± 3 (SD) Worker Bees Olefin

29 ng/bee

4.5 Di-OHImidacloprid 6-Chloronicotinic acid

Pre-treatment

(Schmuck et al., 2001

Not stated Not stated

Starvation 2 hours Not stated Not stated Starvation 2 hours

Worker Bees

Starvation 2 hours

>1000 ng/bee

Worker Bees

Starvation 2 hours

>1000 ng/bee

Worker Bees

Starvation 2 hours

Neonicotinoid pesticides and bees Report to Syngenta Ltd

± 3 (SD)

Reference (Schmuck et al., 2001 (Schmuck et al., 2001 (Schmuck et al., 2001

Page 33 of 133

(Laurino et al., 2011) (Laurino et al., 2011) (Suchail et al., 2001a) (Suchail et al., 2001b) (Laurino et al., 2010) (Suchail et al., 2001a) (Suchail et al., 2001a) (Suchail et al., 2001a) (Suchail et al., 2001a)

Insecticide Urea

Concentration >1000 ng/bee

Range

Bee Type Worker Bees

Pre-treatment Starvation 2 hours

Clothianidin

4.21 ng/bee

3.8 – 4.5 (95% CI)

Not stated

Clothianidin

2.608 ng/bee

Thiamethoxam

4.316 ng/bee

Forager Bee

Not stated

Thiamethoxam

2.88 ng/bee

1.938-3.293 (95% CI) 3.517-5.154 (95% CI) 2.6 – 3.1 (95% CI)

Apis mellifera ligustica Forager Bee

Apis mellifera ligustica

Not stated

Worker Bees

CO2 anaesthesia

(Nauen et al., 2001; Schmuck et al., 2003

Not stated

(Laurino et al., 2011) (Laurino et al., 2011)

LD 50 – 72 hours - Contact (applied directly to the bee) Imidacloprid 104 ng/bee 83.0-130 (95% CI)

LD50 – 72 hours – Indirect Contact (contact with contaminated surface) Clothianidin 2.667 2.121-3.156 (95% Forager Bee CI) Thiamethoxam 2.462 ng/µl 2.156-2.903 (95% Forager Bee CI) LD50 – 96 hours – Oral Imidacloprid 37 ng/bee ± 10 (SD) Worker Bees (1- 1000 ng/bee) Imidacloprid 50 ng/bee Worker Bees (1 – 1000 ng/bee) 5-OH-Imidacloprid 222 ng/bee ± 25 (SD) Worker Bees Olefin

23 ng/bee

4.5 Di-OHImidacloprid

>1000 ng/bee

Neonicotinoid pesticides and bees Report to Syngenta Ltd

± 6 (SD)

Not stated

Not stated

Starvation 2 hours Not stated Starvation 2 hours

Worker Bees

Starvation 2 hours

Worker Bees

Starvation 2 hours Page 34 of 133

Reference (Suchail et al., 2001a) (Laurino et al., 2010) (Laurino et al., 2011) (Laurino et al., 2011) (Laurino et al., 2010)

(Suchail et al., 2001a) (Suchail et al., 2001b) (Suchail et al., 2001a) (Suchail et al., 2001a) (Suchail et al., 2001a)

Insecticide 6-Chloronicotinic acid Urea

Concentration >1000 ng/bee >1000 ng/bee

LC50 – Oral (duration not stated) Thiamethoxam 0.047 ng/mL Thiamethoxam

0.074 ng/mL

Thiamethoxam

0.081 ng/mL

Thiamethoxam

0.101 ng/mL

LC50 – Contact (duration not stated) Thiamethoxam 3.21 mg/mL Thiamethoxam

3.50 mg/mL

Thiamethoxam

4.51 mg/mL

Thiamethoxam

>5.0 mg/mL

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Range

Bee Type Worker Bees

Pre-treatment Starvation 2 hours

Worker Bees

Starvation 2 hours

Worker bees (newly emerged) Worker bees ( 7 days old) Worker bees (14 days old) Worker bees (21 days old)

Not stated

Worker bees (newly emerged) Worker bees ( 7 days old) Worker bees (14 days old) Worker bees (21 days old)

Not stated

Not stated Not stated Not stated

Not stated Not stated Not stated

Page 35 of 133

Reference (Suchail et al., 2001a) (Suchail et al., 2001a) (Hashimoto et al., 2003) (Hashimoto et al., 2003) (Hashimoto et al., 2003) (Hashimoto et al., 2003) (Hashimoto et al., 2003) (Hashimoto et al., 2003) (Hashimoto et al., 2003) (Hashimoto et al., 2003)

2.3 Multiple exposure to pesticides (including substances used in bee medication) and potential additive and cumulative effects. See EFSA 2012 http://www.efsa.europa.eu/en/supporting/pub/340e.htm. The effect of exposure on the scale of synergy is important as many of the laboratory studies have been undertaken with high doses of synergists, e.g. 3-10 µg/bee and at more realistic exposure levels such high increases of toxicity have not been observed even under laboratory conditions (Defra report PS2368). Contact and oral dosing with combinations of a range of EBI fungicides at more realistic exposure levels and neonicotinoid insecticides showed only low levels of synergy at these lower fungicide exposures (Table 9 and Table 10). This is confirmed by semi-field studies with field rates of thiacloprid and tebuconazole and of acetamiprid and triflumizole in which no increase in mortality was observed (Iwasa et al., 2004b; Schmuck, Stadler et al. 2003). Based on the exposure scenarios identified in http://www.efsa.europa.eu/en/supporting/pub/340e.htm. the 90th percentile exposure for a forager bee from a spray application of 1 Kg/ha fungicide would be 1.53 µg/bee from spray application and 3.63 µg/bee/day from nectar therefore for many of the EBI fungicides the maximum EU application rate is 250 g ai/Ha resulting in a 90th percentile exposure of 0.38 µg/bee from spray and potentially a total including oral exposure of 1.3 µg/bee on the day of application. Defra project PS2368 assessed the impact of joint contact:contact and oral:oral exposures to a range of neonicotinoid insecticides (clothianidin, thiamethoxam, imidacloprid and thiacloprid) and EBI fungicides (flusilazole, myclobutanil, propiconazole and tebuconazole) at realistic fungicide exposure rates (extrapolated from Koch and Weisser, 1997). This showed only lower order increases in the toxicity of the neonicotinoids (up to 3 fold) but differences between effects following contact and oral exposures (Table 9 and Table 10). Four other non-EBI fungicides have been assessed for their effects on the toxicity of thiacloprid (Schmuck, Stadler et al. 2003). Cyprodinil (an anilinopyrimidine fungicide) at 8 µg/bee and tolyfluanid (a phenylsulfamide fungicide) at 11 µg/bee slightly increased the mortality assocated with a contact dose of 2 µg thiacloprid /bee from 3 to 20% and 3 to 13% respectively. Mancozeb (a dithiocarbamate fungicide) at 8 µg/bee and azoxystrobin (a methoxyacrylate stobilurin fungicide) at 3 µg/bee had no effect on the toxicity of a contact dose of 2 µg thiacloprid /bee.

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 36 of 133

Table 9. Contact toxicity of combinations (from Defra project PS2368) LD50 (µg/bee) (95% CL)

Insecticide

+ Flusilazole (0.358 µg/bee)

+ Myclobutanil (0.161 µg/bee)

+ Propiconazole (0.224 µg/bee)

+ Tebuconazole (0.447 µg/bee)

LD50 (µg/bee)

95% CL

synergism ratio

LD50 (µg/bee)

95% CL

synergism ratio

LD50 (µg/bee)

95% CL

synergism ratio

LD50 (µg/bee)

95% CL

synergism ratio

Clothianidin

0.0350 (0.01550.0607)

0.0295

0.02300.0367

1.19

0.0451

0.03630.0559

0.78

0.0312

0.02390.0393

1.12

0.00287

0.02130.0368

1.22

Imidacloprid

0.0671 (0.04380.1018)

0.0475

0.01870.0912

1.41

0.0409

0.02050.0663

1.64

0.0585

0.03790.0867

1.15

0.0347

0.01610.0568

1.93

Thiacloprid

122.4 (90.56238.9)

439.3

157.371052.1

0.28

635.8

184.135100000

0.19

434.9

156.865908.9

0.28

266.8

128.93738.9

0.46

Thiamethoxam

0.124 (0.07680.3280)

0.0538

0.02540.1203

2.30

0.0979

0.08040.1223

1.27

0.0638

0.05210.0788

1.94

0.0479

0.03020.0757

2.59#

Synergism ratio = .

Insecticide LD50

.

Insecticide + fungicide LD50

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 37 of 133

Table 10: Oral toxicity of combinations (from Defra project PS2368) LD50 (µg/bee) (range)

+ Flusilazole (0.358 µg/bee)

+ Myclobutanil (0.161 µg/bee)

+ Propiconazole (0.224 µg/bee)

+ Tebuconazole (0.447 µg/bee)

Insecticide LD50 (µg/bee)

95% CL

synergism ratio

LD50 (µg/bee)

95% CL

synergism ratio

LD50 (µg/bee)

95% CL

synergism ratio

LD50 (µg/bee)

95% CL

synergism ratio

Clothianidin

0.00739 (0.006070.00903)

0.00441

0.002670.00762

1.68

0.00597

0.00493.000732

1.24

0.00572

0.004670.00710

1.29

0.00389

0.003050.00489

1.90#

Imidacloprid

0.536 (0.3391.184)

1.180

0.60945.878

0.45

1.075

0.56674.426

0.50

1.501

0.697214.44

0.36

0.893

0.43814.50

0.59

Thiacloprid

22.59 (16.3937.42)

25.88852.8

0.41

25.67

19.0140.70

0.88

47.01

27.45166.1

0.48

36.19

20.99134.1

0.62

Thiamethoxam

0.0112 (0.009150.0135)

0.008010.0136

1.09

0.00742

0.004480.01123

1.51

0.00830

-

1.35

0.00852

0.006880.01037

1.31

Synergism ratio = .

54.65

0.0103

Insecticide LD50

.

Insecticide + fungicide LD50

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 38 of 133

2.3.1 Conclusions Pesticides widely used both in the agricultural and urban environment (pest control and home and garden uses) as well as by beekeepers to control pests, e.g. fluvalinate, amitraz, coumaphos to control varroa, are detectable in bees and hive matrices. Exposure of honeybees to any single pesticide application may occur over the short term or, unlike many organisms, over a longer period if residues are present in pollen and/or nectar stored within the colony or due to migration of lipophilic compounds into wax. These more persistent residues are likely to be available to the colony over a period of time depending on the active ingredient and the frequency of use, e.g. multiple applications. Bees may be exposed to mixtures of products applied to plants on which they forage. Recent data indicates the extent of mixing of formulations that occur on arable, vegetable orchards and soft fruit crops in the UK and includes tank mixing of EBI fungicides with neonicotinoid insecticides. In addition to the application of products as tank mixes, the increasing use of seed treatments raises the possible scenario of nectar, pollen or guttation water containing active ingredients also being contaminated with sprays applied during the flowering period, e.g. oilseed rape. Although many reports of residues in pollen being returned to the hive by foragers are published the majority of these are based on individual pesticide residues rather than assessments of the total pesticide residue levels and the data are often not reported in sufficient detail to determine the residue levels of the individual components within multiple detections.. Data reported for residues of chemicals applied by beekeepers in honeybee colonies have primarily been directed at single varroacides with some limited data after antibiotic dosing. Those that are available show that very high levels of varroacides may be present within colonies and are regularly detected in live bees The risk from most mixtures can be assessed using the additive approaches of concentration addition (or dose addition) and independent action (IA). In identifying the relevance of synergy in determining the toxicity of mixtures it is important to understand the route of metabolism of pesticides in honeybees and the effects of age, season etc on this. The role of oxidative metabolism in detoxification of the cyano-substituted neonicotinoids in bees is highlighted by the increase in toxicity of acetamiprid and thiacloprid in combination with the EBI fungicides. The majority of synergistic effects observed in honeybees have been ascribed to inhibition rather than induction of P450s involved in pesticide metabolism. The level of exposure to the synergist also affects the scale of the synergy. The effect of exposure on the scale of synergy is important as many of the laboratory studies have been undertaken with high doses of synergists, e.g. 3-10 µg/bee and at more realistic exposure levels such high increases of toxicity have not been observed even under laboratory conditions. Contact and oral dosing with combinations of a range of EBI fungicides at more realistic exposure levels with neonicotinoid insecticides showed only low levels of synergy. There are no reports of interactions between varroacides and neonicotinoid pesticides but recent data suggest that antibiotics used in colonies may affect susceptibility to both varroacides and other pesticides

Neonicotinoid pesticides and bees Report to Syngenta Ltd

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2.4 Interactions between neonicotinoids and disease See EFSA 2012 http://www.efsa.europa.eu/en/supporting/pub/340e.htm. Honeybees are known to suffer from a wide array of bacterial, fungal and viral pathogens as well as ecto- and endo-parasite. Multiple infections are common and the impact of some pathogens can be far higher in the presence of other associated pests and diseases. Honeybees have a well-developed immune system for coping with bacterial and fungal infections although their immune response to viral pathogens is less well understood and there has been significant interest recently on the potential for pesticides to affect the susceptibility of bees to diseases. This has been highlighted by high pesticide residues reported in honeybee colonies in the USA and the importance of microbial communities within the hive which may be affected by pesticide residues as well as impacting on the bees directly. The dense crowding within social and eusocial bee colonies together with the relatively homeostatic nest environment with stored resources of pollen and nectar/honey results in conditions conducive to increased susceptibility to disease. This has resulted in the evolution of both individual and social immunity in the honeybee and bumble bee Factors other than pesticides can impact on the immune system in honeybees and increase susceptibility to disease, e.g. other diseases, the immunsuppressive effects of Varroa destructor, antibiotics, sulphonamides and metals and immunostimulators have been proposed. Confinement of colonies can result in immune suppression and oxidative stress in colonies and poor habitat quality may result in lowered immune response. One key factor is that although colonies show qualitatively similar immune responses the colony is a significant factor in the level of the response, i.e. there are large variations between colonies on the level of the response. There have also been suggestions that stress, e.g. isolation, weakens individual immunocompetence. This may explain why some immune competence effects are evident in under laboratory conditions but not in colonies. There have been conflicting reports over the interactions between imidacloprid and Nosema which are confounded by the effects of Nosema on the energetic of individuals and results in significantl;y increased food intake. A similar issue was identified in an increase in mortality was observed when bees (five days after emergence) were infected with 125,000 spores of N ceranae and after a further 10 days were exposed to sublethal (LD50/100) doses of thiacloprid or fipronil for 10 days. The bees clearly showed energetic stress after Nosema infection by their increased consumption of sucrose with infected bees consuming approximately twice the amount of sucrose compared with uninfected bees. Although there was no apparent difference in overall daily intake when exposed to the insecticides, there were large difference in intake on day 1 of the pesticide exposure period There was one report (Aufauvre et al 2012) identified which suggests that N ceranae may increase the susceptibility of bees to fipronil but there were inconsistencies in the reported results. The data do show the variability of mortality caused by N ceranae: N ceranae mortality ranged between 22 and 39% when dosed on day 0 and between 22 and 37% when dosed on day 7 and fipronil resulted in 29-31% mortality whether dosed from day 0 or day 7. It is also vital to understand the disease status of individuals used in pesticide assessments since both the honeybee (Apis mellifera) and the bumble-bee (Bombus terrestris) perform poorly in proboscis extension reflex (PER) memory tests when their immune systems were challenged by lipopolysaccharide. Neonicotinoid pesticides and bees Report to Syngenta Ltd

