Neonicotinoid Pesticides and Bees - The Food and Environment ...

Neonicotinoid Pesticides and Bees 

Report to Syngenta Ltd 

January 2013 

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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 

Neonicotinoid pesticides and bees Page 2 of 133 


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 



(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 



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 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. 

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 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. 

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 



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 



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. 



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 



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. 



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? 



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: 



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 Ca 2+ 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) 



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 



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 [C 14 ] 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 



(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 

[ 14 C]-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 



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 



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) 



Table 1: Oral and contact 48 LD50 values for neonicotinoid pesticides in honeybees from 

ANSES Agritox database (http://www.dive.afssa.fr/agritox/php/fiches.php) or 1 EPA factsheets (United 

States Environmental Protection Agency) 

Insecticide Exposure Route 48 LD50 

Acetamiprid 

Clothianidin 

Dinotefuran 

Imidacloprid 

Thiacloprid 

Thiamethoxam 

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 


Oral 17.32 µg/bee 

Contact 38.83 µg/bee 

Oral 5 ng/bee 




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 



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 24 112.7 5 191.0 – 3.2 

48 44.3 14 109.6 – 3.7 

72 64.0 5 97.4 – 31 

96 43.5 2 50 - 37 

Contact 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 



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 Imidacloprid Thiamethoxam 

Mean 95% CI Mean 95% CI Mean 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 49.55 – 95.15 2.88 2.59 – 3.13 

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). 



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. 

ug/g bee 

10000 

1000 

100 

10 

1 

0.01 1 100 10000 

0.1 

0.01 

Apis mellifera ug/ g bee 

B. Terrestris 210 mg 

B lucorum 210 mg 

B agrorum (pascuorum) 

120 mg 

M rotundata 86.6mg 

O lignaria 90mg 

Nomia melanderi 30.8mg 

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 



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). 



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

Pesticide Species µg a.i./ bee µg ai/g Ref 

(95%CL) bee 

Acetamiprid B ignites 0.0023 (0.0021- 

Wu et al (2010) 

Mosplian (3% ai) 48 hr oral 0.0024) 

Acetamiprid B hypocrite 0.0028 (0.0018- 

Wu et al (2010) 


Acetamiprid B patagiatus 0.0021 (0.0020- 

Wu et al (2010) 


Imidacloprid B terrestris 0.02 0.095 Marletto et al 

(formulation) 72 hour contact 

(2003) 


(formulation) 72 hour oral 

(2003) 

Imidacloprid B terrestris 0.445 2.119 Bortolotti et al 

Confidor (17.8% ai) 24 hour contact 

(2001) 



(2001) 



(2001) 


Confidor (17.8% ai) 24 hour oral 

(2001) 



(2001) 



(2001) 

Imidacloprid A indica 0.0025 0.0362 Kumar and 

24 hr contact 

Regupathy (2005) 

Imidacloprid A florae 0.0022 0.0767 Kumar and 

24 hr contact 


Imidacloprid T irridipenis 0.0020 0.5275 Kumar and 

24 hr contact 


Imidacloprid N perilampoides 0.0011 0.13 Valdovinos-Nunez 

24 hr contact 

et al 2009 


(formulation) 24 hour contact 

(2003) 

Thiacloprid N perilampoides 0.007 0.85 Valdovinos-Nunez 

24 hr contact 

et al 2009 

Thiamethoxam A indica 0.0056 0.0819 Kumar and 

24 hr contact 


Thiamethoxam A florae 0.0056 0.1905 Kumar and 

24 hr contact 


Thiamethoxam T irridipenis 0.0051 1.3381 Kumar and 

24 hr contact 


Thiamethoxam N perilampoides 0.004 0.49 Valdovinos-Nunez 



. 

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) 

imidacloprid 

thiacloprid 

A 

mellifera 

(India) 

B A 

T 

N 

terrestris 

0.025indica 

A florea irridipenis perilampoides 

0.19 0.036 0.077 0.53 0.13 0.028 0.81 

A 

mellifera 

thiamethoxam 0.082 0.191 1.34 0.49 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 



0.85 

B ignitus B hypocrite B patagiatus Bterrestris A mellifera 

acetamiprid 0.01 0.013 0.01 

Conclusions 

0.022- 

0.095 145 

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. 

388

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

Insecticide Concentration Range Bee Type Pre-treatment Reference 

LD50 – 24 hours Oral 

Imidacloprid 5.4 ng/bee 5.2-1.6 (95% CI) Worker Bees (A. Starvation 2 hr (Suchail et al., 

m. mellifera) 

2000) 


m. caucasica) 

2000) 

Imidacloprid 183.78 ng/bee 174.5 – 190.7 (95% Apis mellifera Not stated (Laurino et al., 

(0.15 – 150 ppm) 

CI) 

ligustica 

2010) 

Clothianidin 2.844 ng/bee 1.733-4.045 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 

Clothianidin 4.48 ng/bee 4.0 – 4.9 (95% CI) Apis mellifera Not stated (Laurino et al., 

ligustica 

2010) 

Thiamethoxam 4.679 ng/bee 3.862-5.552 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 

Thiamethoxam 3.54 ng/bee 2.8 – 4.4 (95% CI) Apis mellifera Not stated (Laurino et al., 

ligustica 

2010) 

LD50 – 24 hours – Contact (applied directly to the bee) 

Acetamiprid 7.07 µg/bee 4.57-11.2 (95% CI) Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 

IM-2-1 

(Acetamiprid 

metabolite) 

>50 µg/bee (Iwasa et al., 2004) 

IC-0 (Acetamiprid 

metabolite) 

>50 µg/bee (Iwasa et al., 2004) 

IM-O (Acetamiprid 

metabolite) 

>50 µg/bee 

Imidacloprid 

(dose range to 

give 8 – 100% 

mortality) 

17.9 ng/bee 9.2-31.5 (95% CI) Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 




Imidacloprid 6.7 ng/bee 5.2-8.2 (95% CI) Worker Bees (A. CO2 anaesthesia (Suchail et al., 

m. mellifera) 

2000) 

Imidacloprid 23.8 ng/bee 22.3-25.3 (95% CI) Worker Bees (A. CO2 anaesthesia (Suchail et al., 

m. mellifera) 

2000) 

Imidacloprid 15.1 ng/bee 11.9-18.3 (95% CI) Worker Bees (A. 

m. caucasica) 

CO2 anaesthesia (Suchail et al., 

2000) 

Imidacloprid 27 ng/bee Apis cerana indica CO2 anaesthesia (Jeyalakshmi et al., 

17.8% SL (5 – 52 

ng/bee) 

2011) 

Nitenpyram 0.138 µg/bee 0.0717-0.259 (95% 

CI) 

Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 

Thiacloprid 14.6 µg/bee 9.53-25.4 (95% CI) Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 

Dinotefuran 75.0 ng/bee 62.8-89.6 (95% CI) Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 

Clothianidin 21.8 ng/bee 10.2-46.5 (95% CI) Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 

Clothianidin 50% 14 ng/bee Apis cerana indica CO2 anaesthesia (Jeyalakshmi et al., 

WDG 

2011) 

Thiamethoxam 29.9 ng/bee 20.8-42.9 (95% CI) Worker Bees CO2 anaesthesia (Iwasa et al., 2004) 

Thiamethoxam 26 ng/bee Apis cerana indica CO2 anaesthesia (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 Not stated (Laurino et al., 

CI) 

2011) 

Imidacloprid 0.10 ppm 0.015-0.63 (Fiducial Worker bees Starvation 2 hours (Singh and 

(dose range to 

give 10-90% 

mortality after 24 

hrs) 

Limits) 

Karnatak, 2005) 

Thiamethoxam 5.200 ng/µl 4.302-6.227 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 

Thiamethoxam 15.16 ppm 2.84-97.00 (Fiducial Worker bees Starvation 2 hours (Singh and 



Page 29 of 133


Limits) Karnatak, 2005) 

LC50 - 24 hours - Contact (applied directly to the bee) 

Imidacloprid 

(0.00008 – 1.0% 

sol n ) 

LD50 – 48 hours – Oral 

Imidacloprid 

(0.1 to 81 ng/bee) 

2.2 (%w/v x10 -3 ) 1.5-2.6 (Fiducial 

limits x10 -3 ) 

Forager Bees CO2 anaesthesia (Bailey et al., 2005) 

41 ng/bee Worker Bees Starvation up to 2 

hr 



(Nauen et al., 2001; 

Schmuck et al., 

2003) 


m. mellifera) 

2000) 


m. caucasica) 

2000) 

Imidacloprid 70 ng/bee Worker Bees Not stated (Suchail et al., 

(1 - 1000 ng/bee) 

2001b) 

Imidacloprid 57 ng/bee ± 28 (SD) Worker Bees Starvation 2 hours (Suchail et al., 

(1 – 1000 ng/bee) 