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Honeybees are the target of a large number of viruses with a total of 18 identified to date. Often virus vectoring by Varroa, which is a significant stressor in honeybee colonies by feeding on the haemolymph causes a variety of physical and physiological effects on the colony, and results in infections from viruses which are otherwise present as covert infections resulting in severe disease and mortality within the colony. In addition viruses can be transmitted within the colony by trophallaxis, contact, faeces and salivary gland secretions. However to date there are no reports of interactions between neonicotinoids and viruses

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2.5 Exposure of bees to pesticides See EFSA 2012 http://www.efsa.europa.eu/en/supporting/pub/340e.htm. Conclusions Bees are exposed to pesticides via a number of routes and the relative importance of each depends on the life stage of the insect and the mode of application of the pesticide. Adults may be exposed directly to pesticides through direct overspray or flying through spray drift, by consumption of pollen and nectar (which may contain directly over-sprayed or systemic residues), by contact with treated surfaces (such as resting on recently treated leaves or flowers), by contact with dusts generated during drilling of treated seeds, or by exposure to guttation fluid potentially as a source of water or as dried residues on the surface of leaves. The exposure of larvae is primarily via processed pollen and nectar in brood food. Data available in the literature includes residues in pollen, wax and nectar within colonies, pollen and nectar residues from plants, in pollen loads on bees returning to the hives and in adult workers. Such data also includes the residues of veterinary medicines detected and the distribution of chemicals around the hive. The routes of exposure of bees to pesticides has been assessed and recently reviewed in an EFSA Scientific Opinion particularly in relation to quantifying uptake and extended to include other non-Apis species where data were available. The exposure of bumble bees to pesticides has also been reviewed and showed there are key times in the year when exposure of queens may be particularly important in determining the fate of a colony. A review of residues in bees after pesticide applications in the EFSA review (2012) provides evidence of the exposure of bees to applications aggregated through all routes of exposure, i.e. through direct overspray, foraging on treated crops and consumption of treated food and water as samples were collected over time after exposure. This showed peak residues in the first sample after application with declines for spray applications over the following week. No data from systemic seed or soil application field studies were available but residues of imidacloprid and its metabolite 6-chloronitoctinic acid and fipronil and its metabolites were detected at low levels in monitoring studies Studies on residues deposited as dust containing neonicotinoids are summarised obviously this does not take into account recent EU requirements to limit dust emissions through the use of professionally treated seeds and deflectors. Drift during agricultural treatment determines the deposition of pesticides within a small distance from the field edge. What is less obvious is how to calculate the drift onto flowering weeds in the field margin as dust drift is unlikely to behave in the same way as spray drift due to the wide variations in particle size. Unfortunately the deposition data generated to date for grasses and flowering weeds in flower margins have limitations in terms of the reporting of sowing rates and seed treatment rates. Contact of bees with treated surfaces may occur through resting on treated leaves or during foraging on treated flowers. The major issue is that bees do not come into contact with the treated plant over the entire surface of their body but primarily through their feet; however

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during resting and cleaning they may transfer residues from their feet to other parts of their bodies. The exposure of bees to pesticides in pollen depends on both the residues present and the amounts of pollen collected by the bees. The amount of pollen collected by a colony per day is highly variable and depends on pollen availability, crop species and the needs of the colony. On oilseed rape the amount of pollen collected varied with the stage of flowering with most collected in the latter stage. Bee bread is pollen processed from the pollen loads by bees for storage by combining with nectar or honey and addition of antimicrobial agents. This results in higher residues in bee bread than in pollen which may relate to differences in availability for residue analysis following processing of the pollen by bees. Flower morphology is an important factor in the pesticide content of nectar: flowers in which the nectar is deeper, such as clover, were less contaminated than shallower flowers such as cabbage and nectar yield/flower was less important in determining pesticide content. To date, there are no reports of pesticide residues in aphid honeydew after spray application but the intake by bees may be expected to be similar to that of nectar sources. Residues in honey formed from contaminated nectar and stored within the hive will depend on the concentration of nectar through evaporation of water to produce honey and degradation of residues through biological and chemical factors in honey. Both factors are slow and counter each other to some extent and there are differences between honey contained in open and sealed cells. The residues of neonicotinoids pesticides detected in stored nectar and honey in field studies and available monitoring data for samples taken directly from colonies are summarised. Monitoring data for processed honey has been excluded as honey is combined from a large number of colonies and therefore residues may be diluted. For pesticides (not acaracides) the residues detected in the monitoring studies are lower than those reported in field studies. Water is collected by honeybees to dilute thickened honey, to produce brood food from stored pollen, to maintain humidity within the hive and to maintain temperature within the brood area. Water is not stored in combs by temperate bee colonies. The amount of water required depends on the outside air temperature and humidity, the strength of the colony and the amount of brood present. The production of water by evaporation of nectar to form honey may address at least some of this need. Water consumption by honeybee colonies has been assessed using confined of colonies provided with a source of water within the hive. To date there have been no published studies that demonstrate significant exposure of bees to guttating crops as a source of water in the field. Guttation fluid is unlikely to be identified by honeybees as a source of sugar due to the low levels present. Bees are less subject to dessication than most terrestrial insects due to their nectar diet and high metabolic water production Beeswax is produced by worker bees within the colony to house stores of nectar and pollen and for brood production. Production begins when the worker is slightly less than one week old, peaking at around two weeks and then reducing. It takes between 24 and 48 hours for any particular honeybee worker to produce a moderate-sized wax scale. If unchanged by a beekeeper wax within the colony may accumulate lipophilic residues over time both from Neonicotinoid pesticides and bees Report to Syngenta Ltd

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contaminated pollen and nectar brought into the hive and from chemicals used within the hive, e.g. varroacides. There are no reports of neonicotinoids in beeswax from colonies Propolis is collected by bees as resin from trees, e.g. buds, primarily poplars and pine trees and is used within the hives to block small gaps and as a defense at the hive entrance against ants etc. and also as an anti-bacterial antifungal agent within the hive. The main propolis plants in Europe are poplar, birch, oak, alder, willow and hazel. Foragers collect the resin in their pollen baskets to return it to the hive and can carry approximately 10 mg. The chemical composition of propolis varies between sources but is a mixture of resins, terpenes and volatiles. Due to the range of sources of propolis and storage within the hive it can contain a range of contaminants but only a small number of reports exist of trace residues of pesticides present in propolis collected from colonies and propolis tinctures prepared from this and no reports of neonicotinoid pesticides There are three possible sources of inhalation exposure of bees to pesticides. During applications of pesticides (is a similar manner to flying through spray), through vapour generated from residues on the crop after application and from stored pollen and nectar within the hive (and potentially water evaporated within the hive). There are no reports of exposure associated with inhalation of neonicotinoid pesticide residues. Nectar collected by foragers from plants is transferred to in-hive bees at the colony entrance which then to further bees for transport to storage or brood combs. During spring and summer large quantities of nectar are stored for use in periods of shortage, e.g. during breaks in nectar flow, periods of poor weather, or for over-wintering. Nectar is placed both in storage combs and also in brood combs close to larvae so it is readily available for brood rearing. The majority of published studies relate to in-hive treatments with varroacides and antibiotics and solely measured residues in honey intended for human consumption. However, there are a small number of studies which specifically address the distribution of incoming contaminated nectar within hives, including that releasing just six foragers fed with radiolabelled sμgar into a colony resulted in about 20% of the workers in the brood area receiving some labelled food within 3.5 hours and this included nurse bees which demonstrated the potential exposure of brood. The nectar delivered to brood comb is used rapidly by nurse bees to feed larvae. For spray applications the residue per unit dose (RUD) can be calculated and used to determine the relative amounts of a pesticide available through each routes of exposure. The data for all routes of exposure is currently limited and would benefit feom a larger dataset. For seed treatments and soil applications the data available for calculation of an RUD approach is far more limited and there are a number of issues which require additional research, e.g. crop dependence, concentration dependence and active ingredient dependency of the RUD. Overspray can be related to the surface area of the bee which suggests the RUD for honeybees should be increased but although the surface area of bumble bees is likely to increase significantly due to their greater size they are also far more variable in size making any predictions unreliable. For bumble bees intake data are far more limited than for honeybees but some data are available for adults from queenless microcolonies under laboratory conditions. For larvae intake of sucrose is unclear but an approximation is available. However, the intake of foragers is not reported and therefore the data only relate to intake for metabolic Neonicotinoid pesticides and bees Report to Syngenta Ltd

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requirements. There data can be used to identify possible RUDs but limited confidence can be held in these. There was insufficient data available to assess the exposure of solitary bee species.

2.6 Sublethal and chronic effects of neonicotinoids A large number of studies have been undertaken ion the sublethal and chronic effects of neonicotinoid pesticides on bees. These are summarised in Tables 37-47 and show the wide range of dosing approaches and endpoints reported in both Apis and non-Apis (primarily Bombus) species. Data for fipronil are included in the tables for completeness but as there are no data available for residues in nectar the data are not discussed further. 2.6.1 Honeybees The reported effects of neonicotinoids on honeybees under laboratory conditions is shown in Table 11 to Table 14. By far the majority of the reported literature relates to imidacloprid.. Table 15 to Table 18 summarise the semi-field and field studies reported with neonicotinoid insecticides and fipronil again the majority of the studies relate to imidacloprid given the concerns about the limited dataset for the RUD the rates used have been compared in Figure 3 and Figure 4. These show that a large number of dosing studies have been conducted at dose rates and concentrations in excess of the reported maximum concentrations for imidacloprid and thiamethoxam in nectar following use as seed treatments. Where effects were observed at or below rates close to this value (studies 4, 11,19 and 31 for imidacloprid) three were related to biomarkers such as acinus diameter in the hypopharangela gland, the proboscis extension reflex to sucrose, and one (Belien et al 2010 a short summary paper where detailed data were not available) was associated with an adverse colony level effect at 1 µg/Kg. There were far fewer studies with thiamethoxam and none reported effects at or below the maximum field nectar residue reported (5.2 µg/Kg) following seed treatment. 2.6.2 Bumblebees Table 19 to Table 21 summarise studies undertaken with non-Apis species. The vast majority have been undertaken with bumble bees and Figure 5 shows that in many imidacloprid was used at concentrations in excess of the maximum rate reported in nectar although the study by Whitehorn et al (2012) using colonies dosed in the laboratory and Laycock et al (2012) using worker only microcolonies does report effects following dosing at field realistic rates. Figure 6 highlights that only a small number of studies have been undertaken in bumble bees with clothianidin and thiamethoxam and they do not show effects at field realistic rates. 2.6.3 Conclusion A large number of studies have been undertaken on the sublethal and chronic effects of neonicotinoid pesticides on bees using a number of different exposure scenarios and endpoints and by far the majority of the reported literature relates to imidacloprid that a large number of dosing studies have been conducted at dose rates and concentrations in excess of the reported maximum concentrations for imidacloprid and thiamethoxam in nectar following use as seed treatments. Where effects were observed at or below rates of imidaclorpid close to the maximum reported in nectar only one appears to be a nonbiomarker effect at the colony level. There were far fewer studies with thiamethoxam and Neonicotinoid pesticides and bees Report to Syngenta Ltd

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none reported effects at or below the maximum field nectar residue reported following seed treatment. The vast majority of studies with non-Apis species have been undertaken with bumble bees and again in the majority imidacloprid was used at concentrations in excess of the maximum rate reported in nectar although 2 studies report effects following continuous dosing at field realistic rates. Only a small number of studies have been undertaken in bumble bees with clothianidin and thiamethoxam and they have not been reported to show effects at field realistic rates.

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Figure 3 Comparison of reported effect and no effect levels for imidacloprid in laboratory, semi-field and field honeybee dosing studies with a) maximum reported concentrations in nectar following use as a seed treatment (1.9 µg/Kg) or b) with calculated exposure of foragers based on this value (0.14 ng/bee) (intake rates per day have been divided by the expected 10 foraging trips per day (Rortais et al 2005) to allow comparison with exposure to a single dose (foragers do not consume pollen). (Study numbers refer to Table 12 and Table 16)

a) 1000000 100000

ug imidacloprid/Kg

10000

1000 no effect

100

effect

10

field nectar max

1 0.1 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

0.01 0.001

study

b) 10000

ng imidacloprid/bee

1000

100 no effect

10

effect 1

field nectar max

0.1 0.01

41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 study

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Figure 4. Comparison of reported effect and no effect levels for thiamethoxam in laboratory, semi-field and field honeybee dosing studies with a) maximum reported concentrations in nectar following use as a seed treatment (5.2 µg/Kg) or b) with calculated exposure of foragers based on this value (0.4 ng/bee)(intake rates per day have been divided by the expected 10 foraging trips per day (Rortais et al 2005)) (foragers do not consume pollen) to allow comparison with exposure to a single dose. (study numbers refer to Table 13 and Table 17)

a)

ug thiamethoxam/Kg

10000

1000

no effect

100

effect field nectar max

10

1 0

1

2

3

4

5

6

7

study

b) 3.5

ng thiamethoxam/bee

3 2.5 2 no effect

1.5

effect

1

field nectar max

0.5

0 0

1

2

3

4

5

6

7

study

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Figure 5. Comparison of reported effect and no effect levels for imidacloprid in laboratory, semi-field and field bumble bee dosing studies with maximum reported concentrations in nectar(1.9 µg/Kg) and pollen (36 µg/Kg) following use as a seed treatment to allow comparison with exposure to a single dose (study numbers are shown in Table 20). Limited amounts of pollen are used by workers to construct nests and feed larvae 100000

imidacloprid ug/Kg

10000 1000

no effect

100

effect 10

field nectar max

1

field pollen max 0

2

4

6

8

10

0.1 0.01

study

Figure 6 Comparison of reported effect and no effect levels for thiamethoxam/clothianidin in laboratory, semi-field and field bumble bee dosing studies with maximum reported concentrations in nectar(5.2 µg/Kg) and pollen (51 µg/Kg) following use as a seed treatment to allow comparison with exposure to a single dose study numbers are shown inTable 21). Limited amounts of pollen are used by workers to construct nests and feed larvae. . thiamethoxam/clothianidn ug/Kg

1000

100 no effect effect 10

field nectar max field pollen max

1 0

1

2

3

4

study

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Table 11 Overview of laboratory studies of the effects of sublethal and chronic exposure to acetamiprid and thiacloprid on Apis bees

Expected maximum exposure of foragers (EFSA 99th percentile based on 20% sugar in nectar expressed as µg/bee/day) if the application is at 100 g ai/ha is also provided for comparison Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

/ Notes

Reference

Acetamiprid ‘Bees’

-

-

-

-

Effects on communication and finding food reported

Maccagnani et al (2008)

Acetamiprid (99%) Apis mellifera

Lab study / oral + contact / adults

1, 0.1 µg/bee/d (oral, contact)

11d (from emergence)

Behavioural functions

Acetamiprid (99%) Honeybees

Lab study / oral + contact exposures / adults

0.1, 0.5, 1µg/bee

Locomotor activity 1h after exposure

(i) Locomotor activity

Contact: 1 µL (10% solvent)

Sucrose sensitivity: 1 h before and after treatment

Oral dosing at 0.1 µg/bee significantly increased responsiveness to water. No other significant effects were seen and acetamiprid induced no effect on learning and memory (ii) Significant increase in distance moved for contact applications at 0.1 and 0.5 µg/bee only.

Information from abstract, paper in Italian Doses selected to fall between 1/5 and 1/500 of LD50

(ii) Sucrose sensitivity

(ii) Significant decrease in sucrose responsiveness at 0.1 and 0.5 µg/bee following oral exposure only. Significant dose related increased PER to water at all contact doses.

(iii) Olfactory learning

(iii) PER significantly reduced following oral exposure at 0.1 µg/bee after 48h only. No effect of contact exposure. Mortality at 10d post

Oral: individually dosed in 10 µL 40% (w/v) sucrose

Thiacloprid

Lab/oral/15d

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5.1 mg/L (in

Olfactory learning: treatment 3 h before conditioning and recording 1 h, 24 h, 48 h after

10d

Mortality rate

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Aliouane et al (2009).

El Hassani (2008)

Vidau et al

Compound tested Species

Study type

Test dose

Test duration

Apis mellifera

postemergence (10d post infection)

50% sucrose, 1% protein, 0.1% DMSO)

exposure/observa tion period

[infected with Nosema ceranae 125,000 spores diluted in 3 mL of water - at 5d post emergence]

(1/100 LD50)

[tested both with and without Nosema ceranae infection]

Endpoints

Results

/ Notes

infection (thiacloprid + Nosema) was 71% while for the infected only group (no pesticide) the mortality was 47%. Thiacloprid only bees (no infection) did not differ from untreated controls.

Table 12 Overview of laboratory studies of the effects of sublethal and chronic exposure to imidacloprid on Apis bees Compound tested Study type Test dose Test Endpoints Results Notes (study duration reference in figure Species 20) Imidacloprid ‘Chronic feeding ‘Sublethal’ Sensitivity to No indications of Information from ‘Bees’ study’ imidacloprid significant differences in abstract, only when also sensitivity to imidacloprid abstract available stressed by between bees under other (1) Varroa stressors and control destructor, bees Nosema apis, drugs or lack of pollen supply. Imidacloprid Lab (flight 48µg/kg (i) 4d (i) Syrup (i) Significant reduction (2) (98%) cage)/oral / (syrup) (treated) consumption during treatment phase Apis mellifera workers (ii) 4d (ii) Foraging (ii) Significant reduction in (treated) activity during treatment (mean Neonicotinoid pesticides and bees Report to Syngenta Ltd

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Reference

(2011)

Reference

Wehling et al (2009)

RamirezRomero et al (2005)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Notes (study reference in figure 20)

Reference

(3)

Guez et al (2001)

(4)

Guez et al. (2003)

4.8 visits) and post treatment (mean 20.4 visits) phases c.f. before treatment (mean 23.7 visits).

Imidacloprid Apis mellifera

Imidacloprid metabolites (olefin, 5-hydroxyimidacloprid, 4,5dihydroxyimidacloprid, ureaimidacloprid, denitro-imidacloprid, and 6-chloronicotinic acid) Apis mellifera

Lab study / contact exposure / adults

Lab study / contact exposure / adults

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.1, 1, 10 ng/bee

(i) 1 ng/bee (+ further tests at 0.1ng/bee for olefin and 5hydroxyimidacloprid)

(iii) 2d (treated)

(iii) Olfactory learning

15 min, 1 h, 4 h after application

Habituation (PER)

(i) 15 min, after application

Proboscis extension reflex (PER)

(iii) Non-significant reduction in visits to scented sites during treated period c.f. before and after treatment. In 7d old bees: imidacloprid at 10ng/bee significantly increased the number of trials required In 8-day-old bees imidacloprid decreased the number of trials required at 15min and 1h but increased at 4h (i) In normal conditions PER requires more days in older (8 - 10 days) than younger (4 - 7 days) bees In 7-day-old bees: only olefin increased the number of trials required (also at 0.1ng/bee). In 8-day-old bees only 5hydroxy-imidacloprid significantly decreased the number of trials

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Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Notes (study reference in figure 20)

Reference

(5)

Lambin et al (2001)

(6)

Suchail et al (2001)

required (not significant at 0.1ng/bee) while olefin significantly increased the number of trials required as in 7 d old bees(also at 0.1ng/bee).

Imidacloprid Apis mellifera

Lab study / contact exposure / adults

(ii) 1, 10 ng/bee 5hydroxyimidacloprid

(ii) 1h after application

1µl at 1.25, 2.5, 5, 10, 20 ng/bee

0, 15, 30 and 60 mins after treatment

(i) Gustatory function

(ii) Motor activity

Imidacloprid (Technical >97%) + metabolites

Lab study / oral acute & chronic exposures / adults

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.1, 1, 10 µg/L for 10 days and

10 days 8 days

(iii) Nonassociative learning abilities (PER) Mortality; hyperresponsiveness, hyperactivity,

(ii) Significant increase in number of trials required at both doses (suggests that effect not due tohydroxy-imidacloprid but its metabolites – most likely olefin). (i) 5 ng/bee and above had a significant effect on the gustatory function (ii) 2.5ng/bee and above significantly impaired motor activity in open field (these effects are amplified over time). Motor activity was significantly enhanced at 1.25ng/bee (iii) PER habituation was significantly facilitated at doses below 2.5 ng/bee 50 % mortality after 8 days

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Compound tested Species

Study type

5hydroxymidacloprid; 4,5dihydroxyimidaclopri d; Desnitroimidacloprid , 6-chloronicotinic acid; Olefin; Urea derivative Apis mellifera

Test dose

Test duration

cumulative dose 0.01; 0.1; 1 ng/bee after 8 days (in 50% w/v sucrose solution)

Endpoints

Results

trembling

Imidacloprid LD50 = 57 ng/bee after 48 h, 37 ng/bee after 72 & 96 h

Notes (study reference in figure 20)

Reference

(7)

Decourtye et al (2003)

LD50 5hydroxymidacloprid > LD50 imidacloprid (258, 206, 222ng/bee) LD50 Olefine < LD50 imidacloprid (28, 29, 23ng/bee) Toxicity of other metabolites >1000ng/bee 48, 72, 96h

Imidacloprid (99.4%) Apis mellifera ligustica

Lab study / oral exposure / adults

(5-OH-imidacloprid (99.4%) also tested)

1.5, 3, 6, 12, 24, 48µg/kg in 50% sucrose solution (extra dose of 96µg/kg used in experiment on summer bees) Concentration s based on intake of 33

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Exposed 11d from day 2 to 1415.

Mortality, PER, foraging

All compounds toxic in chronic 10d test with mortality after 72h. Significant increase in mortality after 11d at 48µg/kg in winter bees and 96µg/kg No significant effect on reflex response in winter bees. Significant reduction in response at 48and 96µg/kg Learning performance of winter bees significantly

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Compound tested Species

Study type

Test dose

Test duration

Endpoints

µL/bee/d 2 - 32 ng/bee

Imidacloprid (98%) Apis mellifera L.

Lab study / oral exposure / adults

12 and 0.12 ng/bee (25mg/L and 250µg/L in 30% sucrose solution with 0.5µL fed to each 15-6d old bee with micropipette)

Imidacloprid Apis mellifera carnica

Lab study / oral exposure / adults

Imidacloprid (Confidor) Apis mellifera

Lab study / oral exposure / adults

Imidacloprid

1 ppb (1/10 LD)

Lab study / oral

Neonicotinoid pesticides and bees Report to Syngenta Ltd

100, 500 ppb at single dose (20µL) and ad libitum (in 50% sucrose)

500ng/kg (in

Short (30 s to 3 min) mid (15 to 60 min) and long term (24 h)

Sampled treated 7d old bees 1d and7d after treatment Recorded at 0 - 30 mins; 30 - 60 mins; 1 - 2 h; 6.5 7 h; 23 23.5h

Exposure24

Olfactory learning (PER)

Results

affected at 48µg/kg. In summer bees significant impairment occurred at 12µg/kg and all higher doses Significant increase of cytochrome oxydase (CO) in mushroom bodies.

Notes (study reference in figure 20)

Reference

(8)

Decourtye et al (2004a)

(9)

Heylen et al (2011)

(10)

Medrzycki et al (2003)

(11)

Smodis Skerl

No significant on PER to sucrose solution alone.

Size and morphology of HPG on 8- and 14-days-old bees Activity

Hypopharyngeal

Impairment of medium term olfactory learning at highest dose No effect on short (0 s - 3 min) and long (24 h) term of OL No effects

Mobility significantly reduced with effects starting at 30 - 60 min after ingestion and disappearing after a few hours Suggested effects could result in reduced capacity to communicate. Significant HPG acinus

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Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

(Gaucho) Apis mellifera carnica

exposure / adults

35% sugar water)

, 48 or 72 h

diameter variations at all treatment periods.

Imidacloprid metabolites (urea NTN and 6-CAN) Apis mellifera

Lab study / oral exposure / Adults of two age cohorts (emerging versus foragers) Lab/contact exposure/worker bees

0.1, 1 and 10 µg/L (in 50% sucrose)

24 h, 48 h, 10 days

glands (HPG) acinus diameter in 1-6, 7-12, 1318, 19-32d old bees Mortality, knock down, staggering, responsiveness

No significant effects found.

(12)

Schmuck (2004)

(i) Topical application (1µl) 1.25, 2.50, 5ng/bee

(i) 15, 30, 60mins

(i) Gustatory threshold

(i) No effects at 1.25 or 2.50 ng/bee Loss of sensitivity after 60mins at 5ng/bee

(13)

Armengaud et al (2002)

(ii) Topical application (1µl) 1.25, 2.50, 5ng/bee

(ii) 15, 30, 60mins

(ii) Locomotion

(ii) Increase in displacements at 1.25ng/bee Significant increase in locomotion at 2.50ng/bee after 15min Significant decrease in displacements at 5ng/bee after 30mins (loss of motor coordination with no behavioural recovery after 2h).

(iii) Topical application (1µl) 1.25ng/bee

(iii) 15, 30, 60mins

(iii) Habituation (proboscis extension reflex - PER)

(iv) 30mins

(iv) CO

Imidacloprid (98%) Apis mellifera

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(iii) Fewer trials to display PER at 1.25ng/bee. No effect of time.

Page 56 of 133

Notes (study reference in figure 20)

Reference

and Gregorc (2010)

Compound tested Species

Study type

Test dose

Test duration

(iv Intracranial injection of 0.5µl (10-8, 10-6 or 10-4M imidacloprid) at the brain surface

Endpoints

Results

histochemistry

(iv) Glomeruli. Significant increase in staining (+8% to +17%) of two regions of glomeruli in line with dose.

Notes (study reference in figure 20)

Reference

(14)

Alaux et al (2010)

α lobe. Reduction of CO labelling at 10-8M, increased labelling at other doses with significant increase at 104M (maximum increase of 23% for dorsal layer B1) Calyces. Significant reduction in labelling at 10-8M at lip and basal ring. Significant reduction at 10-6M in basal ring only. Significant increase for both at 10-4M.

Imidacloprid Apis mellifera

Lab/oral/adult

0.7, 7, 70µg/kg Exposure method not defined.

Neonicotinoid pesticides and bees Report to Syngenta Ltd

10d

(i) individual energetic demands

Central body. Significant increase in staining in both upper and lower divisions at 10-4M. Opposite effects at lower doses. (i) Imidacloprid alone - no effect Imidacloprid + Nosema – significant increase in energy stress (sucrose

Page 57 of 133

Compound tested Species

Study type

Test dose

Test duration

Endpoints

(ii) individual immunity (iii) social immunity

Lab/oral/adult

0.05% to 0.00005%

Imidacloprid Apis mellifera ligustica L.)

Lab/oral/adults

LD50/100LD50/10

Imidacloprid Honeybees

Lab/oral/workers

4 and 8 µg/L in syrup (500g/L

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Notes (study reference in figure 20)

Reference

(15)

Moise et al (2003)

Deltamethrin also tested (16) Information taken from abstract, paper in Chinese Mean consumption of syrup per bee

Song et al (2011)

consumption) over control, imidacloprid and Nosema only groups

(control, imidacloprid only, Nosema only, Nosema + imidacloprid)

Imidacloprid Apis mellifera carpatica

Results

2 to 300min

Behaviour (mortality)

Olfactory sensitivity Proboscis extension reflex (PER) 60d (chronic)

Survival time

(ii) No effect on haemocyte number or phenoloxidase. (iii) Activity of glucose oxidase was significantly decreased by the combination of both factors compared with control, Nosema or imidacloprid groups, suggesting a synergistic interaction and reduced ability to sterilize colony and brood food. Negative effects reported included apathy, laboured breathing, un-coordination and convulsion reported but no details of doses etc. except for mortality. No effect on olfactory sensitivity assays but increased the waterinduced PER

Significant reduction in survival time. Survival

Page 58 of 133

Dechaume Moncharmont

Compound tested Species

Study type

Test dose

Test duration

Endpoints

sucrose) [ Imidacloprid Apis mellifera

Imidacloprid (analytical standard) Apis mellifera ligustica

Lab/oral/workers

Lab/oral/workers

(i) 4, 8, 40ppb in diet

(i) 11d

(i) Olfactory conditioning (PER)

(ii) 50 ppb imidacloprid in 50% saccharose solution 0.21ng/bee (24ppb) or 2.16ng/bee (241ppb) in 56% sucrose

(ii) 13d exposure

(ii) Flight activity (flight cage)

1h

Sucrose response (PER)

(each bee fed 7µL imidacloprid in 2.0mol/L sucrose solution using micropipette)

Imidacloprid (analytical standard) Apis mellifera

Lab/oral/workers (hives placed in

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.21ng/bee (24ppb) in 56%

Results

time was 28.3±5.6d (mean ± standard error) at 4 µg/L and 31.3±4.1d at 8 µg/L (i) Significantly higher mortality at 8 and 40ppb. Significantly lower performance in conditioning test at all doses.

Notes (study reference in figure 20) was 20±0.95 µl/d (17)

Reference

(18)

Decourtye et al (2001)

(19)

Eiri and Nieh (2012)

(20)

Eiri and Nieh (2012)

et al (2003)

(ii) Decreased flight activity and olfactory discrimination. Nectar foragers: significant increase in sucrose response threshold (SRT) at both dose levels Pollen foragers: significant effect on SRT at highest dose only.

24h

(i) Visits to feeder

Significant reduction in total PER/bee at both doses in nectar foragers and highest dose in pollen foragers. (i) No significant treatment related effect.

Page 59 of 133

Compound tested Species

Study type

Test dose

ligustica

temperature controlled room with sucrose feeder (50% w/w) 1.5m from hive entrance)

sucrose

Imidacloprid Apis mellifera L.

Imidacloprid Apis mellifera L.