2001a) 

Imidacloprid 

(0.2 – 3.2 mg/L -1 30.6 ng/bee 26.7-36.3 (95% CI) Worker Bees (Decourtye et al., 

) 

2003) 

Imidacloprid 3.7 ng/bee 2.6-5.3 (95% CI) Worker Bees (Schmuck et al., 

2001) 

Imidacloprid >21.0 ng/bee Worker Bees (Schmuck et al., 

2001) 

Imidacloprid 40.9 ng/bee Worker Bees (Schmuck et al., 

2001) 

Imidacloprid (as 11.6 ng/bee 7.3-18.3 (95% CI) Worker Bees (Schmuck et al., 

formulation 

WG70) 

2001) 

Imidacloprid (as 21.2 ng/bee 15.0-29.6 (95% CI) Worker Bees (Schmuck et al., 

formulation 

2001)

Insecticide 

SC200) 

Concentration Range Bee Type Pre-treatment Reference 

Imidacloprid 104.12 ng/bee 99.5 – 109.0 (95% Apis mellifera Not stated (Laurino et al., 

(0.15 – 150 ppm) 

CI) 

ligustica 

2010) 

Olefin 28 ng/bee ± 19 (SD) Worker Bees Starvation 2 hours (Suchail et al., 

2001a) 

Olefine >36 ng/bee Worker Bees Starvation up to 2 (Nauen et al., 2001; 

hr 


2003) 

5-OH-Imidacloprid 159 ng/bee Worker Bees Starvation up to 2 (Nauen et al., 2001; 

hr 


2003) 

5-OH-Imidacloprid 258 ng/bee ± 7 (SD) Worker Bees Starvation 2 hours (Suchail et al., 

2001a) 

5-OH-imidacloprid 153.5 125.9-196.9 (95% Worker Bees (Decourtye et al., 

CI) 

2003) 

Di-OH- 

>49 ng/bee Worker Bees Starvation up to 2 (Nauen et al., 2001; 

Imidacloprid 

hr 


2003) 

4.5 Di-OH- >1000 ng/bee Worker Bees Starvation 2 hours (Suchail et al., 

Imidacloprid 

2001a) 

Urea metabolite >99500 ng/bee Worker Bees Starvation up to 2 (Nauen et al., 2001; 

hr 


2003) 

Urea metabolite >1000 ng/bee Worker Bees Starvation 2 hours (Suchail et al., 

2001a) 

6-Chloronicotinic >121500 ng/bee Worker Bees Starvation up to 2 (Nauen et al., 2001; 

acid 

hr 


2003) 

6-Chloronicotinic >1000 ng/bee Worker Bees Starvation 2 hours (Suchail et al., 

acid 

2001a) 





CI) 

2011) 


ligustica 

2010) 


CI) 

2011) 


ligustica 

2010) 

LD50 48 hours – Contact (applied directly to the bee) 

Imidacloprid 61.0 ng/bee 26.0-90.0 (95% CI) Worker Bees CO2 anaesthesia (Nauen et al., 2001; 

(40-154 ng/bee) 


2003 

Imidacloprid 

(40-154 ng/bee) 

50.0 ng/bee 9.1-71.0 (95% CI) Worker Bees CO2 anaesthesia (Nauen et al., 2001; 


2003 

Imidacloprid 42.0 ng/bee 20.0–59.0 (95% CI) Worker Bees CO2 anaesthesia (Nauen et al., 2001; 

(40-154 ng/bee) 


2003 


(40-154 ng/bee) 


2003 


(40-154 ng/bee) 


2003 


m. mellifera) 

CO2 anaesthesia (Suchail et al., 2000 


m. mellifera) 



m. caucasica) 





Imidacloprid 81.0 ng/bee 55.0-119.0 (95% CI) Worker Bees (Schmuck et al., 

2001 

Imidacloprid 230.3 ng/bee Worker Bees (Schmuck et al., 

Imidacloprid (as 

formulation 

WG70) 

Imidacloprid (as 

formulation 

(SC200) 

242.6 ng/bee 173.3-353.4 (95% 

CI) 



2001 

Worker Bees (Schmuck et al., 

2001 

59.7 ng/bee 39.1-92.7 (95% CI) Worker Bees (Schmuck et al., 

2001 

LD50 – 48 hours Indirect Contact (contact with contaminated surface) 

Clothianidin 2.967 ng/µl 2.398-3.467 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 

Thiamethoxam4 3.313 ng/µl 2.786-3.806 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 



(1 - 1000 ng/bee) 

2001a) 


(1 - 1000 ng/bee) 

2001b) 

Imidacloprid 72.94 ng/bee 49.5 – 95.1 (95% CI) Apis mellifera Not stated (Laurino et al., 

(0.15 – 150 ppm) 

ligustica 

2010) 


2001a) 


2001a) 


Imidacloprid 

2001a) 


acid 

2001a)


Urea >1000 ng/bee Worker Bees Starvation 2 hours (Suchail et al., 

2001a) 


ligustica 

2010) 


CI) 

2011) 


CI) 

2011) 


ligustica 

2010) 

LD 50 – 72 hours - Contact (applied directly to the bee) 

Imidacloprid 104 ng/bee 83.0-130 (95% CI) Worker Bees CO2 anaesthesia (Nauen et al., 2001; 


2003 

LD50 – 72 hours – Indirect Contact (contact with contaminated surface) 

Clothianidin 2.667 2.121-3.156 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 

Thiamethoxam 2.462 ng/µl 2.156-2.903 (95% Forager Bee Not stated (Laurino et al., 

CI) 

2011) 



(1- 1000 ng/bee) 

2001a) 


(1 – 1000 ng/bee) 

2001b) 


2001a) 


2001a) 


Imidacloprid 

2001a) 



Page 34 of 133



acid 

2001a) 

Urea >1000 ng/bee Worker Bees Starvation 2 hours (Suchail et al., 

2001a) 

LC50 – Oral (duration not stated) 

Thiamethoxam 0.047 ng/mL Worker bees Not stated (Hashimoto et al., 

(newly emerged) 

2003) 

Thiamethoxam 0.074 ng/mL Worker bees ( 7 Not stated (Hashimoto et al., 

days old) 

2003) 

Thiamethoxam 0.081 ng/mL Worker bees (14 Not stated (Hashimoto et al., 

days old) 

2003) 

Thiamethoxam 0.101 ng/mL Worker bees (21 Not stated (Hashimoto et al., 

days old) 

2003) 

LC50 – Contact (duration not stated) 

Thiamethoxam 3.21 mg/mL Worker bees Not stated (Hashimoto et al., 

(newly emerged) 

2003) 

Thiamethoxam 3.50 mg/mL Worker bees ( 7 Not stated (Hashimoto et al., 

days old) 

2003) 

Thiamethoxam 4.51 mg/mL Worker bees (14 Not stated (Hashimoto et al., 

days old) 

2003) 

Thiamethoxam >5.0 mg/mL Worker bees (21 Not stated (Hashimoto et al., 

days old) 

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 90 th 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 

90 th 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. 



Table 9. Contact toxicity of combinations (from Defra project PS2368) 

LD50 (µg/bee) 

(95% 

+ Flusilazole (0.358 µg/bee) + Myclobutanil (0.161 µg/bee) + Propiconazole (0.224 µg/bee) + Tebuconazole (0.447 µg/bee) 

CL) 

Insecticide 

Clothianidin 

Imidacloprid 

Thiacloprid 

Thiamethoxam 

0.0350 

(0.0155- 

0.0607) 

0.0671 

(0.0438- 

0.1018) 

122.4 

(90.56- 

238.9) 

0.124 

(0.0768- 

0.3280) 

LD 50 

(µg/bee) 

0.0295 

0.0475 

439.3 

0.0538 

95% 

CL 

0.0230- 

0.0367 

0.0187- 

0.0912 

157.3- 

71052.1 

0.0254- 

0.1203 

synergism 

ratio 

Synergism ratio = . Insecticide LD50 . 