Lab/oral/workers

Lab/oral/workers 12d old at testing

(each bee fed 7µL imidacloprid in 2.0mol/L sucrose solution using micropipette) 48ng/g in treated pollen (water, honey and pollen 1:2:7 by weight)

Test duration

7d exposure

48ng/g in treated pollen (water, honey and pollen 1:2:7 by weight)

7d exposure, 1d starvation, 1d test

Imidacloprid (99.8%) Apis mellifera ligustica

Lab/oral/workers

4 or 8µg/L in sucrose solution

60d

Imidacloprid Apis mellifera L.

Lab/oral/workers

0.05, 0.5, 5.0, 50 or 500ppb in sucrose

26h

(Video-tracking study) Neonicotinoid pesticides and bees Report to Syngenta Ltd

Endpoints

Results

(ii) Average number of dance circuits per nest visit

(ii) Significant reduction in waggle dances in imidacloprid treated group

(iii) Unloading wait time

(iii) No treatment related effect on wait time

(i) Mortality during chronic exposure

(i) No significant treatment related effect

(ii) Feeding behaviour

(ii) Significantly reduced pollen consumption over 7d (i) Significantly fewer successful bees un treated group.

(i) Visual learning capacity (Tmaze) (ii) Conditioned PER Sucrose consumption

(i) Activity

(ii) No significant effects on PER No significant effect of treatment (although significantly reduced survival time at both concentrations compared to control). (i) Significant reduction in distance travelled at 50 and 500ppb. No significant effect at lower

Page 60 of 133

Notes (study reference in figure 20)

Reference

(21)

Han et al (2010)

(22)

Han et al (2010)

(23)

Moncharmont et al (2003)

(24)

Teeters et al (2012)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Notes (study reference in figure 20)

Reference

doses. (ii) Time spent in food zone

(iii) Time interacting

(ii) Increased with dose from a mean of 78.98min at 0.05ppb to 587.62 at 500ppb although significantly different from controls (114.68min) only at 50 and 500ppb. (iii) Non significant dose related decrease in interaction time from 106.29min at 0.05ppb to 69.91min at 500ppb (control 147.44min).

Table 13 Overview of laboratory studies of the effects of sublethal and chronic exposure to thiamethoxam on Apis bees Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Thiamethoxam (97%) Apis mellifera

Lab study / oral + contact exposures / adults

1, 0.1 ng/bee/d (oral, contact)

11d (from emergence)

Behavioural functions

Contact exposure induced either a significant decrease of olfactory memory 24 h after learning at 0.1 ng/bee or a significant impairment of learning performance with no effect on memory at 1 ng/bee. Responsiveness to antennal sucrose stimulation was

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 61 of 133

Notes (study reference in figure 21) Doses selected to fall between 1/5 and 1/500 of LD50 (1)

Reference

Aliouane et al (2009).

Compound tested Species

Thiamethoxam (97%) Honeybees

Thiamethoxam Apis mellifera

Thiamethoxam

Study type

Lab study / oral + contact exposures / adults

Lab study / oral exposure / adults

Lab/oral/workers

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Test dose

0.1, 0.5, 1ng/bee

Test duration

Contact: 1 µL

Locomotor activity 1h after exposure

Oral: individually dosed in 10 µL 40% (w/v) sucrose

Sucrose sensitivity: 1 h before and after treatment

6 µg/mL, 3 µg/mL, 1.5 µg/mL, 0.5 µg/mL, 0.05 µg/mL, 0.005 µg/mL

Olfactory learning: treatment 3 h before conditioning and recording 1 h, 24 h, 48 h after 0-, 7-, 14and 21-dayold bees in boxes tests with feeder for 24 h

(mixed with honey 1:1) 3 ng bee

-

Endpoints

Results

(i) Locomotor activity

significantly decreased for high sucrose concentrations (1 ng/bee). (i) No effects on locomotor activity

(ii) Sucrose sensitivity

(ii) No effects on sensitivity

(iii) Olfactory learning

Notes (study reference in figure 21)

Reference

(2)

El Hassani (2008)

(3)

Falco et al (2010)

(4)

Decourtye and

(iii) No effects on ulfacory learning

(i) Mortality,

(i) Age and dose related effect on mortality with higher mortality rates in younger bees

(ii) Acceptance / rejection of food,

(ii) Rejection at high dose and acceptance at low dose

Associative

Only 38% of the treated

Page 62 of 133

Compound tested Species

Study type

Test dose

Test duration

Honeybees

Endpoints

Results

learning between a visual mark and a reward (sugar solution) in a complex maze.

bees negotiated the maze with no mistakes compared to 61% in the control group.

Notes (study reference in figure 21)

Reference

Devillers (2010)

Table 14 Overview of laboratory studies of the effects of sublethal and chronic exposure to fipronil on Apis bees Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Fipronil Apis mellifera

Lab study / contact exposure / adults

0.5ng/bee

3-24h

Learning (PER)

Decreased acquisition success.

Notes

Reference

Bendahou et al (2009)

(c. 1/10 LD50) Subsequent memory performances lowered. No effect on distribution of responses to the tactile stimuli between sides. Fipronil Honeybees

Lab study / exposure through injection on thorax / adults

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.1 or 0.5ng/bee at +20 and +60min

1h, 24h, 48h

Olfactory learning (PER)

Olfactory learning significantly impaired at 0.1ng/bee (1h and 24h only), but not impaired at 0.5ng/bee.

Page 63 of 133

El Hassani et al. (2009)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Notes

Reference

Fipronil (98.5%) Apis mellifera

Lab study / oral + contact exposures / adults

0.1, 0.01 ng/bee/d (oral, contact)

11d (from emergence)

Behavioural functions

Mortality at 0.1ng/bee after 1 week

Doses selected to fall between 1/5 and 1/500 of LD50

Aliouane et al (2009).

Contact treatment at 0.01ng/bee, caused bees to spend significantly more time immobile in an open-field apparatus and to ingest significantly more water.

Fipronil Apis mellifera

Lab study / oral + contact exposures / adults

0.1, 0.5, 1ng/bee (+0.01ng/bee oral for PER)

1 h before and 1 h after treatment

(i) Locomotor activity (ii) Sucrose sensitivity

Contact: 1 µL Oral: individually dosed in 10 µL 40% (w/v) sucrose

Fipronil Apis mellifera

Lab study / oral + contact exposures / adults

Acute:1, 0.5, 0.1 ng/bee Chronic: 0.1,

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(iii) Olfactory learning

Foraging bees (acute toxicity) and emerging

Memory, learning, odour sensitivity

Oral and contact exposure at 0.01ng/bee did not affect learning performance. (i) Oral or contact: no effect on locomotor activity

El Hassani et al (2005)

(ii) Contact: 1 ng/bee significantly decreased sucrose sensitivity 1 h after treatment (iii) Contact exposure at 0.5 ng/bee significantly impaired olfactory learning Contact: acute: 1ng/bee decreases sensitivity to sugar solution (low concentrated)

Page 64 of 133

Gauthier et al (2009)

Compound tested Species

Study type

Test dose

Test duration

0.01 ng/bee

bees (chronic) for 11 days

Endpoints

Results

Notes

Reference

0.5ng/bee affect learning and memory (PER) Chronic exposure at 0.1ng/bee leads to 100% mortality, at 0.01ng/bee (contact) reduction in locomotion and increase in water consumption;

Fipronil (98.5%) Apis mellifera ligustica

Fipronil (Reagent grade) Africanized Apis. mellifera

Lab study / oral exposure / adults

0.075, 0.15, 0.3ng/bee/d (2.2, 4.5, 9 µg/L in sucrose solution allowing 33µL/bee/d)

Lab study / oral exposure / larvae

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Highest dose 1/20 LD50 0.1, 1 µg/g (in food, 10g royal jelly,7.4ml distilled water, 1.4g Dglucose, 1.4g D-fructose and 0.2g yeast

2 to 14-15day-old bees treated daily during 11d

Proboscis Extension Response PER on 14-15d bees

3d

Larvae mortality, morphological alterations of midgut

0.01ng/bee (contact or oral) decreases odour discrimination Dose related mortality 40.6, 87.3 and 91.1% (control 6.6%).

Decourtye et al (2005)

Significantly lower conditioned PER response at middle dose for test 4 (highest dose not tested).

No significant effect on survival Morphological alterations in mid-gut.

Page 65 of 133

Cruz et al (2010)

Compound tested Species

Study type

Test dose

Fipronil Apis mellifera

Lab/oral/15d postemergence (10d post infection)

extract) 1 µg/L (in 50% sucrose, 1% protein, 0.1% DMSO)

[infected with Nosema ceranae 125,000 spores diluted in 3 mL of water - at 5d post emergence]

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(1/100 LD50)

Test duration

Endpoints

Results

10d exposure/o bservation period

Mortality rate

Mortality at 10d post infection (fipronil + Nosema) was 82% while for the infected only group (no pesticide) the mortality was 47%. Fipronil only bees (no infection) did not differ from untreated controls.

Page 66 of 133

Notes

Reference

Vidau et al (2011)

Table 15: Overview of semi-field and field studies of the effects of sublethal and chronic exposure to acetamiprid and thiacloprid pesticides on Apis bees Compound tested Study type Test dose Test Endpoints Results Reference Notes Species duration Acetamiprid [Epik], Imidacloprid [Confidor], Thiacloprid [Calypso], Thiamethoxam [Actara] Apis mellifera ligustica

Thiacloprid (480g/L SC) Apis mellifera L.

Semifield /field (spray)/workers (Five tunnels of 19.8m2, one included for the control, sown with a Phacelia tanacetifolia. Each tunnel divided into six plots of 3.3m2. One hive of about 7000±500 bees was positioned inside some days before the spray.) Semi-field (Tunnel) / field / workers

Neonicotinoid pesticides and bees Report to Syngenta Ltd

The investigations were conducted applying one spray per treatment during bloom on three of the six plots in a randomized design.

-

144g/ha (oilseed rape)

Monitored for 7-9d

Repellency to foraging honeybees Mortality and health condition of broods.

The investigation showed a ‘relevant difference’ of the selectivity level of the neonicotinoids. Acetamiprid (Epik) was the least toxic to honeybees.

(i) Foraging activity

(i) Transitory reduction in foraging activity. Normal activity by 48h.

96g/ha (Phacelia tanacetifolia)

(ii) Returning bees

(ii) Returning be numbers reduced for 24-48h after application.

(spray)

(iii) Hive vitality

(iii) No systematic differences found.

Page 67 of 133

Information from English abstract, paper in Italian

Fanti et al (2006)

Schmuck et al (2003)

Table 16 Overview of semi-field, dosed in field and field studies of the effects of sublethal and chronic exposure to imidacloprid pesticides on Apis bees Compound tested Study type Test dose Test Endpoints Results Notes (reference Reference Species duration to study numbers in figures 20 and 21) Imidacloprid Dosed in field 20–100 μg/kg Foraging and Foraging and dances Data from EFSA Kirchner Honeybee waggle dance affected at 20 μg/kg report and reported (1999) in Cure et al (2001) as Kirchner WH (1988) The effects of sublethal doses of imidacloprid on the foraging behaviour and orientation ability of honeybees. Unpublished report, Konstanz, 13pp.) (25) Imidacloprid Dosed in field / 10, 20, 50, Not Communication Deviation from correct Data from Kirchner Cure et al (Gaucho) oral / workers 100ppb (w/v) recorded behaviour of angle information not WH (1988) The (2001) Honeybees in saccharose individually affected. effects of sublethal solution marked bees doses of (feeders 500m Communicated distance imidacloprid on the from hives) information substantially foraging behaviour reduced at 50ppb and and orientation higher. ability of honeybees. Number (%) of bees Unpublished report, performinging trembling Konstanz, 13pp. dances greatly increased (26) at 20ppb and higher Number (%) of bees performing waggle Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 68 of 133

Compound tested Species

Imidacloprid (powder form) Apis mellifera carnica

Study type

Dosed in field / oral exposure (bees treated in lab) / adults

Test dose

0.15, 1.5, 3, 6ng/bee (dosed in 10µL of 2M sucrose solution)

Test duration

Immediately after treatment for 3h and 24 and 48h

Endpoints

Foraging behaviour: number of trips from hive to feeder, duration of trips, time interval between trips

Results

dances substantially reduced at 20ppb and higher At 3ng/bee 95% of bees return to the hive versus 25% at 6ng (among these 5% at 3ng/bee and 75% at 6ng/bee trembling, reduced mobility, cleaning). Visit frequency to feeder significantly reduced immediately after treatment at 1.5 and 3ng/bee with no visits at 6ng/bee. No significant effect at 24, 48h. At 1.5 and 3ng/bee foraging trip duration, flight time to feeder, duration of feeder stay and flight time to hive, interval between foraging trips all significantly increased immediately after treatment but mostly recovered by 24h. Time in hive after treatment significantly extended in 3ng/bee group.

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 69 of 133

Notes (reference to study numbers in figures 20 and 21)

Reference

(27)

Schneider et al (2012)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Imidacloprid (95% TG) Apis mellifera L.

Dosed in field / oral exposure (lab treatment) / adults

40, 50, 100, 200, 400, 600, 800, 1200, 1600, 3000, 4000, 6000µg/L (in 50% sucrose solution)

Continuous recording of feeding intervals for 1h before treatment and for 1.5h after treatment

Time interval between foraging visits.

In controls, time interval is < 300 s; in treated bees time interval is > 300 s at all concentrations above 50µg/L (after 20 min at 50µg/L and after 10 min at 100µg/L). 100% abnormal behaviour at 1200µg/L and above

Returning bees.

Some bees did not return to the feeding site after treatment for at least 1.5 h. At 600, 800, 1,200, and 3,000, 4,000 and 6,000µg/L, the percentages of missing bees were 34.4, 50, 68, 93.3, and 96.9, 100 and 100% respectively. This recovered on the following day at 1600 µg/L and below but were 77.4, 63.6, and 48.4% missing at 3,000, 4,000 and 6,000 µg/L. Calculation of consumption showed possible behavioural disruption at 1.82 - 4.33 ng/bee Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 70 of 133

Notes (reference to study numbers in figures 20 and 21) (28)

Reference

Yang et al (2008)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Imidacloprid (Confidor) Apis mellifera

Dosed in field / oral exposure / adults

100 ppb, 500 ppb, 1000 ppb (in 50% sucrose solution)

Observation s 0-2h, 4-5, and 24h (for 1h) after release

Feeding, foraging and homing behaviours

Feeders avoided at 5001000ppb

Imidacloprid (Confidor 200 SL) Apis mellifera carnica

Dosed in field / oral exposure with syrup in hive / adults + brood

3.55 ng a.i./L

Every 2-7d, after several weeks after treatment

Mortality Number of active bees inside hive

Reference

Fenoxycarb, Indoxacarb also tested (30)

Beliën et al (2009)

Bortolotti et al (2003)

At 100ppb, 2h observation 57% returned to hive, 3% returned to feeder (control 72-80% return, 31-33% feeder). At 5h observation 57% returned to hive, 7% returned to feeder (control 79-87% return, 76-77% feeder). At 24h observation 84% returned to hive, 73% returned to feeder (control 87-90% return). At 500 and 1000ppb none of the bees returned to hive or feeder at any observation. No significant differences between treated and control hives or in foraging activity.

Brood development Colony weight

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Notes (reference to study numbers in figures 20 and 21) (29)

Page 71 of 133

Compound tested Species

Study type

Test dose

Test duration

Imidacloprid Apis mellifera

Dosed in field / oral exposure with syrup in hive / adults + brood

1 ppb?

Every 2 weeks for 10 weeks; foraging behaviour was measured on individual bees of 13, 15, 18, 20 and 25 days-old -

Imidacloprid Honeybee

Imidacloprid Apis mellifera mellifera

Dosed in field /Larvae exposure in hive

Dosed in field /oral/adults

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.4, 24, 200, 2000, 4000, 6000, 8000ng a.i./larva

0.5or 5.0µg/L in saccharose syrup (50g saccharose+5 0ml water)

Exposure 3 times per week 12/07 to 14/08 (last check 21/03)

Endpoints

Results

Notes (reference to study numbers in figures 20 and 21)

Reference

First 6 weeks: normal population size and drop during the next 4 weeks, significantly after 6 weeks. Total number of active bees and caped brood cells decreased after 6 weeks.

Fenoxycarb, Indoxacarb also tested (31)

Beliën et al (2010).

(32)

Yang et al (2011)

Pupation rate

Significant reduction in capped brood, pupation and eclosion rates at 2000ng/larva and above

Eclosion rate (i) Activity (bees/min)

(i) Non significant increase

(33)

Faucon et al (2005)

(ii) Pollen carrying

(ii) Significant increase

(iii) Occupied inter-frames

(iii) No significant effect

(iv) Capped brood area

(iv) No significant effect

Foraging activity Colony parameters (active & dead bees; surface of capped brood, colony weight). Foraging behaviour (phototaxis)

Capped brood rate

Page 72 of 133

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Notes (reference to study numbers in figures 20 and 21)

Reference

(34)

Mayer and Lunden (1997)

(35)

Lu et al (2012)

(v) Hive weight Imidacloprid Honeybee

Dosed in field /oral/adults

(i) 0, 2, 10, 50, 100. 500 ppm (imidaclprid 240 FS in syrup)

(i) Visits to feeders (choice test)

(ii) 4h

(ii) Foraging on dandelions in orchard

(ii) Significant reduction in foraging at higher dose at 0.5h and 1h (59 and 60% reduction respectively). Significant increase in foraging at higher dose at 4h (149% increase).

(iii) 6h

(iii) Foraging on trees and dandelions in orchard

(iii) No significant effect.

13wks exposure

(i) Number of sealed broods during exposure period

(i) Significantly lower than controls but not concentration related

(ii) Colony survival (colony collapse)

(ii) Hive loss began at 18 weeks post exposure. At 23 weeks post exposure only one treated hive remained alive, only one

(ii) 0.05 and 0.112kg a.i./ha (imidaclprid 240 FS in syrup)

Imidacloprid (Technical) Apis mellifera L.

Dosed in field /oral/colony

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(iii) 0.112kg a.i./ha (imidaclprid 240 FS in syrup) 0.1, 1.1, 5.3 or 10.5 µg/kg high fructose corn syrup (HFCS). 2.6kg supplied weekly for 4 wks followed by 9 wks at 20, 40, 200 or 400

(v) No significant effect (i) Avoidance - visits reduced 7% at lower dose and 85% at higher

(i) 2d

23wks observation period

Page 73 of 133

Compound tested Species

Study type

Test dose

Test duration

Endpoints

µg/kg HFCS Imidacloprid Apis mellifera

Dosed in field /oral/colony

10 or 20ppb in Megabee® protein patties

Imidacloprid (98%) Apis mellifera ligustica

Dosed in semi field; (flight cage) studies + feeder / oral (feeding + foraging) and contact (PER) exposures / adults

24 μg/kg (in 50% sucrose solution)

Dosed in semifield (Tunnel) / oral exposure / adults

6 µg/kg (in 40% sucrose solution)

Imidacloprid (analytical) Apis mellifera

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(LOEC for olfactory learning following chronic oral exposure)

Colony exposed for 10 weeks. Newly emerged bees collected at 5 and 8 weeks and fed sucrose containing Nosema spores. Recording visits at scented/uns cented sites every 30s for 5min; bee counter to measure colony activity at the hive entrance in June-July 8 control colonies during 5 days at

Development of Nosema over 12d in newly emerged bees from imidacloprid treated vs. untreated colonies.

Results

control hive (of 4) had died Significantly greater number of Nosema spores per bee in bees from imidacloprid exposed colonies compared to controls

(i) Foraging activity

(i) Decrease in foraging activity on the food source and activity at the hive entrance.

(ii) Associative learning

(ii) Significant effects found in both semi-field and laboratory conditions

Foraging activity at feeders in tunnels

No significant effects on attendance at feeder. Significant decrease in

Page 74 of 133

Notes (reference to study numbers in figures 20 and 21)

Reference

(36)

Pettis et al (2012)

(37)

Decourtye et al (2004b)

(38)

Colin et al (2004)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

different times of the season and 3 colonies before contaminati on and during 4 days after Imidacloprid ‘Bees’

Dosed in semifield (tunnel)/oral/adults (foraging)

(ii) quantity of syrup taken

25µg/kg and higher imidacloprid in sucrose

Imidacloprid (60%, Gaucho FS) Apis mellifera ligustica

Field study / oral exposure / adults + brood

100µg/kg to 3ug/kg imidacloprid in sucrose Seeds treated with 0.24 mg a.i./seed (Sunflower treated at 600 ml/100 kg of

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(iii) duration of visits

Long-term (226 days: 10 days in the field and observation s on the remaining

Notes (reference to study numbers in figures 20 and 21)

Reference

(39)

Colin et al (2001)

the proportion of active bees at the feeder

Foraging activity (i) Frequency of visits to feeding station

50µg/kg and higher imidacloprid in sucrose

Results

Population development and honey production (hive weight, nectar, pollen, brood, honey

(i) Number of bees fell to 0 during the imidacloprid treated phase (1h)

(ii) Quantity of syrup taken declined during the treated phase (1h)

(iii) Affected at all concentrations

No significant difference for their development regarding pollen entrance and pollen in the hives, nectar and mortality. Treated hives were

Page 75 of 133

Stadler et al (2003)

Compound tested Species

Imidacloprid (Gaucho) Honeybees

Study type

Field test / field / workers

Imidacloprid Bee

Field/field application/adults

Imidacloprid (200 SL) Bees Imidacloprid (Confidor 200 SL) Honeybees

Field/field exposure/adults Field/field exposure/workers

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Test dose

Test duration

Endpoints

Results

seed, 60.000 seeds/ha)

216 days)

production, foraging activity, pollen entrance and mortality)

significantly more productive (higher weight, honey production, increased foraging activity, brood).

Not recorded but states recommended rate is 0.7mg a.i./plant (sunflower). (Equivalent to 50g a.i./ha at 70,000 plants/ha.) 0.0178% (??)

140, 168 or 196ml product/ha 46g a.i./ha leaf wall area (calculated taking height

Not recorded

Number of bees entering hive. Number of bees visiting flowers

High proportion of sunflower pollen in both treated and controls No effects on any parameter. No behaviourally impaired bees observed.

Notes (reference to study numbers in figures 20 and 21)

Reference

Cure et al (2001)

Hive weight development

5d

Bee visits to crop

Kumar and Singh (2012)

Number of foraging bees

Reduced number of visits compared to controls (in line with data for other insecticides, endosulfan, malathion) No effect on number of foraging bees.

NR

Flower visits following spraying at either green bud

No effects on visits to flowers following either treatment therefore no repellency observed.

Gobin et al (2008)

Page 76 of 133

Singh and Singh (2004)

Compound tested Species

Study type

Test dose

Test duration

and length of sprayed plot)

Imidacloprid (200g/L) + Oliocin (Confidor 200 SL) Honeybees

Field/oral exposure/colony

Orchards (3 x Jonagold, 1 x Golden delicious) Confidor 200 SL 600-800ml product/h (120-160g imidacloprid/h a) (Both control and treated plots treated with Oliocin at 36-48L product/ha)

Endpoints

Notes (reference to study numbers in figures 20 and 21)

Reference

or 10% open flower stages.

14d

(i) Weight of hives

(ii) Development of colony size (iii) Determination of the percentage of cells containing pollen, nectar, larvae and pupae, number of cells that have eggs in them or are empty on both sides of comb. (iv) Percentage of combs with pollen, nectar or

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Results

(i) No significant difference 1995. In 1998 mean increase 7.3% treated, 4.8% in controls. (not significant). (ii) Increase in worker bees greater in treated plots

(iii) Normal development of colonies in both treatments

(iv) No significant differences between

Page 77 of 133

Cantoni et al (2001)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

brood cells.

control and treated

(v) Number of bees returning to hives

(vi) Percentage of bees returning with pollen

Notes (reference to study numbers in figures 20 and 21)

Reference

(v) In 1995 there were no significant differences. In 1998 mean numbers were lower in the treated crop but relative differences were variable and not significant. (vi) In both years there were no significant differences between plots.

(vii) Visits to flowers

Imidacloprid Confidor SL 200 Honeybees

Semi-field (Cage trial)/field exposure/workers

0.6, 1.2, 2, 4, 9 and 14g a.i./ha (on rape plants)

4d (after treatment)

(i) Foraging intensity

(vii) There were no significant differences between plots (1995). (i) No effect on foraging at 2g/ha or less. At 4 and 9g/ha feeding was significantly reduced on the day after treatment only. At 14g/ha foraging was significantly lower for the first two days after treatment.

(ii) Mortality (ii) No increase in mortality compared to controls. Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 78 of 133

Schnier et al (2003)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Imidacloprid (technical) Apis mellifera

Semi-field (cages on field) / oral / workers

0.002, 0.005, 0.010 and 0.020mg/kg (in sunflower honey)

39d (chronic)

Feeding activity

No concentration related effects on any of the endpoints.

Wax/comb production

Imidacloprid (Gaucho 70 WS) Honeybees

Semi-field (Tunnel test) / field exposure / workers

Semi-field (tunnels) /field exposure/adults

Field/field exposure/adults

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Reference

Schmuck et al (2001)

Breeding performance

(no replication of tests doses) Imidacloprid (Gaucho) Honeybees

Notes (reference to study numbers in figures 20 and 21)

Colony vitality Increase in bee numbers

Between 0.35 and 1.05mg a.i./plant (sunflower)

Not recorded

No effects of treatment on these parameters. No records of behaviourally impaired bees. Fertilisation rates unaffected (i) No negative effect, 9.0 bees/min vs. 8.2 bees/min (control)

0.005g/cm2

(i) 5d

Number of foraging bees per 100 (i) Foraging activity (returning foragers)

(ii) -

(ii) Orientation

(ii) No effect

(iii) -

(iii) Honey sac weight

(iii) No negative effect, 26mg/bee vs. 25mg/bee (control)

(iv) -

(iv) Effects on larvae

(iv) None recorded

(v) 4 dates

(v) Foraging activity on flower clusters

(v) No negative effect, 3.2 bees/inflorescence vs. 8.2 bees/ inflorescence

Page 79 of 133

French testing guideline CEB 129 (adapted for seed treatment)

Cure et al (2001)

Wallner (2001)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Imidacloprid (Gaucho, 70% a.i.) Apis melliferea ligustica L. x A. m. caucasica L.

Semi-field/oral exposure/adults

0, 0.35, 0.7 mg a.i./seed (in tunnels)

-

Imidacloprid (Gaucho, 70% a.i.) ‘Honeybees’

Semi-field/oral exposure/adults

0.35, 0.7, 1.05 mg a.i./seed (in tunnels) 0.7 mg a.i./seed (in field) 20ppb spiked sugar solution (feeding experiments under field conditions)

-

Behaviour, activity, flower foraging, progress of pollination, collection of nectar. Vitality, foraging activity, behaviour

Results

(control) No effects observed on any of the measures

No effects observed on any of the measures.

Notes (reference to study numbers in figures 20 and 21)

Reference

In french

Ambolet et al (1997)

In french

Ambolet et al (1999)

No residues of imidacloprid found in sunflower nectar (LOQ 10ppb)

Table 17: Overview of semi-field and field studies of the effects of sublethal and chronic exposure to clothianidin and thiamethoxam on Apis bees Compound tested Study type Test dose Test Endpoints Results Notes Reference Species duration Thiamethoxam

Dosed in field

1.34ng in 20 µl/bee

3 days after dosing

Return to hive

Clothianidin (powder form)

Dosed in field / oral exposure

0.05, 0.5, 1, 2ng/bee

Immediately after

Foraging behaviour:

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Significant reduction in numbers of treated foragers returning particularly from unfamiliar landscapes At 1ng/bee 73.8% returned to the hive

Page 80 of 133

(5)

Henry et al 2012

(6)

Schneider et al (2012)

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Apis mellifera carnica

(bees treated in lab) / adults

(dosed in 10µL of 2M sucrose solution)

treatment for 3h and 24 and 48h

number of trips from hive to feeder, duration of trips, time interval between trips

versus 20.6% at 2ng/bee.

Clothianidin (Prosper FL, Poncho FS) Honeybees

Field study / oral exposure / adults + brood

(control seed treated with blank (no clothianidin) Prosper Fl and Poncho FS)

Neonicotinoid pesticides and bees Report to Syngenta Ltd

400 g a.i./100kg seed 80kg seed/ha 32g a.i./ha Seed treatment slurry contained Prosper FL (9.64%

From spring to spring (1 year)

Colony weight gain, honey production ; adult mortality, brood development, longevity

Notes

Reference

Significantly reduction in feeder visits at 0.5, 1 and 2ng/bee immediately after treatment No significant effect at 24h. At 0.5, 1 and 2ng/bee foraging trip duration, duration of feeder stay and flight time to hive, interval between foraging trips all significantly increased immediately after treatment but mostly recovered by 24h except at the highest dose. Time in hive after treatment significantly extended in 1 and 2ng/bee groups. No significant effects of treatment

Page 81 of 133

Cutler and Scott-Dupree (2007)

Compound tested Species

Thiamethoxam (Cruiser 350g a.i./L) Honeybees

Study type

Field/dust/workers

[seed also treated with Celest xl (fludioxonil and metalaxyl-M at 0.525 and 0.21 g/ha respectively)]

Test dose

clothianidin, plus thiram, carboxin, and metalaxyl) at 1,375.0 ml/100 kg seed, and Poncho 600 FS (48.96% clothianidin) at 458.7 ml/ 100 kg seed. 7.35g a.i./ha (corn dressed at 100mL product/100 kg of seeds, 70,000 seeds/ha, mean seed weight 0.3g, 21 kg seeds/ha,).

Test duration

Endpoints

Results

15d observation after sowing.

(i) Direct mortality in hive area

(i) Significant increase in mortality in period after sowing compared presowing and controls

(ii) Foraging activity

(ii) Reduction of foraging in all hives after sowing but significantly greater reduction in hives next to treated fields.

Notes

Reference

Tremolada et al (2010)

[calculated as 0.105mg/seed based on above] Table 18: Overview of semi-field and field studies of the effects of sublethal and chronic exposure to fipronil on Apis bees Compound tested Study type Test dose Test Endpoints Results Notes Species duration Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 82 of 133

Reference

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

Fipronil Apis mellifera

Dosed in field /oral/workers

2, 10, 50, 100, 500ppm in syrup – Choice tests (50% sucrose solution and honey mixed 3:1 v/v)

4h on two test days with feeders placed at 1030 and checks at 1100, 1130, 1200 and 1230

Visits to feeders (honeybees /5s /dish)

Visits and consumption significantly reduced compared to control dish (untreated syrup) only at 100 and 500ppm.