Insecticide + fungicide LD50 

LD 50 

(µg/bee) 

1.19 0.0451 

1.41 0.0409 

0.28 635.8 

2.30 0.0979 

95% CL 

0.0363- 

0.0559 

0.0205- 

0.0663 

184.1- 

35100000 

0.0804- 

0.1223 

synergism 

ratio 

LD 50 

(µg/bee) 

0.78 0.0312 

1.64 0.0585 

0.19 434.9 

1.27 0.0638 



95% 

CL 

0.0239- 

0.0393 

0.0379- 

0.0867 

156.8- 

65908.9 

0.0521- 

0.0788 

synergism 

ratio 

LD 50 

(µg/bee) 

1.12 0.00287 

1.15 0.0347 

0.28 266.8 

1.94 0.0479 

95% 

CL 

0.0213- 

0.0368 

0.0161- 

0.0568 

128.9- 

3738.9 

0.0302- 

0.0757 

synergism 

ratio 

1.22 

1.93 

0.46 

2.59#

Insecticide 

Clothianidin 

Imidacloprid 

Thiacloprid 

Thiamethoxam 

Table 10: Oral toxicity of combinations (from Defra project PS2368) 

LD 50 

(µg/bee) 

(range) 

0.00739 

(0.00607- 

0.00903) 

0.536 

(0.339- 

1.184) 

22.59 

(16.39- 

37.42) 

0.0112 

(0.00915- 

0.0135) 

LD 50 

(µg/bee) 

0.00441 

1.180 

54.65 

0.0103 

+ Flusilazole (0.358 µg/bee) + Myclobutanil (0.161 µg/bee) + Propiconazole (0.224 µg/bee) + Tebuconazole (0.447 µg/bee) 

95% CL 

0.00267- 

0.00762 

0.6094- 

5.878 

25.88- 

852.8 

0.00801- 

0.0136 

synergism 

ratio 

Synergism ratio = . Insecticide LD50 . 

Insecticide + fungicide LD50 

LD 50 

(µg/bee) 

1.68 0.00597 

0.45 1.075 

0.41 25.67 

1.09 0.00742 

95% CL 

0.00493- 

.000732 

0.5667- 

4.426 

synergism 

ratio 

LD 50 

(µg/bee) 

1.24 0.00572 

0.50 1.501 



19.01- 

40.70 

0.00448- 

0.01123 

0.88 47.01 

95% 

CL 

0.00467- 

0.00710 

0.6972- 

14.44 

27.45- 

166.1 

synergism 

ratio 

LD 50 

(µg/bee) 

1.29 0.00389 

0.36 0.893 

0.48 36.19 

1.51 0.00830 - 1.35 0.00852 

95% CL 

0.00305- 

0.00489 

0.438- 

14.50 

20.99- 

134.1 

0.00688- 

0.01037 

synergism 

ratio 

1.90# 

0.59 

0.62 

1.31

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 



2.4 Interactions between neonicotinoids and disease 


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. 



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 



2.5 Exposure of bees to pesticides 


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 



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 



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 



equirements. 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 



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. 



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) 

b) 

ng imidacloprid/bee 

ug imidacloprid/Kg 

1000000 

100000 

10000 

1000 

100 

10 

1 

0.1 

0.01 

0.001 

10000 

1000 

100 

10 

1 

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 

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 

no effect 



effect 

field nectar max 

no effect 

effect 

field nectar max

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) 

b) 

ug thiamethoxam/Kg 

ng thiamethoxam/bee 

10000 

1000 

3.5 

100 

3 

2.5 

2 

1.5 

1 

0.5 

0 

10 

1 

0 1 2 3 4 5 6 7 

study 

0 1 2 3 4 5 6 7 

study 

no effect 



effect 


no effect 

effect 

field nectar max

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 

imidacloprid ug/Kg 

100000 

10000 

1000 

100 

10 

1 

0.1 

0.01 

0 2 4 6 8 10 

study 

no effect 



effect 


field pollen max 

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 

10 

1 

0 1 2 3 4 

study 

no effect 

effect 


field pollen max

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 99 th 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’ 

Acetamiprid 

(99%) 

Apis mellifera 

Acetamiprid 

(99%) 

Honeybees 

- - - - Effects on communication 

and finding food reported 

Lab study / 

oral + contact 

/ adults 

Lab study / 

oral + contact 

exposures / 

adults 

1, 0.1 

µg/bee/d 

(oral, 

contact) 

0.1, 0.5, 

1µg/bee 

Contact: 1 µL 

(10% 

solvent) 

Oral: 

individually 

dosed in 10 

µL 40% (w/v) 

sucrose 

11d (from 

emergence) 

Locomotor activity 

1h after exposure 

Sucrose 

sensitivity: 1 h 

before and after 

treatment 

Olfactory learning: 

treatment 3 h 

before 

conditioning and 

recording 1 h, 24 

h, 48 h after 

Behavioural 

functions 

(i) Locomotor 

activity 

(ii) Sucrose 

sensitivity 

(iii) Olfactory 

learning 

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. 

(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) PER significantly 

reduced following oral 

exposure at 0.1 µg/bee 

after 48h only. No effect 

of contact exposure. 



Information from 

abstract, paper in 

Italian 

Doses selected to 

fall between 1/5 

and 1/500 of LD50 

Maccagnani et 

al (2008) 

Aliouane et al 

(2009). 

El Hassani 

(2008) 

Thiacloprid Lab/oral/15d 5.1 mg/L (in 10d Mortality rate Mortality at 10d post Vidau et al


Species 


[tested both with 

and without 

Nosema ceranae 

infection] 

Study type Test dose Test duration Endpoints Results / Notes Reference 

postemergence 

(10d post 

infection) 

[infected with 

Nosema 

ceranae - 

125,000 

spores diluted 

in 3 mL of 

water - at 5d 

post 

emergence] 

50% 

sucrose, 1% 

protein, 0.1% 

DMSO) 

(1/100 LD50) 

exposure/observa 

tion period 

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 


Species 

Imidacloprid 


Imidacloprid 

(98%) 


Study type Test dose Test 

duration 

‘Chronic feeding 

study’ 

Lab (flight 

cage)/oral / 

workers 

‘Sublethal’ - Sensitivity to 

imidacloprid 

when also 

stressed by 

Varroa 

destructor, 

Nosema apis, 

drugs or lack of 

48µg/kg 

(syrup) 

(i) 4d 

(treated) 

(ii) 4d 

(treated) 

Endpoints Results Notes (study 

reference in figure 

pollen supply. 

(i) Syrup 

consumption 

(ii) Foraging 

activity 

No indications of 

significant differences in 

sensitivity to imidacloprid 

between bees under other 

stressors and control 

bees 

(i) Significant reduction 

during treatment phase 

(ii) Significant reduction in 

during treatment (mean 



20) 


abstract, only 

abstract available 

(1) 

(2011) 

Reference 

Wehling et al 

(2009) 

(2) Ramirez- 

Romero et al 

(2005)


Species 

Imidacloprid 


Imidacloprid 

metabolites (olefin, 

5-hydroxyimidacloprid,4,5dihydroxyimidacloprid,ureaimidacloprid, 

denitro-imidacloprid, 

and 6-chloronicotinic 

acid) 



duration 

Lab study / contact 

exposure / adults 



0.1, 1, 10 

ng/bee 

(i) 1 ng/bee (+ 

further tests at 

0.1ng/bee for 

olefin and 5hydroxyimidacloprid) 

(iii) 2d 

(treated) 

15 min, 1 h, 

4 h after 

application 

(i) 15 min, 

after 

application 




learning 

Habituation 

(PER) 

Proboscis 

extension reflex 

(PER) 

4.8 visits) and post 

treatment (mean 20.4 

visits) phases c.f. before 

treatment (mean 23.7 

visits). 

(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 


(also at 0.1ng/bee). 

In 8-day-old bees only 5hydroxy-imidacloprid 

significantly decreased 

the number of trials 



20) 

Reference 

(3) Guez et al 

(2001) 

(4) Guez et al. 

(2003)


Species 

Imidacloprid 


Imidacloprid 

(Technical >97%) 

+ metabolites 


duration 



Lab study / oral 

acute & chronic 

exposures / adults 

(ii) 1, 10 

ng/bee 5hydroxyimidacloprid 

1µl at 1.25, 

2.5, 5, 10, 20 

ng/bee 

0.1, 1, 10 µg/L 

for 10 days 

and 

(ii) 1h after 

application 

0, 15, 30 

and 60 mins 

after 

treatment 

10 days 

8 days 



(i) Gustatory 

function 

(ii) Motor activity 

(iii) Nonassociative 

learning abilities 

(PER) 

Mortality; hyperresponsiveness, 

hyperactivity, 

required (not significant at 

0.1ng/bee) while olefin 

significantly increased the 


as in 7 d old bees(also at 

0.1ng/bee). 

(ii) Significant increase in 


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 



20) 

Reference 

(5) Lambin et al 

(2001) 

(6) Suchail et al 

(2001)


Species 

5hydroxymidacloprid;4,5dihydroxyimidaclopri 

d; 

Desnitroimidacloprid 

, 6-chloronicotinic 

acid; 

Olefin; 

Urea derivative 


Imidacloprid 

(99.4%) 


ligustica 

(5-OH-imidacloprid 

(99.4%) also tested) 


duration 



cumulative 

dose 0.01; 0.1; 

1 ng/bee after 

8 days (in 50% 

w/v sucrose 

solution) 

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 

Exposed 

11d from 

day 2 to 14- 

15. 



trembling Imidacloprid LD50 = 57 

ng/bee after 48 h, 37 

ng/bee after 72 & 96 h 

Mortality, PER, 

foraging 

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 

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 



20) 

Reference 

(7) Decourtye et 

al (2003)


Species 

Imidacloprid 

(98%) 

Apis mellifera L. 