Mayer and Lunden (1999)

Fipronil (98%) Apis mellifera L.

Dosed in semifield (Outdoor flight cage) + feeder / oral exposure / adults

1µg/kg (in sucrose solution)

Orientation capacities

Significant effect on number of foragers entering maze and finding correct path. This was reduced from 86-89% before and after treatment to 60% for fipronil treated bees

Decourtye et al (2009)

Fipronil (98%) Apis mellifera ligustica

Dosed in semifield (tunnel) / oral exposure / adults

Fipronil

Dosed in semi-

0.06 and 0.3 ng/bee

6 - 7 h out of the tunnel

(6 and 30 µg/kg in 50% w/w sucrose solution and providing 10 µL per bee)

Neonicotinoid pesticides and bees Report to Syngenta Ltd

2 µg/kg (in

8 control

Foraging with RFID (time spent in the hive, at the feeder, between the feeder and hive, number of entries and exits from the hive and at the feeder Foraging activity

Notes

Reference

4% of bees do not find the path within 5min in control and 34% in treated 0.3 ng/bee significantly reduced the number of foraging flights and prolonged the duration of homing flights over 3d

Decourtye et al (2011)

Significant effects on

Colin et al

Page 83 of 133

Compound tested Species

Study type

Test dose

Test duration

Endpoints

Results

(analytical) Apis mellifera

field / oral exposure / adults

40% sucrose solution)

colonies during 5 days at different times of the season and 3 colonies before contaminati on and during 4 days after (i) 2d

at feeders in tunnels

attendance at feeder.

(i) Visits to crop (honybees /30s /9.1m)

(i) Visits not significantly different from untreated (control) field

(ii) 3d

(ii) Mortality (Mean no. dead honeybees /colony in Todd traps)

(ii) Mortality not significantly different from untreated (control) field

Fipronil (80WG) Apis mellifera

Field/field/workers

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.014 or 0.028kg a.i./ha sprayed on canola (Brassica napus cv. Legend)

Notes

Reference

(2004)

Significant decrease in the proportion of active bees at the feeder. Clinical signs of intoxication

Page 84 of 133

Mayer and Lunden (1999)

Table 19 Overview of studies of the effects of sublethal and chronic exposure to acetamiprid and thiacloprid on non-Apis bees Compound tested Study type Test dose Test Endpoints Results Notes duration species/subspecies

Reference

Acetamiprid Bombus terrestris

Very little detail given. Comparison of two treatments rather than with control

Fanigliulo et al (2009)

This study reports the development of a new bioassay to assess the impact of sublethal concentrations on the bumblebee foraging behavior under laboratory conditions.

Mommaerts et al (2010)

Little detail

Little detail

(tomato plants)

(maximum label rate?)

Pollination observed at 3, 8 and 12 d postreatment

(i) Pollination rate

(i) Pollination slightly reduced compared to Flonicamid (Teppeki) treatment

(ii) Hive status

(ii) No detailed effects on hives reported (‘generally homogeneous’) Without foraging 100% mortality at 120 ppm after 11 weeks. Mortality at to 60, 12, 1.2 and 0.12 ppm and 12 ppb was 78, 41, 39, 17 and 0%, respectively

14d

Thiacloprid (Calypso 48% SC) Bombus terrestris

Lab (artificial nestbox) /Oral exposure/adults

From the MFRC to several dilutions: 120 (MFRC), 60, 12, 1.2, 0.12 ppm and 12 ppb(in sugar water) 12 ppm (1/10 MFRC) for foraging behaviour

Up to 11 weeks (chronic exposure)

Mortality, drone production and foraging behavior

Chronic LC50 = 18 ppm; Total reproductive failure at 120 and 60ppm. Significant effect at 12ppm with reproduction reduced by 36% relative to control. EC50 = 12 ppm.

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Page 85 of 133

Table 20 Overview of studies of the effects of sublethal and chronic exposure to imidacloprid on non-Apis bees Compound tested Study type Test dose Test Endpoints Results Notes (study species/subspecies duration reference figure 22) Imidacloprid Field (lab Low: 8 weeks Colony growth Significant effect on (1) Bombus terrestris exposure) /oral 6µg/kg (pollen) colony weight with low /colony and 0.7µg/kg and high dose 8 and 12% (sugar water) smaller than controls respectively. High: 12µg/kg Significant reduction in (pollen) and queen production with 1.4µg/kg means of 13.72, 2.00 and (sugar water) 1.4 in control, low and high respectively. Imidacloprid Lab /oral /workers 10ppb (in 40% 28d Effects at Worker production Effects of λBombus terrestris sucrose (exposure) colony level significantly lower cyhalothrin alone solution) (relative to and imidacloprid+ controls) Brood number λ-cyhalothrin also significantly lower tested (2) Nest structure mass unaffected

Reference

Whitehorn et al (2012)

Gill et al (2012)

Worker mortality unaffected Worker loss significantly greater

Imidacloprid Bombus terrestris

Field / field / workers

Neonicotinoid pesticides and bees Report to Syngenta Ltd

0.7mg/seed (sunflower)

(i) 9d

(i) Loss of workers

No colony losses (0/10 failures) (iii) Losses were 33.5% in treated field and 23.1% in control (not significant)

Page 86 of 133

53% of foragers visited sunflowers in the control field, compared with 62%

Tasei et al (2001a) Tasei et al

Compound tested species/subspecies

Study type

Test dose

Test duration

Endpoints

(ii) 26d

(ii) Population increase

Imidacloprid (97.5% TG) Osmia lignaria

Field / Oral exposure (pollen)/ larvae

low (3ppb), intermediate (30ppb), or high (300ppb) in pollen provisions

Total developme nt period

Mortality rate, Development duration, adult weight

Imidacloprid Bombus terrestris

Field /oral /workers

10ppb (in 40% sucrose solution)

28d (exposure)

Effects on individual behaviour (relative to controls)

Results

(iii) No significant difference. 3.3 (treated field) and 3.0 (control field) workers/d/colony. Queens per colony were 17 (control) and 24 (treated). Mating ability was 71 and 74% respectively. No lethal effects Significant sublethal effects on larval development with greater developmental time at the intermediate (30 ppb) and high (300 ppb) doses. Number of foragers significantly increased Foraging bout frequency unaffected

Notes (study reference figure 22) in the treated field.

Reference

(2001b)

(Same data reported in both papers)

Abbott et al (2008)

Effects of λcyhalothrin alone and imidacloprid+ λ-cyhalothrin also tested (3)

Gill et al (2012)

(Same data reported in both papers)

Tasei et al (2001a)

Amount of pollen collected significantly reduced Duration of pollen foraging bouts + Imidacloprid Bombus terrestris

Greenhouse / field / workers

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Not recorded – assumed 0.7mg/seed

4d

(i) Total number of foragers visiting the

(i) No significant differences between control and treated plants.

Page 87 of 133

Compound tested species/subspecies

Study type

Test dose

Test duration

based on field study (sunflower)

Endpoints

Lab (artificial nestbox) /Oral exposure/adults

From the MFRC to several dilutions: 200 (MFRC), 20, 2 and 0.2 ppm and 20 and 10 ppb (in sugar water) for chronic and foraging assessments

Up to 11 weeks (chronic exposure)

Mortality, drone production and foraging behavior

Reference

Tasei et al (2001b)

(ii) Numbers of short (50s) visits not significantly different. Without foraging 100% mortality at 0.2ppm and higher, 15 and 0% at 20 and 10ppb. Significant effect on drone production at 0.2ppm and higher. Chronic toxicity LC50 = 59 ppb (NOEC for survival = 10 ppb); EC50 = 37 ppb (NOEC for reproduction = 20 ppb); With foraging 100% worker mortality at 200, 20, 2 and 0.2 ppm observed after a few hours, 7, 14 and 49 days respectively. 20 ppb caused 50% mortality after 49 days with none at 10ppb. Significant effect on reproduction at 200, 20, 2 and 0.2 ppm and 20

Neonicotinoid pesticides and bees Report to Syngenta Ltd

Notes (study reference figure 22)

heads at each blooming stage (ii) Mean visit duration/head was estimated for 75 foragers

Imidacloprid (Confidor 20% SC) Bombus terrestris

Results

Page 88 of 133

This study reports the development of a new bioassay to assess the impact of sublethal concentrations on the bumblebee foraging behaviour under laboratory conditions. (4)

Mommaerts et al (2010)

Compound tested species/subspecies

Study type

Test dose

Test duration

Endpoints

Results

Notes (study reference figure 22)

Reference

Use of an artificial flower foraging array proved to be a sensitive method for detecting sublethal response of bees to pesticides

Morandin and Winston (2003)

(5)

Tasei et al (2000)

and10 ppb with 0, 0, 0, 4.8,7.0 and 10.8 drones produced respectively (c.f. 28.4 in controls.) LC50 = 20ppb (NOEC for survival = 10 ppb); EC50 = 3.7ppb (NOEC for reproduction = single application 2nd hive: less clear differences (i) 92% mortality at 3 weeks following

Page 99 of 133

Sechser et al (2002)

Bombus terrestris

oral/adults

tent either immediatel y spray dry or 1wk later

(ii) 100g a.i./ha

(ii) 28d observation , Hives placed in tent either immediatel y spray dry or 14d later

(iii) 40g a.i./ha (iii) 24d observation . Sprayed 1, 2, 7 or 14d before exposure

Thiamethoxam (Actara WG 25) Bombus terrestris

Semi-field (tunnel)/ contact and oral/adults

Neonicotinoid pesticides and bees Report to Syngenta Ltd

(Phacelia)

immediate introduction. 68% mortality if introduced after 1wk. Control mortality 50%

(ii) Effects of foliar application (tomatoes)

(ii) 93% mortality at 3 weeks following immediate introduction. 94% mortality if introduced after 14d.

(iii) Effects of foliar application (tomatoes)

(iii) Mortality following 1, 2, 7 or 14d delay before introduction was 79, 80, 88, and 78% respectively. Control mortality 50% (iv) 50% mortality, 58% in controls

(iv) Effects of single drench application on tomatoes

(iv) 150g a.i./ha

(iv) 28d

(i) 10g a.i./ha

(i) 28d

(i) Effects of single application via irrigation (Trial 1)

(ii) 10g a.i./ha (50ppm in 70% sugar

(ii) 35d

(ii) Effects of single application via

(i) No negative effects on hives observed

(ii) No negative effects on hives observed

Page 100 of 133

Sechser et al (2002)

solution) Thiamethoxam (Actara WG 25) Bombus terrestris

Semi-field (tunnel)/ contact and oral/colony

Neonicotinoid pesticides and bees Report to Syngenta Ltd

150 and 161g a.i./ha (via irrigation system – single drip application)

Hives opened 1336d after application

irrigation (Trial 2) Impact on broods

No negative impact deteceted.

Page 101 of 133

Sechser and Freuler (2003)

5. References References in bold are used in the text Aajoud A, Raveton M, Aouadi H, Tissut M, and Ravanel P, 2006. Uptake and xylem transport of fipronil in sunflower. Journal of Agricultural and Food Chemistry 54, 5055-5060. Abbott VA, Nadeau JL, Higo HA and Winston ML (2008). Lethal and sublethal effects of imidacloprid on Osmia lignaria and clothianidin on Megachile rotundata (Hymenoptera: megachilidae). Journal of Economic Entomology, 101:784-796. Adler LS, 2000. The ecological significance of toxic nectar. Oikos 91, 409-420. AFSSA, 2007. OPINION of the French Food Safety Agency (Afssa. on the conclusions of the THE DIRECTOR GENERAL Cruiser assessment regarding the long-term risk to bee colonies AFSSA Request no. 2007-SA-0393 subject related to no. 2007-3845 – Cruiser AFSSA, 2008. Weakening, collapse and mortality of bee colonies, AFSSA. pp. 155. Alarcon AL, Canovas M, Senn R and Correia R (2005) The safety of thiamethoxam to pollinating bumble bees (Bombus terrestris L.) when applied to tomato plants through drip irrigation. Communications in Agricultural and Applied Biological Sciences 70(4): 569-579. Alaux C, Brunet JL, Dussaubat C, Mondet F, Tchamitchan S, Cousin M, Brillard J, Baldy A, Belzunces LP, and Le Conte Y, 2010b. Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera. Environmental Microbiology 12, 774-782. Alaux C, Ducloz F, Crauser D, and Le Conte Y, 2010a. Diet effects on honeybee immunocompetence. Biology Letters. Al-Fattah MA, and El-Shemy AAM, 1990. Eight methods for ventilating confined honeybee colonies during the application of insecticides. Journal of Apicultural Research 29, 214-220. Alferis KA, and Jabaji S, 2011. Metabolomics - a robust bioanalytical approach for the discovery of modes of action of pesticides, A review. Pesticide Biochemistry and Physiology 100, 105-117. Alghamdi A, Dalton L, Phillis A, Rosato E, and Mallon EB, 2008. Immune response impairs learning in free-flying bumble-bees. Biology Letters 4, 479-481. Aliouane Y, El Hassani AK, Gary V, Armengaud C, Lambin M and Gauthier M (2009) Subchronic exposure of honeybees to sublethal doses of pesticides: Effects on behaviour. Environmental Toxicology and Chemistry, 28(1):113-122. Alptekin S, Bass C, and Paine M, 2011. Microarray analysis of P450 upregulation in honey bee following neonicotinoid treatment. Current Opinion in Biotechnology 225, G17, S152. Ambolet B, Crevat JF and Schmidt HW (1997) Recherche d’eventuels effets secondaires d’un traitement de semences a base d’imidaclopride sur le comportement des abeilles domestique sur les fleurs de tournesol [Research on possible side effects of a seed treatment imidacloprid based on the behavior of domestic bees on sunflowers]. Annales ANPP - 4eme Conference Internationale sur Les Ravageurs en Agriculture, Montpelier 6-7-8 Janvier 1997. 103-110. Ambolet B, Crevat JF, Cure G, Schmuck R and Vincinaux C (1999) Etude au Champ des Effets de l’Imidaclopride sur Abeilles [Influence Under Field Conditions of Imidacloprid on Neonicotinoid pesticides and bees Page 102 of 133 Report to Syngenta Ltd