Imidacloprid 


carnica 

Imidacloprid 

(Confidor) 



duration 







µL/bee/d 

2 - 32 ng/bee 

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) 

1 ppb 

(1/10 LD) 

100, 500 ppb 

at single dose 

(20µL) and ad 

libitum (in 50% 

sucrose) 

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 



Olfactory 

learning (PER) 

Size and 

morphology of 

HPG on 8- and 

14-days-old 

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. 

No significant on PER to 

sucrose solution alone. 

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 



bees 

Activity 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. 

20) 

Reference 


al (2004a) 

(9) Heylen et al 

(2011) 

(10) 

Medrzycki et al 

(2003) 

Imidacloprid Lab study / oral 500ng/kg (in Exposure24 Hypopharyngeal Significant HPG acinus (11) Smodis Skerl


Species 

(Gaucho) 


carnica 

Imidacloprid 

metabolites (urea 

NTN and 6-CAN) 


Imidacloprid 

(98%) 



duration 

exposure / adults 35% sugar 

water) 


exposure / Adults 

of two age cohorts 

(emerging versus 

foragers) 

Lab/contact 

exposure/worker 

bees 

0.1, 1 and 10 

µg/L (in 50% 

sucrose) 

(i) Topical 

application 

(1µl) 1.25, 

2.50, 5ng/bee 

(ii) Topical 

application 

(1µl) 1.25, 

2.50, 5ng/bee 

(iii) Topical 

application 

(1µl) 

1.25ng/bee 



, 48 or 72 h glands (HPG) 

acinus diameter 

in 1-6, 7-12, 13- 

18, 19-32d old 

24 h, 48 h, 

10 days 

(i) 15, 30, 

60mins 

(ii) 15, 30, 

60mins 

(iii) 15, 30, 

60mins 

(iv) 30mins 



bees 

Mortality, knock 

down, 

staggering, 

responsiveness 

(i) Gustatory 

threshold 

(ii) Locomotion 

(iii) Habituation 

(proboscis 


- PER) 

(iv) CO 

diameter variations at all 

treatment periods. 

No significant effects 

found. 

(i) No effects at 1.25 or 

2.50 ng/bee 

Loss of sensitivity after 

60mins at 5ng/bee 

(ii) Increase in 

displacements at 

1.25ng/bee 


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) Fewer trials to display 

PER at 1.25ng/bee. No 

effect of time. 

20) 

Reference 

and Gregorc 

(2010) 

(12) Schmuck 

(2004) 

(13) Armengaud et 

al (2002)


Species 

Imidacloprid 



duration 

(iv Intracranial 

injection of 

0.5µl (10-8, 

10-6 or 10-4M 

imidacloprid) 

at the brain 

surface 

Lab/oral/adult 0.7, 7, 70µg/kg 

Exposure 

method not 

defined. 

10d (i) individual 

energetic 

demands 



histochemistry (iv) Glomeruli. Significant 

increase in staining (+8% 

to +17%) of two regions of 

glomeruli in line with 

dose. 

α lobe. Reduction of CO 

labelling at 10-8M, 

increased labelling at 

other doses with 

significant increase at 10- 

4M (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. 

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 



20) 

Reference 

(14) Alaux et al 

(2010)


Species 

Imidacloprid 


carpatica 

Imidacloprid 


ligustica L.) 

Imidacloprid 

Honeybees 


duration 

(control, 

imidacloprid 

only, Nosema 

only, Nosema 

+ imidacloprid) 

Lab/oral/adult 0.05% to 

0.00005% 

Lab/oral/adults LD50/100- 

LD50/10 

Lab/oral/workers 4 and 8 µg/L in 

syrup (500g/L 



(ii) individual 

immunity 

(iii) social 

immunity 

2 to 300min Behaviour 

(mortality) 

60d 

(chronic) 

Olfactory 

sensitivity - 

Proboscis 


(PER) 

consumption) over 

control, imidacloprid and 

Nosema only groups 

(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 

Survival time Significant reduction in 

survival time. Survival 



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) 

Dechaume 

Moncharmont


Species 

Imidacloprid 


Imidacloprid 

(analytical standard) 


ligustica 

Imidacloprid 

(analytical standard) 



duration 

sucrose) 

Lab/oral/workers (i) 4, 8, 40ppb 

in diet 

[ 

(ii) 50 ppb 

imidacloprid in 

50% 

saccharose 

solution 

Lab/oral/workers 0.21ng/bee 

(24ppb) or 

2.16ng/bee 

(241ppb) in 

56% sucrose 

Lab/oral/workers 

(hives placed in 

(each bee fed 

7µL 


2.0mol/L 

sucrose 

solution using 

micropipette) 

0.21ng/bee 

(24ppb) 

in 56% 

(i) 11d 

(ii) 13d 

exposure 



(i) Olfactory 

conditioning 

(PER) 

(ii) Flight activity 

(flight cage) 

1h Sucrose 

response (PER) 

24h (i) Visits to 

feeder 

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. 

(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. 

Significant reduction in 

total PER/bee at both 

doses in nectar foragers 

and highest dose in pollen 

foragers. 

(i) No significant treatment 

related effect. 

20) 

was 20±0.95 µl/d 

(17) 

Reference 

et al (2003) 


al (2001) 

(19) Eiri and Nieh 

(2012) 

(20) Eiri and Nieh 

(2012)


Species 

ligustica temperature 

controlled room 

with sucrose 

feeder (50% w/w) 

1.5m from hive 

entrance) 

Imidacloprid 


Imidacloprid 


Imidacloprid 

(99.8%) 


ligustica 

Imidacloprid 



duration 



12d old at testing 

sucrose 

(each bee fed 

7µL 


2.0mol/L 

sucrose 

solution using 

micropipette) 

48ng/g in 

treated pollen 

(water, honey 

and pollen 

1:2:7 by 

weight) 

48ng/g in 

treated pollen 

(water, honey 

and pollen 

1:2:7 by 

weight) 

Lab/oral/workers 4 or 8µg/L in 

sucrose 

solution 


(Video-tracking 

study) 

0.05, 0.5, 5.0, 

50 or 500ppb 

in sucrose 

7d 

exposure 

7d 

exposure, 

1d 

starvation, 

1d test 



(ii) Average 

number of 

dance circuits 

per nest visit 

(iii) Unloading 

wait time 

(i) Mortality 

during chronic 

exposure 

(ii) Feeding 

behaviour 

(i) Visual 

learning 

capacity (Tmaze) 

(ii) Conditioned 

PER 

60d Sucrose 

consumption 

26h (i) Activity 


waggle dances in 

imidacloprid treated group 

(iii) No treatment related 

effect on wait time 

(i) No significant treatment 

related effect 

(ii) Significantly reduced 

pollen consumption over 



7d 

(i) Significantly fewer 

successful bees un 

treated group. 

(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 

20) 

Reference 

(21) Han et al 

(2010) 

(22) Han et al 

(2010) 

(23) 

Moncharmont 

et al (2003) 

(24) Teeters et al 

(2012)


Species 


duration 



20) 

(ii) Time spent in 

food zone 

(iii) Time 

interacting 

doses. 

(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 


Species 

Thiamethoxam 

(97%) 



duration 

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 



21) 




(1) 

Reference 

Reference 


(2009).


Species 

Thiamethoxam 

(97%) 

Honeybees 

Thiamethoxam 



duration 



/ adults 



0.1, 0.5, 

1ng/bee 


Oral: 

individually 

dosed in 10 µL 

40% (w/v) 

sucrose 

6 µg/mL, 

3 µg/mL, 

1.5 µg/mL, 

0.5 µg/mL, 

0.05 µg/mL, 

0.005 µg/mL 

Locomotor 

activity 1h 

after 

exposure 

Sucrose 

sensitivity: 1 

h before 

and after 

treatment 

Olfactory 

learning: 

treatment 3 

h before 

conditioning 


recording 1 

h, 24 h, 48 

h after 

0-, 7-, 14- 

and 21-dayold 

bees in 

boxes tests 

with feeder 

for 24 h 



(i) Locomotor 

activity 

(ii) Sucrose 

sensitivity 


learning 

(i) Mortality, 

significantly decreased for 

high sucrose 

concentrations (1 ng/bee). 

(i) No effects on 

locomotor activity 

(ii) No effects on 

sensitivity 

(iii) No effects on ulfacory 

learning 

(i) Age and dose related 

effect on mortality with 

higher mortality rates in 

younger bees 



21) 

Reference 

(2) El Hassani 

(2008) 

(3) Falco et al 

(2010) 

(ii) Acceptance / (ii) Rejection at high dose 

rejection of food, and acceptance at low 

(mixed with 

honey 1:1) 

dose 

Thiamethoxam Lab/oral/workers 3 ng bee - Associative Only 38% of the treated (4) Decourtye and


Species 


duration 

Honeybees 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. 

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


Species 

Fipronil 


Fipronil 

Honeybees 


duration 



Lab study / 

exposure through 

injection on thorax 

/ adults 

0.5ng/bee 

(c. 1/10 LD50) 

0.1 or 

0.5ng/bee at 

+20 and 

+60min 



21) 

Reference 

Devillers 

(2010) 

Endpoints Results Notes Reference 

3-24h Learning (PER) Decreased acquisition 

success. 

1h, 24h, 

48h 

Olfactory 

learning (PER) 

Subsequent memory 

performances lowered. 

No effect on distribution of 

responses to the tactile 

stimuli between sides. 

Olfactory learning 

significantly impaired at 

0.1ng/bee (1h and 24h 

only), but not impaired at 

0.5ng/bee. 

Bendahou et 

al (2009) 

El Hassani et 

al. (2009)


Species 

Fipronil 

(98.5%) 


Fipronil 


Fipronil 



duration 



/ adults 



/ adults 



/ adults 

0.1, 0.01 

ng/bee/d 

(oral, contact) 

0.1, 0.5, 

1ng/bee 

(+0.01ng/bee 

oral for PER) 


Oral: 

individually 

dosed in 10 µL 

40% (w/v) 

sucrose 

Acute:1, 0.5, 

0.1 ng/bee 

Chronic: 0.1, 

11d (from 

emergence) 

1 h before 

and 1 h 

after 

treatment 

Foraging 

bees (acute 

toxicity) and 

emerging 




Behavioural 

functions 

(i) Locomotor 

activity 

(ii) Sucrose 

sensitivity 


learning 

Memory, 

learning, odour 

sensitivity 

Mortality at 0.1ng/bee 

after 1 week 

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. 

Oral and contact 

exposure at 0.01ng/bee 

did not affect learning 

performance. 

(i) Oral or contact: no 

effect on locomotor 

activity 

(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) 





(2009). 

El Hassani et 

al (2005) 

Gauthier et al 

(2009)


Species 

Fipronil 

(98.5%) 


ligustica 

Fipronil 

(Reagent grade) 

Africanized Apis. 

mellifera 


duration 




exposure / larvae 

0.01 ng/bee bees 

(chronic) for 

11 days 

0.075, 0.15, 

0.3ng/bee/d 

(2.2, 4.5, 9 

µg/L in 

sucrose 

solution 

allowing 

33µL/bee/d) 

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 

0.5ng/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; 

0.01ng/bee (contact or 

oral) decreases odour 

discrimination 

Dose related mortality 

40.6, 87.3 and 91.1% 

(control 6.6%). 

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. 

Decourtye et 

al (2005) 

Cruz et al 

(2010)


Species 

Fipronil 



duration 

Lab/oral/15d postemergence 

(10d 

post infection) 

[infected with 

Nosema ceranae - 

125,000 spores 

diluted in 3 mL of 

water - at 5d post 

emergence] 

extract) 

1 µg/L (in 50% 

sucrose, 1% 

protein, 0.1% 

DMSO) 

(1/100 LD50) 

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. 

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 


Species 

Acetamiprid 

[Epik], 

Imidacloprid 

[Confidor], 

Thiacloprid 

[Calypso], 

Thiamethoxam 

[Actara] 


ligustica 

Thiacloprid 

(480g/L SC) 



duration 

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 

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) 

96g/ha 

(Phacelia 

tanacetifolia) 

(spray) 

- Repellency to 

foraging 

honeybees 

Monitored 

for 7-9d 




Mortality and 

health condition 

of broods. 

(i) Foraging 

activity 

(ii) Returning 

bees 

(iii) Hive vitality 

The investigation showed 

a ‘relevant difference’ of 

the selectivity level of the 

neonicotinoids. 

Acetamiprid (Epik) was 

the least toxic to 

honeybees. 

(i) Transitory reduction in 

foraging activity. Normal 

activity by 48h. 

(ii) Returning be numbers 

reduced for 24-48h after 

application. 

(iii) No systematic 

differences found. 


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 


Species 

Imidacloprid 

Honeybee 

Imidacloprid 

(Gaucho) 

Honeybees 


duration 

Dosed in field 20–100 μg/kg - Foraging and 

waggle dance 

Dosed in field / 

oral / workers 

10, 20, 50, 

100ppb (w/v) 

in saccharose 

solution 

(feeders 500m 

from hives) 

Not 

recorded 

Endpoints Results Notes (reference 

to study numbers 

in figures 20 and 

Communication 

behaviour of 

individually 

marked bees 

Foraging and dances 

affected at 20 μg/kg 

Deviation from correct 

angle information not 

affected. 

Communicated distance 

information substantially 

reduced at 50ppb and 

higher. 

Number (%) of bees 

performinging trembling 

dances greatly increased 

at 20ppb and higher 

Number (%) of bees 

performing waggle 



21) 

Data from EFSA 

report and reported 

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) 

Data from Kirchner 

WH (1988) The 

effects of sublethal 

doses of 

imidacloprid on the 

foraging behaviour 

and orientation 

ability of 

honeybees. 

Unpublished report, 

Konstanz, 13pp. 

(26) 

Reference 

Kirchner 

(1999) 

Cure et al 

(2001)


Species 

Imidacloprid 

(powder form) 


carnica 


duration 


oral exposure 

(bees treated in 

lab) / adults 

0.15, 1.5, 3, 

6ng/bee 

(dosed in 10µL 

of 2M sucrose 

solution) 

Immediately 

after 

treatment 

for 3h and 

24 and 48h 




Foraging 

behaviour: 

number of trips 

from hive to 

feeder, duration 

of trips, time 

interval between 

trips 

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 


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. 



21) 

Reference 

(27) Schneider et al 

(2012)


Species 

Imidacloprid 

(95% TG) 



duration 


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. 

Returning bees. 

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 

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 



21) 

Reference 

(28) Yang et al 

(2008)


Species 

Imidacloprid 

(Confidor) 


Imidacloprid 

(Confidor 200 SL) 


carnica 


duration 


oral exposure / 

adults 


oral exposure with 

syrup in hive / 

adults + brood 

100 ppb, 500 

ppb, 1000 ppb 

(in 50% 

sucrose 

solution) 

3.55 ng a.i./L 

Observation 

s 0-2h, 4-5, 

and 24h (for 

1h) after 

release 

Every 2-7d, 

after 

several 

weeks after 

treatment 




Feeding, 

foraging and 

homing 

behaviours 

Mortality 

Number of 

active bees 

inside hive 

Brood 

development 

Colony weight 

Feeders avoided at 500- 

1000ppb 

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. 



21) 

Reference 

(29) Bortolotti et al 

(2003) 

Fenoxycarb, 

Indoxacarb also 

tested (30) 

Beliën et al 

(2009)


Species 

Imidacloprid 


Imidacloprid 

Honeybee 

Imidacloprid 


mellifera 


duration 


oral exposure with 

syrup in hive / 

adults + brood 

Dosed in field 

/Larvae exposure 

in hive 


/oral/adults 

1 ppb? Every 2 

weeks for 

10 weeks; 

foraging 

behaviour 

was 

measured 

on 

individual 

bees of 13, 

15, 18, 20 

and 25 

0.4, 24, 200, 

2000, 4000, 

6000, 8000ng 

a.i./larva 

0.5or 5.0µg/L 

in saccharose 

syrup (50g 

saccharose+5 

0ml water) 

days-old 




21) 

Foraging activity 

Colony 

parameters 

(active & dead 

bees; surface of 

capped brood, 

colony weight). 

Foraging 

behaviour 

(phototaxis) 

- Capped brood 

rate 

Exposure 3 

times per 

week 12/07 

to 14/08 

(last check 

21/03) 

Pupation rate 

Eclosion rate 

(i) Activity 

(bees/min) 

(ii) Pollen 

carrying 

(iii) Occupied 

inter-frames 

(iv) Capped 

brood area 

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. 


capped brood, pupation 

and eclosion rates at 

2000ng/larva and above 

(i) Non significant 

increase 

(ii) Significant increase 

(iii) No significant effect 

(iv) No significant effect 



Fenoxycarb, 

Indoxacarb also 

tested (31) 

Reference 

Beliën et al 

(2010). 