Honeybees]. Annales ANPP - 5eme Conference Internationale sur Les Ravageurs en Agriculture, Montpelier 7-8-9 Decembre 1999. 617-624. Andersen KE, Sheehan TH, Eckholm BJ, Mott BM, and DeGrandi-Hoffman G, 2011. An emerging paradigm of colony health, microbial balance of the honey bee and hive (Apis mellifera.. Insectes Sociaux, 431-444. Anon, 2008. Pesticide build-up could lead to poor honey bee health. American Bee Journal 148, 861-861. Antúnez K, Martín-Hernández R, Prieto L, Meana A, Zunino P, and Higes M, 2009. Immune suppression in the honey bee (Apis mellifera following infection by Nosema ceranae (Microsporidia). Environmental Microbiology 11. Armengaud C, Lambin M and Gauthier M (2002) Effects of imidacloprid on the neural processes of memory in honeybees. In: Honey Bees: Estimating the Environmental Impact of Chemicals (Devillers J and Pham-Delège MH Eds.). Taylor & Francis, London. pp. 85-100. Aronstein KA, and Murray KD, 2010. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology 103, S20-29. Aufauvre, J., D. G. Biron, et al. (2012). "Parasite-insecticide interactions: a case study of Nosema ceranae and fipronil synergy on honeybee." Scientific Reports 2. Babendrier D, Kalberger N, Romeis J, Fluri P, and Bigler F, 2004. Pollen consumption in honeyb bee larvae, a step forward in the risk assessment of transgenic plants. Apidologie 35, 293-300. Bacandritsos N, Granato A, Budge G, Papanastasiou I, Roinioti E, Caldon M, Falcaro C, Gallina A, and Mutinelli F, 2010. Sudden deaths and colony population decline in Greek honey bee colonies. Journal of Invertebrate Pathology 105, 335-340. Bailey, J., C. Scott-Dupree, et al. (2005). "Contact and oral toxicity to honey bees (Apis mellifera) of agents registered for use for sweet corn insect control in Ontario, Canada." Apidologie 36(4): 623-633. Barbara, G. S., B. Gruenewald, et al. (2008). "Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains." Invertebrate Neuroscience 8(1): 19-29. Barbara, G. S., C. Zube, et al. (2005). "Acetylcholine, GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, Apis mellifera." Journal of Comparative Physiology A Neuroethology Sensory Neural & Behavioral Physiology 191(9): 823-836. Bekesi L, 2005. Infection and immunity of the honeybee (Apis mellifera.. Literature review. Magyar Allatorvosok Lapja 127, 594-602. Belien T, Kellers J, Heylen K, Billen J, Arckens L, Huybrechts R and Gobin B (2010). Identification and evaluation of sublethal effects of crop protection products on honey bees (Apis mellifera). IOBC/WPRS Bulletin, 55:55-59. Belien T, Kellers J, Heylen K, Keulemans W, Billen J, Arckens L, Huybrechts R and Gobin B (2009). Effects of sublethal doses of crop protection agents on honey bee (Apis mellifera) global colony vitality and its potential link with aberrant foraging activity. Communications in agricultural and applied biological sciences, 74(1):245-253. Benzidane, Y., B. Lapied, et al. (2011). "Neonicotinoid insecticides imidacloprid and clothianidin affect differently neural Kenyon cell death in the cockroach Periplaneta americana." Pesticide Biochemistry and Physiology 101(3): 191-197.

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Szymas B, and Jedruszuk A, 2003. The influence of different diets on haemocytes of adult worker honey bees, Apis mellifera. Apidologie 34, 97-102. Tapparo A, Marton D, Giorio C, Zanella A, Solda L, Marzaro M, Vivan L, and Girolami V, 2012. Assessment of the environmental exposure of honeybees to particulate matter containing neonicotinoid insecticides coming from corn coated seeds. Environmental Science and Technology. Tasei JN, and Aupinel P, 2008. Nutritive value of 15 single pollens and pollen mixes tested on larvae produced by bumblebee workers (Bombus terrestris, Hymenoptera, Apidae.. Apidologie 39, 397-409. Tasei JN, Lerin J and Ripault G (2000). Sub-lethal effects of imidacloprid on bumblebees, Bombus terrestris (Hymenoptera: Apidae), during a laboratory feeding test. Pest Management Science, 56, 784-788. Tasei JN, Ripault G and Rivault E (2001a) Hazards of imidacloprid seed coating to Bombus terrestris (Hymenoptera : Apidae) when applied to sunflower. Journal of Economic Entomology 94(3): 623-627. Tasei JN, Ripault G and Rivault E (2001b) Effects of Gaucho [R] seed coating on bumblebees visiting sunflower. In: Hazards of Pesticides to Bees. Belzunces LP, Pelissier C and Lewis GB (eds). Inst Natl Recherche Agronomique (INRA), Paris, pp.207-212. Teeters BS, Johnson RM, Ellis MD and Siegfried BD. (2012). Using video-tracking to assess sublethal effects of pesticides on honey bees (Apis mellifera L.). Environmental Toxicology and Chemistry 31(6):1349-1354. Thany, S. H. (2010). Electrophysiological studies and pharmacological properties of insect native nicotinic acetylcholine receptors. Insect Nicotinic Acetylcholine Receptors. S. H. Thany. Heidelberger Platz 3, D-14197 Berlin, Germany, Springer-Verlag Berlin. 683: 53-63. Thany, S. H. (2010). Insect Nicotinic Acetylcholine Receptors. Insect Nicotinic Acetylcholine Receptors. S. H. Thany. Heidelberger Platz 3, D-14197 Berlin, Germany, SPRINGER-VERLAG BERLIN. Thany, S. H. (2012). Neonicotinoid Insecticides. Historical Evolution and Resistance Mechanisms. Insect Nicotinc Acetylcholine Receptors. S. H. Thany. Heidelberger Platz 3, D-14197 Berlin, Germany, Springer-Verlag Berlin. 683: 75-83. Thany, S. H. and H. Tricoire-Leignel (2011). "Emerging Pharmacological Properties of Cholinergic Synaptic Transmission: Comparison between Mammalian and Insect Synaptic and Extrasynaptic Nicotinic Receptors." Current Neuropharmacology 9(4): 706-714. Thany, S. H., G. Lenaers, et al. (2007). "Exploring the pharmacological properties of insect nicotinic acetylcholine receptors." Trends in Pharmacological Sciences 28(1): 14-22. Thany, S. H., H. Tricoire-Leignel, et al. (2010). Identification of cholinergic synaptic transmission in the insect nervous system. Insect Nicotinic Acetylcholine Receptors. S. H. Thany. Heidelberger Platz 3, D-14197 Berlin, Germany, Springer-Verlag Berlin. 683: 1-10. Thompson HM, 2001. Assessing the exposure and toxicity of pesticides to bumblebees (Bombus sp... Apidologie 32, 305-321. Thompson HM, 2010. Risk assessment for honey bees and pesticides - recent developments and 'new issues'. Pest Management Science 66, 1157-1162. Tome HVV, Martins GF, Lima MAP, Campos LAO and Guedes RNC (2012) ImidaclopridInduced Impairment of Mushroom Bodies and Behavior of the Native Stingless Bee Melipona quadrifasciata anthidioides. Plos One 7(6), Article No. e38406. Tomizawa, M. and I. Yamamoto (1992). "Binding of nicotinoids and the related compounds to the insect nicotinic acetylcholine receptor." Journal of Pesticide Science 17(4): 231-236. Neonicotinoid pesticides and bees Page 119 of 133 Report to Syngenta Ltd

Tomizawa, M. and I. Yamamoto (1993). "Structure activity relationships of niotinoids and imidacloprid analogs." Journal of Pesticide Science 18(1): 91-98. Tomizawa, M., H. Otsuka, et al. (1995). "Pharmacological characteristics of insect nicotinic acetylcholine receptor with its ion channel and the comparison if the effect of nicotinoids and neonicotinoids." Journal of Pesticide Science 20(1): 57-64. Traver BE, Williams MR, and Fell RD, 2012. Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies. Journal of Invertebrate Pathology 109, 187-193. Tremolada P, Bernardinelli I, Colombo M, Spreafico M, and Vighi M, 2004. Coumaphos distribution in the hive ecosystem, Case study for modeling applications. Ecotoxicology 13, 589-601. Tremolada P, Bernardinelli I, Rossaro B, Colombo M, and Vighi M, 2011. Predicting pesticide fate in the hive (part 2., development of a dynamic hive model. Apidologie 42, 439-456. Tremolada P, Mazzoleni M, Saliu F, Colombo M and Vighi M (2010) Field Trial for Evaluating the Effects on Honeybees of Corn Sown Using Cruiser(A (R)) and Celest xl(A (R)) Treated Seeds. Bulletin of Environmental Contamination and Toxicology 85(3): 229-234. Tricoire-Leignel, H. and S. H. Thany (2010). Identification of Critical Elements Determining Toxins and Insecticide Affinity, Ligand Binding Domains and Channel Properties. Insect Nicotinic Acetylcholine Receptors. S. H. Thany. Berlin, Springer-Verlag Berlin. 683: 45-52. Vacante V (1997) The effect of imidacloprid on the pollination of tomato by Bombus terrestris. l’Informatore Agrario 53(43): 68-70. Valdovinos-Nunez, G. R., J. J. G. Quezada-Euan, et al. (2009). "Comparative toxicity of pesticides to stingless bees (Hymenoptera: Apidae: Meliponini)." Journal of Economic Entomology 102(5): 1737-1742. Van der Steen, J. J. M. (2001). "Review of the methods to determine the hazard and toxicity of pesticides to bumblebees." Apidologie 32(5): 399-406. vanEngelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E, Nguyen BK, Frazier M, Frazier J, Cox-Foster D, Chen Y, Underwood R, Tarpy DR, and Pettis JS, 2009b. Colony Collapse Disorder, A Descriptive Study. PLoS One 4, Article-No, e6481. vanEngelsdorp D, Speybroeck N, Evans JD, Nguyen BK, Mullin C, Frazier M, Frazier J, CoxFoster D, Chen Y, Tarpy DR, Haubruge E, Pettis JS, and Saegerman C, 2010. Weighing Risk Factors Associated With Bee Colony Collapse Disorder by Classification and Regression Tree Analysis. Journal of Economic Entomology 103, 1517-1523. Verbruggen EMJ, and Van den Brink PJ, 2010. Review of recent literature concerning mixture toxicity of pesticides to aquatic organisms Vidau C, Diogon M, Aufauvre J, Fontbonne R, Vigues B, Brunet JL, Texier C, Biron DG, Blot N, El-Alaoui H, Belzunces LP and Delbac F (2011) Exposure to Sublethal Doses of Fipronil and Thiacloprid Highly Increases Mortality of Honeybees Previously Infected by Nosema ceranae. Plos One 6(6): Article No. e21550. Viuda-Martos M, Ruis-Navajas Y, Fernandez-L´Opez J, and Perez-´Alvarez JA, 2008. Functional Properties of Honey, Propolis, and Royal Jelly. Journal of Food Science 73, R117-R124. Vo, D. T., W. H. Hsu, et al. (2010). "Insect nicotinic acetylcholine receptor agonists as flea adulticides in small animals." Journal of Veterinary Pharmacology and Therapeutics 33(4): 315322. Wahl O, and Ulm K, 1983. Influence of Pollen Feeding and Physiological Condition on Pesticide Sensitivity of the Honey Bee Apis-Mellifera-Carnica. Oecologia 59, 106-128. Neonicotinoid pesticides and bees Page 120 of 133 Report to Syngenta Ltd

Walker CH, 1998. The use of biomarkers to measure the interactive effects of chemicals. Ecotoxicology and Environmental Safety 40, 65-70. Waller GD, Barker RJ, and Martin JH, 1979. Effects of dimethoate on honey bee foraging. Chemosphere, 461-463. Wallner K, 1997. Honeybee intoxication caused by chemical plant protection in vineyards. Mitteilungen der Deutschen Gesellschaft fur Allgemeine und Agewandte Entomologie, Band 11, Heft 1-6, Dezember 1997, Entomologists Conference 11, 205-209. Wallner K, 2009. Sprayed and seed dressed pesticides in pollen, nectar and honey of oilseed rape. Julius Kuhn Archive 423, 152-153. Wallner K. (2001). Tests regarding effects of imidacloprid on honey bees. In: Hazards of Pesticides to Bees. Belzunces LP, Pelissier C and. Lewis GB (eds). Paris, Inst Natl Recherche Agronomique: 91-94. Walorczyk S, and Gnusowski B, 2009. Development and validation of a multi-residue method for the determination of pesticides in honeybees using acetonitrile-based extraction and gas chromatography-tandem quadrupole mass spectrometry. Journal of Chromatography A 1216, 6522-6531. Wang C-J, Qiu L-H, Zheng M-Q, Tao C-J, Jiahg H, Zhang W-J, and JLi X-F, 2006. Safety evaluation of abamectin and its mixtures to honey bees (Apis mellifera L... Journal of AgroEnvironment Science 25, 229-231. Warne MSJ, 2003. A Review of the ecotoxicity of mixtures, approaches to, and recommendations for, their management., in, A. Langley, et al, Eds.., Proceedings of the 5th national workshop on the assessment of site contamination, NEPC, Adelaide. pp. 253276. Wehling M, Ohe Wvd, Brasse D, and Forster R, 2009 Colony losses - interactions of plant protection products and other factors, in, P. A. T. H. M. Oomen (Ed.., Hazards of pesticides to bees. 10th International Symposium of the ICP-Bee Protection Group. Bucharest, Romania, 8-10 October, 2008., JKI, Germany. pp. 153-154. Whitehorn PR, O’Connor S, Wackers FL and Goulson D (2012) Neonicotinoid pesticide reduces bumble bee colony growth and queen production. Science 336(6079):351-352 Wiest L, Bulete A, Giroud B, Fratta C, Amic S, Lambert O, Pouliquen H, and Arnaudguilhem C, 2011. Multi-residue analysis of 80 environmental contaminants in honeys, honeybees and pollens by one extraction procedure followed by liquid and gas chromatography coupled with mass spectrometric detection. Journal of Chromatography A 1218, 5743-5756. Wilkinson CF, Christoph GR, Julien E, Kelley JM, Kronenberg J, McCarthy J, and Reiss R, 2000. Assessing the risks of exposures to multiple chemicals with a common mechanism of toxicity, How to cumulate? Regulatory Toxicology and Pharmacology 31, 30-43. Wilson K, Knell R, Boots M, and Koch-Osborne J, 2003. Group living and investment in immune defence, an interspecific analysis. Journal of Animal Ecology 72, 133-143. Wilson-Rich N, Dres ST, and Starks PT, 2008. The ontogeny of immunity, Development of innate immune strength in the honey bee (Apis mellifera.. Journal of Insect Physiology 54, 1392-1399. Winterlin W, Walker G, and Luce A, 1973. Carbaryl residues in bees, honey, and bee bread following exposure to carbaryl via the food supply. Archives of Environmental Contamination and Toxicology 1, 362-374. Woyciechowski M, 2007. Risk of water collecting in honeybee (Apis mellifera. workers (Hymenoptera, Apidae.. Scociobiology 50, 1059-1068. Neonicotinoid pesticides and bees Report to Syngenta Ltd

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Wu JY, Anelli CM, and Sheppard WS, 2011. Sub-lethal effects of pesticide residues in brood comb on worker honey bee (Apis mellifera. development and longevity. PLoS One 6, e14720. Wu JY, Smart MD, Anelli CM, and Sheppard WS, 2012. Honey bees (Apis mellifera. reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia. infection. Journal of Invertebrate Pathology. Wu, J., J. Li, et al. (2010). "Sensitivities of three bumblebee species to four pesticides applied commonly in greenhouses in China." Insect Science 17(1): 67-72 Wustenberg, D. G. and B. Grunewald (2004). "Pharmacology of the neural nicotinic acetylcholine receptor of cultured Kenyon Cells of the honeybee, Apis mellifera." Journal of Comparative Physiology A Neuroethology Sensory Neural & Behavioral Physiology 190: 807. Wykes G.R (1953) The sugar content of nectars Biochem J 53 294-296 Yamamoto I, Tomizawa M, Saito T, Miyamo to T, Walcott EC, and Sumikawa K, 1988. Structural factors contributing to insecticidal and selective actions of neonicotinoids. Archives of Insect Biochemistry and Physiology 37, 24-32. Yamamoto, I., G. Yabuta, et al. (1995). "Molecular mechanism for selective toxicity of nicotinoids and neonicotinoids." Journal of Pesticide Science 20: 33-40. Yang EC, Chuang YC, Chen, YL and Chang LH (2008). Abnormal Foraging Behavior Induced by sublethal dose of imidacloprid in the honey bee (Hymenoptera: Apidae) J Econ Enotomology 101 (6) 1743-1748 Yang EC, Wu PS, Chang HC and Chen YW (2011) Effect of sub-lethal dosages of insecticides on honeybee behavior and physiology. Proceedings of International Seminar on Enhancement of Functional Biodiversity Relevant to Sustainable Food Production in ASPAC — In association with MARCO — November 9-11, 2010 Tsukuba, Japan. Ye S, Dun Y, and Feng M, 2005. Time and concentration dependent interactions of Beauveria bassiana with sublethal rates of imidacloprid against the aphid pests Macrosiphoniella sanborni and Myzus persicae. pp. 459-468. Yu SJ, Robinson FA, and Nation JL, 1984. Detoxication capacity in the honey bee, Apis mellifera L. Pesticide Biochemistry and Physiology 22, 360-368.