(32) Yang et al 

(2011) 

(33) Faucon et al 

(2005)


Species 

Imidacloprid 

Honeybee 

Imidacloprid 

(Technical) 



duration 


/oral/adults 


/oral/colony 

(i) 0, 2, 10, 50, 

100. 500 ppm 

(imidaclprid 

240 FS in 

syrup) 

(ii) 0.05 and 

0.112kg a.i./ha 

(imidaclprid 

240 FS in 

syrup) 

(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 

Endpoints 

(v) Hive weight 

Results Notes (reference 



21) 

(v) No significant effect 



(i) 2d 

(ii) 4h 

(iii) 6h 

13wks 

exposure 

23wks 

observation 

period 

(i) Visits to 

feeders 

(choice test) 

(ii) Foraging on 

dandelions in 

orchard 

(iii) Foraging on 

trees and 

dandelions in 

orchard 

(i) Number of 

sealed broods 

during exposure 

period 

(ii) Colony 

survival (colony 

collapse) 

(i) Avoidance - visits 

reduced 7% at lower dose 

and 85% at higher 


foraging at higher dose at 

0.5h and 1h (59 and 60% 

reduction respectively). 


foraging at higher dose at 

4h (149% increase). 

(iii) No significant effect. 

(i) Significantly lower than 

controls but not 

concentration related 

(ii) Hive loss began at 18 

weeks post exposure. At 

23 weeks post exposure 

only one treated hive 

remained alive, only one 

(34) 

Reference 

Mayer and 

Lunden (1997) 

(35) Lu et al (2012)


Species 

Imidacloprid 


Imidacloprid 

(98%) 


ligustica 

Imidacloprid 

(analytical) 



duration 




21) 



Reference 

µg/kg HFCS control hive (of 4) had 

died 

Dosed in field 10 or 20ppb in Colony Development of Significantly greater (36) Pettis et al 

/oral/colony Megabee® exposed for Nosema over number of Nosema 

(2012) 

protein patties 10 weeks. 12d in newly spores per bee in bees 

Newly emerged bees from imidacloprid exposed 

emerged from 

colonies compared to 

bees imidacloprid controls 

collected at treated vs. 

5 and 8 untreated 

weeks and 

fed sucrose 

containing 

Nosema 

spores. 

colonies. 

Dosed in semi 24 μg/kg (in Recording (i) Foraging (i) Decrease in foraging (37) Decourtye et 

field; (flight cage) 50% sucrose visits at activity 

activity on the food source 

al (2004b) 

studies + feeder / solution) scented/uns 

and activity at the hive 

oral (feeding + 

cented sites 

entrance. 

foraging) and (LOEC for every 30s 

contact (PER) olfactory for 5min; (ii) Associative (ii) Significant effects 

exposures / adults learning bee counter learning found in both semi-field 

following to measure 

and laboratory conditions 

chronic oral colony 

exposure) activity at 

the hive 

entrance in 

June-July 

Dosed in semi- 6 µg/kg (in 8 control Foraging activity No significant effects on (38) Colin et al 

field (Tunnel) / oral 40% sucrose colonies at feeders in attendance at feeder. 

(2004) 

exposure / adults solution) during 5 tunnels 

days at 

Significant decrease in


Species 

Imidacloprid 


Imidacloprid 

(60%, Gaucho FS) 


ligustica 


duration 

Dosed in semifield 

(tunnel)/oral/adults 

(foraging) 

Field study / oral 


+ brood 

50µg/kg and 

higher 


sucrose 

25µg/kg and 

higher 


sucrose 

100µg/kg to 

3ug/kg 


sucrose 

Seeds treated 

with 0.24 mg 

a.i./seed 

(Sunflower 

treated at 600 

ml/100 kg of 

different 

times of the 

season and 

3 colonies 

before 

contaminati 

on and 

during 4 

days after 

Long-term 

(226 days: 

10 days in 

the field and 

observation 

s on the 

remaining 




Foraging activity 

(i) Frequency of 

visits to feeding 

station 

(ii) quantity of 

syrup taken 

(iii) duration of 

visits 

Population 

development 

and honey 

production (hive 

weight, nectar, 

pollen, brood, 

honey 

the proportion of active 

bees at the feeder 

(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 



21) 

Reference 

(39) Colin et al 

(2001) 

Stadler et al 

(2003)


Species 

Imidacloprid 

(Gaucho) 

Honeybees 

Imidacloprid 

Bee 

Imidacloprid 

(200 SL) 

Bees 

Imidacloprid 


Honeybees 


duration 

Field test / field / 

workers 

Field/field 

application/adults 

Field/field 

exposure/adults 

Field/field 

exposure/workers 

seed, 60.000 

seeds/ha) 

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 




216 days) production, 

foraging activity, 

pollen entrance 

and mortality) 

Not 

recorded 

Number of bees 

entering hive. 

Number of bees 

visiting flowers 

Hive weight 

development 

5d Bee visits to 

crop 

NR Number of 

foraging bees 

Flower visits 

following 

spraying at 

either green bud 

significantly more 

productive (higher weight, 

honey production, 

increased foraging 

activity, brood). 

High proportion of 

sunflower pollen in both 

treated and controls 

No effects on any 

parameter. No 

behaviourally impaired 

bees observed. 

Reduced number of visits 

compared to controls (in 

line with data for other 

insecticides, endosulfan, 

malathion) 

No effect on number of 

foraging bees. 

No effects on visits to 

flowers following either 

treatment therefore no 

repellency observed. 



21) 

Reference 

Cure et al 

(2001) 

Kumar and 

Singh (2012) 

Singh and 

Singh (2004) 

Gobin et al 

(2008)


Species 

Imidacloprid 

(200g/L) 

+ Oliocin 


Honeybees 


duration 

Field/oral 

exposure/colony 

and length of 

sprayed plot) 

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) 




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 

(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 



21) 

Reference 

Cantoni et al 

(2001)


Species 

Imidacloprid 

Confidor SL 200 

Honeybees 


duration 

Semi-field (Cage 

trial)/field 


0.6, 1.2, 2, 4, 9 

and 14g a.i./ha 

(on rape 

plants) 

4d (after 

treatment) 




21) 

brood cells. control and treated 

(v) Number of 

bees returning 

to hives 

(vi) Percentage 

of bees 

returning with 

pollen 

(vii) Visits to 

flowers 

(i) Foraging 

intensity 

(ii) Mortality 

(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) 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) No increase in 

mortality compared to 

controls. 



Reference 

Schnier et al 

(2003)


Species 

Imidacloprid 

(technical) 


Imidacloprid 

(Gaucho) 

Honeybees 

Imidacloprid 

(Gaucho 70 WS) 

Honeybees 


duration 

Semi-field (cages 

on field) / oral / 

workers 

Semi-field (Tunnel 

test) / field 

exposure / workers 

Semi-field 

(tunnels) /field 


Field/field 


0.002, 0.005, 

0.010 and 

0.020mg/kg (in 

sunflower 

honey) 

(no replication 

of tests doses) 

Between 0.35 

and 1.05mg 

a.i./plant 

(sunflower) 

39d 

(chronic) 

Not 

recorded 

0.005g/cm2 (i) 5d 




Feeding activity 

Wax/comb 

production 

Breeding 

performance 

Colony vitality 

Increase in bee 

numbers 

Number of 

foraging bees 

per 100 

No concentration related 

effects on any of the 

endpoints. 

No effects of treatment on 

these parameters. No 

records of behaviourally 

impaired bees. 

Fertilisation rates 

unaffected 



(ii) - 

(iii) - 

(iv) - 

(v) 4 dates 

(i) Foraging 

activity 

(returning 

foragers) 

(ii) Orientation 

(iii) Honey sac 

weight 

(iv) Effects on 

larvae 

(v) Foraging 

activity on flower 

clusters 

(i) No negative effect, 9.0 

bees/min vs. 8.2 bees/min 

(control) 

(ii) No effect 

(iii) No negative effect, 

26mg/bee vs. 25mg/bee 

(control) 

(iv) None recorded 

(v) No negative effect, 3.2 

bees/inflorescence vs. 8.2 

bees/ inflorescence 

21) 

French testing 

guideline CEB 129 

(adapted for seed 

treatment) 

Reference 

Schmuck et al 

(2001) 

Cure et al 

(2001) 

Wallner (2001)


Species 

Imidacloprid 

(Gaucho, 70% a.i.) 

Apis melliferea 

ligustica L. x A. m. 

caucasica L. 

Imidacloprid 

(Gaucho, 70% a.i.) 

‘Honeybees’ 


duration 

Semi-field/oral 


Semi-field/oral 


0, 0.35, 0.7 mg 

a.i./seed 

(in tunnels) 

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) 




21) 

- Behaviour, 

activity, flower 

foraging, 

progress of 

pollination, 

collection of 

nectar. 

- Vitality, foraging 

activity, 

behaviour 

(control) 

No effects observed on 

any of the measures 

No effects observed on 

any of the measures. 

No residues of 

imidacloprid found in 

sunflower nectar (LOQ 

10ppb) 



Reference 

In french Ambolet et al 

(1997) 

In french Ambolet et al 

(1999) 

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 

Clothianidin 

(powder form) 


oral exposure 

0.05, 0.5, 1, 

2ng/bee 

3 days after 

dosing 

Immediately 

after 

Return to hive Significant reduction in 

numbers of treated 

foragers returning 

particularly from 

Foraging 

behaviour: 

unfamiliar landscapes 

At 1ng/bee 73.8% 

returned to the hive 

(5) Henry et al 

2012 

(6) Schneider et al 

(2012)


Species 


carnica 

Clothianidin 

(Prosper FL, 

Poncho FS) 

Honeybees 

(control seed 

treated with blank 

(no clothianidin) 

Prosper Fl and 

Poncho FS) 


duration 

(bees treated in 

lab) / adults 

Field study / oral 


+ brood 

(dosed in 10µL 

of 2M sucrose 

solution) 

400 g 

a.i./100kg 

seed 

80kg seed/ha 

32g a.i./ha 

Seed 

treatment 

slurry 

contained 

Prosper FL 

(9.64% 

treatment 

for 3h and 

24 and 48h 

From spring 

to spring 

(1 year) 




number of trips 

from hive to 

feeder, duration 

of trips, time 

interval between 

trips 

Colony weight 

gain, honey 

production ; 

adult mortality, 

brood 

development, 

longevity 

versus 20.6% at 2ng/bee. 

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 

Cutler and 

Scott-Dupree 

(2007)


Species 

Thiamethoxam 

(Cruiser 350g a.i./L) 

Honeybees 

[seed also treated 

with Celest xl 

(fludioxonil and 

metalaxyl-M at 

0.525 and 

0.21 g/ha 

respectively)] 


duration 

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. 

Field/dust/workers 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,). 

[calculated as 

0.105mg/seed 

based on 

above] 

15d 

observation 

after 

sowing. 




(i) Direct 

mortality in hive 

area 

(ii) Foraging 

activity 

(i) Significant increase in 

mortality in period after 

sowing compared presowing 

and controls 

(ii) Reduction of foraging 

in all hives after sowing 

but significantly greater 

reduction in hives next to 

treated fields. 

Tremolada et 

al (2010) 

Table 18: Overview of semi-field and field studies of the effects of sublethal and chronic exposure to fipronil on Apis bees 


Species 

duration


Species 

Fipronil 


Fipronil 

(98%) 


Fipronil 

(98%) 


ligustica 


duration 


/oral/workers 


(Outdoor flight 

cage) + feeder / 

oral exposure / 

adults 


(tunnel) / oral 


2, 10, 50, 100, 

500ppm in 

syrup – Choice 

tests 

(50% sucrose 

solution and 

honey mixed 

3:1 v/v) 

1µg/kg 

(in sucrose 

solution) 

0.06 and 0.3 

ng/bee 

(6 and 30 

µg/kg in 50% 

w/w sucrose 

solution and 

providing 10 

µL per bee) 

4h on two 

test days 

with feeders 

placed at 

1030 and 

checks at 

1100, 1130, 

1200 and 

1230 

6 - 7 h out 

of the 

tunnel 




Visits to feeders 

(honeybees /5s 

/dish) 

Orientation 

capacities 

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 

Visits and consumption 


compared to control dish 

(untreated syrup) only at 

100 and 500ppm. 

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 

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 


Lunden (1999) 

Decourtye et al 

(2009) 

Decourtye et al 

(2011) 

Fipronil Dosed in semi- 2 µg/kg (in 8 control Foraging activity Significant effects on Colin et al


Species 

(analytical) 


Fipronil 

(80WG) 



duration 

field / oral 


40% sucrose 

solution) 

Field/field/workers 0.014 or 

0.028kg a.i./ha 

sprayed on 

canola 

(Brassica 

napus cv. 

Legend) 

colonies 

during 5 

days at 

different 

times of the 

season and 

3 colonies 

before 

contaminati 

on and 

during 4 

days after 

(i) 2d 



(ii) 3d 


at feeders in 

tunnels 

(i) Visits to crop 

(honybees /30s 

/9.1m) 

(ii) Mortality 

(Mean no. dead 

honeybees 

/colony in Todd 

traps) 

attendance at feeder. 

Significant decrease in 

the proportion of active 

bees at the feeder. 

Clinical signs of 

intoxication 

(i) Visits not significantly 

different from untreated 

(control) field 

(ii) Mortality not 

significantly different from 

untreated (control) field 

(2004) 


Lunden (1999)

Table 19 Overview of studies of the effects of sublethal and chronic exposure to acetamiprid and thiacloprid on non-Apis bees 


species/subspecies 

duration 

Acetamiprid 

Bombus terrestris 

Thiacloprid 

(Calypso 48% SC) 


Little detail 

(tomato plants) 

Lab (artificial 

nestbox) /Oral 


Little detail 

(maximum 

label rate?) 

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 

Pollination 

observed at 

3, 8 and 12 

d postreatment 



14d 

Up to 11 

weeks 

(chronic 

exposure) 

(i) Pollination 

rate 

(ii) Hive status 

Mortality, drone 

production and 

foraging 

behavior 

(i) Pollination slightly 

reduced compared to 

Flonicamid (Teppeki) 

treatment 

(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 

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. 

Very little detail 

given. Comparison 

of two treatments 

rather than with 

control 

This study reports 

the development of 

a new bioassay to 

assess the impact 

of sublethal 

concentrations on 

the bumblebee 

foraging behavior 

under laboratory 

conditions. 

Fanigliulo et al 

(2009) 

Mommaerts et 

al (2010)

Table 20 Overview of studies of the effects of sublethal and chronic exposure to imidacloprid on non-Apis bees 



Imidacloprid 


Imidacloprid 


Imidacloprid 



duration 

Field (lab 

exposure) /oral 

/colony 

Low: 

6µg/kg (pollen) 

and 0.7µg/kg 

(sugar water) 

High: 

12µg/kg 

(pollen) and 

1.4µg/kg 

(sugar water) 

Lab /oral /workers 10ppb (in 40% 

sucrose 

solution) 

Field / field / 

workers 

0.7mg/seed 

(sunflower) 


reference figure 

8 weeks Colony growth Significant effect on 

colony weight with low 

and high dose 8 and 12% 

smaller than controls 

respectively. 

28d 

(exposure) 

Effects at 

colony level 

(relative to 

controls) 


queen production with 

means of 13.72, 2.00 and 

1.4 in control, low and 

high respectively. 

Worker production 

significantly lower 

Brood number 

significantly lower 

Nest structure mass 

unaffected 

Worker mortality 

unaffected 

Worker loss significantly 

greater 

No colony losses (0/10 

failures) 

(iii) Losses were 33.5% in 

treated field and 23.1% in 

control (not significant) 



(i) 9d 

(i) Loss of 

workers 

22) 

Reference 

(1) Whitehorn et 

al (2012) 

Effects of λcyhalothrin 

alone 

and imidacloprid+ 

λ-cyhalothrin also 

tested 

(2) 

53% of foragers 

visited sunflowers 

in the control field, 

compared with 62% 

Gill et al 

(2012) 

Tasei et al 

(2001a) 

Tasei et al



Imidacloprid 

(97.5% TG) 

Osmia lignaria 

Imidacloprid 


Imidacloprid 



duration 

Field / Oral 

exposure (pollen)/ 

larvae 

Field /oral 

/workers 

Greenhouse / 

field / workers 

low (3ppb), 

intermediate 

(30ppb), or 

high (300ppb) 

in pollen 

provisions 

10ppb (in 40% 

sucrose 

solution) 

Not recorded – 

assumed 

0.7mg/seed 

(ii) 26d 

Total 

developme 

nt period 

28d 

(exposure) 



(ii) Population 

increase 

Mortality rate, 

Development 

duration, adult 

weight 

Effects on 

individual 

behaviour 

(relative to 

controls) 

4d (i) Total number 

of foragers 

visiting the 

(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 

Amount of pollen 

collected significantly 

reduced 

Duration of pollen 

foraging bouts + 

(i) No significant 

differences between 

control and treated plants. 



22) 

in the treated field. 

(Same data 

reported in both 

papers) 

Effects of λcyhalothrin 

alone 

and imidacloprid+ 

λ-cyhalothrin also 

tested (3) 

(Same data 

reported in both 

papers) 

Reference 

(2001b) 

Abbott et al 

(2008) 

Gill et al 

(2012) 

Tasei et al 

(2001a)



Imidacloprid 

(Confidor 20% SC) 



duration 




based on field 

study 

(sunflower) 

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) 



heads at each 

blooming stage 

(ii) Mean visit 

duration/head 

was estimated 

for 75 foragers 



foraging 

behavior 

(ii) Numbers of short 

(50s) 

visits not significantly 

different. 