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6. Annex: Database search terms Databases: BIOSIS Previews , CAB Abstracts , Zoological Record All searches included the terms in the title, abstract and/or keywords

revision 2, 13th Feb 2012 1. agrochemical.mp. or pesticides.sh. or chemical control.sh. or herbicides.sh. or agricultural chemicals.sh. or fungicides.sh. or pesticide residues.sh. or insecticides.sh. 2. plant protection product*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 3. plant protection compound*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 4. plant protection chemical*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 5. (Pesticid* or insecticid* or Acaricid* or Nematicid* or Molluscicid*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 6. (Herbicid* or Fungicid* or antifungal* or anti-fungal*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 7. 1 or 2 or 3 or 4 or 5 or 6 8. veterinary medicine*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 9. veterinary pharmaceutical*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 10. (varroacid* or miticid*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 11. (antibacterial* or antibiotic*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 12. 7 or 8 or 9 or 10 or 11 13. (honeybee* or honey bee*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] Neonicotinoid pesticides and bees Report to Syngenta Ltd

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14. Apis mellifera.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 15. 13 or 14 16. 12 and 15 17. (toxic* or sublethal* or sub-lethal*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 18. (ecotox* or nontarget* or non-target*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 19. ((additiv* or cumulativ* or synergis* or mixture* or sequent*) adj5 effect*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 20. (multiple adj exposur*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 21. (sublethal* or sub-lethal*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 22. 19 or 20 or 21 23. 17 or 18 24. 16 and 23 25. 16 and 22 26. remove duplicates from 25 27. route*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 28. (oral* or pollen* or nectar* or water*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 29. (contact* or spray* or overspray* or systemic*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 30. (dust* or guttation*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 31. (inhation* or vapor* or vapour*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 32. (adult* or larv* or brood*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 33. 27 or 28 or 29 or 30 or 31 or 32 34. 24 and 33 35. remove duplicates from 34 Neonicotinoid pesticides and bees Report to Syngenta Ltd

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36. 35 not 26 37. (insect* or arthropod*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 38. 15 or 37 39. 23 and 38 40. (foulbrood* or bacillus* or leissococcus* or pathogen* or disease* or fungus* or fungal* or bacteria* or biocontrol*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 41. (nosema* or microsporidia* or varroa* or mite* or acarine* or virus* or viral* or parasit*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 42. 40 or 41 43. 38 and 42 44. 12 and 43 45. interact*.mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, tm, tn, ot, hw, nm, rs, ui] 46. 12 and 38 47. 42 and 46 48. 45 and 47 49. "interact*".m_titl. 50. 47 and 49 51. remove duplicates from 50 52. 36 use mesz 53. 51 use mesz

15th February 2012 *** It is now 2012/02/15 16:22:06 *** (Dialog time 2012/02/15 11:22:06) Subaccount is set to W8JZ_HONEYBEES. Notice

= $10.00

? b155,50,5,185,10,203,40,156,76,41,34,434

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SYSTEM:OS - DIALOG OneSearch File 155:MEDLINE(R) 1950-2012/Feb 13 (c) format only 2012 Dialog *File 155: MEDLINE has been reloaded. Please see HELP NEWS154 for details. File 50:CAB Abstracts 1972-2012/Feb W1 (c) 2012 CAB International File 5:Biosis Previews(R) 1926-2012/Feb W1 (c) 2012 The Thomson Corporation File 185:Zoological Record Online(R) 1864-2012/Feb (c) 2012 The Thomson Corp. File 10:AGRICOLA 70-2012/Feb (c) format only 2012 Dialog File 203:AGRIS 1974-2012/Dec Dist by NAL, Intl Copr. All rights reserved File 40:Enviroline(R) 1975-2008/May (c) 2008 Congressional Information Service *File 40: This file is closed and will no longer update. For similar data, please search File 76-Environmental Sciences. File 156:ToxFile 1965-2012/Feb W2 (c) format only 2012 Dialog *File 156: The last daily update of Medline records for 2011 was UD20111114. Updates resumed with the 2012 MeSH with UD20120105. File 76:Environmental Sciences 1966-2012/Jan (c) 2012 CSA. File 41:Pollution Abstracts 1966-2012/Jan (c) 2012 CSA. File 34:SciSearch(R) Cited Ref Sci 1990-2012/Feb W2 Neonicotinoid pesticides and bees Report to Syngenta Ltd

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(c) 2012 The Thomson Corp File 434:SciSearch(R) Cited Ref Sci 1974-1989/Dec (c) 2006 The Thomson Corp

Set S1

Items Description 1690058 AGROCHEMICAL? OR PESTICID? OR CHEMICAL()CONTROL? OR HERBICID? OR AGRICULTURAL()CHEMICAL? OR FUNGICID? OR INSECTICID?

S2

4562 PLANT()PROTECTION()PRODUCT? OR PLANT()PROTECTION()COMPOUND? OR PLANT()PROTECTION()CHEMICAL?

S3

63739 ACARICID? OR NEMATICID? OR MOLLUSCICID?

S4

232227 ANTIFUNGAL? OR ANTI-FUNGAL?

S5

1882500 S1 OR S2 OR S3 OR S4

S6

360277 VETERINARY()MEDICINE? OR VETERINARY()PHARMACEUTICAL?

S7

2530 VARROACID? OR MITICID?

S8

1199988 ANTIBACTERIAL? OR ANTIBIOTIC?

S9

3314429 S5 OR S6 OR S7 OR S8

S10

116545 APIS()MELLIFERA OR HONEYBEE? OR HONEY()BEE?

S11

11752 S9 AND S10

S12 4460623 TOXIC? OR SUBLETHAL? OR SUB-LETHAL? S13

137777 ECOTOX? OR NONTARGET? OR NON-TARGET?

S14 2732942 ADDITIV? OR CUMULATIV? OR SYNERGIS? OR MIXTURE? OR SEQUENT? S15

284151 S14(3N)EFFECT?

S16

23078 MULTIPLE()EXPOSURE? OR REPEATED()EXPOSURE?

S17

78386 SUBLETHAL? OR SUB-LETHAL?

S18

344571 (S12 OR S13) AND (S14 OR S16 OR S17)

S19

123044 (S12 OR S13) AND (S15 OR S16 OR S17)

S20

736 S11 AND S18

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S21 S22

486 S11 AND S19 500316 ROUTE?

S23 8063047 ORAL? OR POLLEN? OR NECTAR? OR WATER? S24 2399654 CONTACT? OR SPRAY? OR OVERSPRAY? OR SYSTEMIC? S25

277125 DUST? OR GUTTATION?

S26

442968 INHALATION? OR VAPOR? OR VAPOUR?

S27 7976748 ADULT? OR LARV? OR BROOD? S28 18030575 S22 OR S23 OR S24 OR S25 OR S26 OR S27 S29

5568 S11 AND S28

S30 397 RD S20 PREVIOUSLY SEARCHED S31

(unique items) – ITEMS PRINTED FROM DATABASES NOT

15336 EXPOSURE?(2N)ROUTE?

S32

57 S29 AND S31

S33

22 RD S32 (unique items) – ALL ITEMS PRINTED

S34 4351537 INSECT? OR ARTHROPOD? S35

941 S9 AND S31 AND S34

S36

35 S35 AND REVIEW?/TI,DE – ALL ITEMS PRINTED

S37 20481533 D-

FOULBROOD? OR BACILLUS? OR LEISSOCOCCUS? OR PATHOGEN? OR

ISEASE? OR FUNGUS? OR FUNGAL? OR BACTERIA? OR BIOCONTROL? S38 5977919 NOSEMA? OR MICROSPORIDIA? OR VARROA? OR MITE? OR ACARINE? OR VIRUS? OR VIRAL? OR PARASIT? S39

6010 S11 AND (S37 OR S38)

S40

72 S39 AND INTERACT?/TI,DE

S41

48 RD S40 (unique items) – ALL ITEMS PRINTED

SearchSave "SD915239102" stored

Temp SearchSave "TF960075806" stored ? b155,5,50,34,185,28,40,73,76,144,156,10,399 Neonicotinoid pesticides and bees Report to Syngenta Ltd

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SYSTEM:OS - DIALOG OneSearch File 155:MEDLINE(R) 1950-2012/Aug 08 (c) format only 2012 Dialog File 5:Biosis Previews(R) 1926-2012/Aug W1 (c) 2012 The Thomson Corporation File 50:CAB Abstracts 1972-2012/Jul W5 (c) 2012 CAB International *File 50: For details on weekly updates in May & June 2012, please see HELP NEWS50. File 34:SciSearch(R) Cited Ref Sci 1990-2012/Aug W1 (c) 2012 The Thomson Corp File 185:Zoological Record Online(R) 1864-2012/Aug (c) 2012 The Thomson Corp. File 28:Oceanic Abstracts 1966-2012/Jul (c) 2012 CSA. File 40:ENVIROLINE(R) 1975-2008/MAY (c) 2008 Cis *File 40: This file is closed and will no longer update. For similar data, please search File 76-Environmental Sciences. File 73:EMBASE 1974-2012/Aug 10 (c) 2012 Elsevier B.V. *File 73: Embase has been enhanced with Conference Abstract records. Please see HELP NEWS072 for information. File 76:Environmental Sciences 1966-2012/Jul (c) 2012 CSA. File 144:Pascal 1973-2012/Aug W1 (c) 2012 INIST/CNRS *File 144: Please see HELP NEWS144 for important information on recent update processing. File 156:ToxFile 1965-2012/Aug W1 (c) format only 2012 Dialog *File 156: Toxfile has been reloaded with the 2012 MeSH Thesaurus. File 10:AGRICOLA 70-2012/Jul (c) format only 2012 Dialog File 399:CA SEARCH(R) 1967-2012/UD=15707 (c) 2012 American Chemical Society *File 399: Use is subject to the terms of your user/customer agreement. IPCR/8 classification codes now searchable as IC=. See HELP NEWSIPCR.

Set Items Description S1 6227 NEONICOTINOID? S2 4953 ACETAMIPRID? OR RN=(135410-20-7 OR 160430-64-8) S3 2498 CLOTHIANIDIN? OR RN=(210880-92-5 OR 205510-53-8) S4 1491 DINOTEFURAN? OR RN=165252-70-0 S5 21253 IMIDACLOPRID? OR RN=138261-41-3 S6 1394 NITENPYRAM? OR RN=(150824-47-8 OR 120738-89-8) S7 2642 THIACLOPRID? OR RN=111988-49-9 S8 5761 THIAMETHOXAM? OR RN=153719-23-4 S9 28561 S1 OR S2 OR S3 OR S4 OR S5 OR S6 OR S7 OR S8 S10 234556 HONEYBEE? OR BEE OR BEES OR APIS OR BOMBUS OR BUMBLEBEE? S11 128067 ANDRENA OR LARANDRENA OR MELANDRENA OR PYROBOMBUS OR THORANeonicotinoid pesticides and bees Report to Syngenta Ltd

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COBOMBUS OR CERATINA OR ZADONTOMERUS OR FLORILEGUS OR HABROPODA OR ACENTRON OR AGAPOSTEMON OR AUGOCHLORELLA OR AUGOCHLOROPSIS OR BEES OR BOREOCOELIOXYS OR COELIOXYS OR COLLETES OR COLLETIDAE OR DIALICTUS OR DIEUNOMIA OR EOXENOGLOSSA OR EUMELISSODES OR EUTRICHARAEA OR EVYLAEUS OR HALICTIDAE OR HALICTUS OR LASIOGLOSSUM OR LITOMEGACHILE OR MEGACHILE OR MELANOSARUS OR MELANOSMIA OR MELISSODES OR ODONTALICTUS OR OSMIA OR PARAUGOCHLOROPSIS OR SAYAPIS OR SCHONNHERRIA OR XENOGLOSSA OR XYLOCOPA OR XYLOCOPOIDES S12 243429 S10 OR S11 S13 1313 S9 AND S12 S14 4268882 SUBLETHAL? OR SUB-LETHAL? OR CHRONIC? OR SUBCHRONIC? OR SUB-CHRONIC? OR MULTIPLE()EXPOSUR? OR REPEATED()EXPOSUR? OR INCREMENT?OR CUMMULATIV? S15 479 RD S13 (unique items) SearchSave "SG960077717" stored ? t15/4/406-479 (records 1-405 are in databases previously interrogated on the WoK and OVID hosts)

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Neonicotinoid pesticides and bees Report to Syngenta Ltd

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DEFRA hereby excludes all liability for any claim, loss, demands or damages of any kind whatsoever (whether such claims, loss, demands or damages were foreseeable, known or otherwise) arising out of or in connection with the preparation of any technical or scientific report , including without limitation, indirect or consequential loss or damage; loss of actual or anticipated profits (including loss of profits on contracts); loss of revenue; loss of business; loss of opportunity; loss of anticipated savings; loss of goodwill; loss of reputation; loss of damage to or corruption of data; loss of use of money or otherwise, and whether or not advised of the possibility of such claim, loss demand or damages and whether arising in tort (including negligence), contract or otherwise. This statement does not affect your statutory rights. Nothing in this disclaimer excludes or limits DEFRA’s liability for: (a) death or personal injury caused by DEFRA’s negligence (or that of its employees, agents or directors); or (b) the tort of deceit; [or (c) any breach of the obligations implied by Sale of Goods Act 1979 or Supply of Goods and Services Act 1982 (including those relating to the title, fitness for purpose and satisfactory quality of goods);] or (d) any liability which may not be limited or excluded by law (e) fraud or fraudulent misrepresentation. The parties agree that any matters are governed by English law and irrevocably submit to the non-exclusive jurisdiction of the English courts.

© Crown copyright 2012 All printed publications and literature produced by Fera are subject to Crown copyright protection unless otherwise indicated.

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