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 



22) 





of sublethal 


the bumblebee 

foraging behaviour 


conditions. (4) 

Reference 

Tasei et al 

(2001b) 

Mommaerts et 

al (2010)



Imidacloprid 

(98%) 

Bombus occidentalis 

(Experiment 1); 

Bombus impatiens 

(Experiment 2); 

Imidacloprid 



duration 



exposurevia 

pollen or 30% 

sucrose 

solution/colony 

Lab / Oral 

(chronic) 

exposure via 

pollen sugar 

solution/adults 

Experiment 1 

Realistic 

residue level in 

pollen: 

7 ng/g pollen; 

Experiment 2 

7 and 30 ng/g 

pollen (in 

sugar water) 

D1 = 10 µg 

a.i./kg in syrup 

and 6 µg 

a.i./kg in 

pollen; 



11 weeks Pollen 

consumption, 

bumble bee 

worker weights, 

colony size, 

amount of 

brood, queens 

and drones 

produced and 

foraging ability 

on artificial 

flowers (only in 

the Exp 2). 

85 days Food 

consumption, 

survival rate, 

brood 

production, 

larval 

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 =



Imidacloprid 

(97.5% TG) 

Osmia lignaria 

Imidacloprid 

(in acetonitrile – 

removed before use) 



duration 

Lab / Oral 

exposure (pollen)/ 

larvae 

D2 was 25 µg 

a.i./kg (2.5 

times higher) 

in syrup and 

16 µg a.i./kg 

(2.7 times 

higher) in 

pollen 

low (3ppb), 

intermediate 

(30ppb), or 

high (300ppb) 

in pollen 

provisions 

Lab/oral/adults 0.08, 0.20, 

0.51, 1.28, 

3.20, 8.00, 

20.00, 50.00, 

125.00µg a.i./L 

(in syrup) 

Total 

developme 

nt period 

1d 

acclimatisat 

ion then 

13d 

exposure to 

treated 

syrup and 



development 

duration. 


Development 


weight 

(i) Worker 

fecundity 

(ii) First 

oviposition date 

(iii) Syrup 

worker survival rate by 

10% during the first 

month, without any doseeffect 

relationship. 

Before egg-laying, daily 

intake of 

imidacloprid from pollen 

and syrup was 2.15ng 

and 4.81ng per worker in 

D1 and D2 respectively. 

Brood production was 

significantly reduced in 

D1. 

No significant effect of D1 

and D2 treatments on the 

duration of larval 

development. 

No lethal effects 

Minor sublethal effects on 

larval development. 

(i) Significantly declined 

with increasing dose. 

(ii) No effect 

(iii) Significantly reduced 



22) 

Reference 

Abbott et al 

(2008) 

(6) Laycock et al 

(2012)



Imidacloprid 

Melipona 

quadrifasciata 

anthidioides 

(Native stingless 

bee) 


duration 

Lab/oral/larvae 0.0056, 

0.014, 0.028, 

0.037, 0.051, 

0.056, 0.08, 

0.112, 0.28, 

0.37, 0.56, 

1.12, 1.75, 

3.50, 7.00, 

14.00, 28.00 

or 56µg 

a.i./bee (fed to 

larvae) 

untreated 

pollen balls 

Exposure 

during 

larval 

period (c. 

41d) 

Behaviour 


morphomet 

ry at 1, 4 

and 8d post 

emergence 



uptake 

(iv) Oocyte size 

(i) Survival of 

larvae 

(ii) Walking 

behaviour of 

emerged 

workers 

(iii) 

Morphometry of 

mushroom 

bodies in 

with increasing dose 

(although imidacloprid 

intake still increased) 

(iv) significantly reduced 

by increasing dose 

(i) At 0.28-28 µg/bee 

larvae survived only 5d. 

At 56 µg/bee larvae 

survived50% only at lowest dose 

with dose related 

reduction. Development 

period and bodyweight 

not significantly affected 

(ii) No effect of dose at 

1d. Dose related effects 

(reductions in distance 

walked, reduced walking 

velocity, increased 

resting, increased number 

of stops in the arena) at 4 

and 8d. 

(iii) No significant effect at 

1d but growth 

compromised by 

imidacloprid exposure in 

dose related manner. At 

highest dose (0.112µg 

/bee) volume reduced by 

36% at 8d. 



22) 

Reference 

Tome et al 

(2012)



Imidacloprid 

(600 g/kg WP) 

(Intercept 60 WP) 


Imidacloprid 




duration 

Lab/oral/workers 0.0192mg 

a.i./g pollen 

Lab/treated 

plants/ adults 

(paste created 

by mixing 

pollen, honey 

and pesticide 

dispersion 

together in a 

ratio of 5:1:1. 

Balls were 

created coated 

with melted 

beeswax. A 2g 

ball was 

provided on 

which brood 

was started 

and remained 

with the colony 

throughout. 

Supplemental 

1g treated ball 

also provided 

which was 

replaced twice 

weekly) 

50ml/hl (on 

flowering 

cucumber 

plants) 

Treated 

pollen was 

provided for 

30d; 

colonies 

were then 

maintained 

on 

untreated 

pollen balls 

for an 

additional 

30 d. 

ELT 

Introduced 

1h after 

treatment, 

Endpoints 

emerged 

Results Notes (study 


22) 

workers 

(i) Number of 

days 

to first 

oviposition, 

(ii) Number of 

ejected larvae 

(iii) Total pollen 

consumption 

(iv) Worker 

lifespan 

(i) Oviposition not initiated 

(ii) No larvae produced 

(iii) Significantly reduced 

pollen consumption 

(iv) Significantly reduced 

lifespan 

Mortality Reduced mortality 

following delayed 

introduction (DELT) 

relative to early 



Complete 

reproductive failure 

(7) 

Reference 

Gradish et al 

(2010) 

Incerti et al 

(2003)



Imidacloprid 


Imidacloprid 

(Confidor 20% SC) 



duration 

Semi-field 

(Greenhouse) 

/exposure route 

not known 

/workers 

[tomato crop] 

Semi-field 

(greenhouse) 

/Oral 


observed at 

24, 48 and 

72h 

DELT 

Introduced 

24h after 

treatment, 

observed at 

24 and 48 h 

after this. 

- - Pollination 

efficiency 

20, 10 and 2ppb 

(in sugar water) 

for chronic and 

foraging 

assessments 

Up to 11 

weeks 

(chronic 

exposure) 





foraging 

behavior 

introduction (ELT) to 

treated plants. 

An interval of 7 days was 

necessary before 

releasing Bombus 

terrestris so that its 

pollination efficiency 

would not be reduced 

All workers affected at 10 

and 20ppb after 2 weeks 

with mortality of 62 and 

92% respectively and all 

survivors apathic. At 

20ppb nearly all dead 

bees found at feeder 

while at 10ppb all dead 

bees found in hive. 

Complete reproductive 

failure at these doses. 

No effects on mortality, 

reproduction or foraging 

behaviour at 2ppb. 



22) 

Information taken 

from abstract 

(paper in Italian) 





of sublethal 


the bumblebee 




Reference 

Vacante 

(1997). 

Mommaerts et 

al (2010)



Imidacloprid 


Imidacloprid 

(SL 200) 


Imidacloprid 



duration 

Semi-field 

(Greenhouse)/fiel 

d 


Semi-field 

(Greenhouse)/fiel 

d-spray/adult 

Semi-field 

(Greenhouses) 

/field exposure/ 

adults 

- 

Treated 7, 14 

and 21d 

before 

introduction of 

bumblebees 

into cold 

greenhouse 

(tomato crop) 

0.05 ml/plant 

(0.75 l/ha) 

[38, 48, 58 and 

68 days after 

transplanting 

of tomato 

plants] 

7d 

exposure 

before 

endpoint 

38, 44, 52, 

59, 66, 73 

and 80 

days after 

transplant 

of plants 

2, 3, 4, 5, 6, 

7 weeks 

1.5 months 

after setting 



Flower visits 

after 7d. 

(i) Pollination 

efficiency (4 

parameters). 

(ii) Flight 

frequencies. 

(iii) Laboratory 

assessments of 

final hive status 

(10 parameters) 

Visits reduced by 88, 50 

and 30% respectively 

compared to controls. 

In 21d before introduction 

group, visits returned to 

normal within 14d 

(i) No significant effect 

(ii) No significant effect 

(iii) No significant effects 

- - Foraging activity No negative effect on B. 

terrestris foraging on 

imidacloprid-treated 

plants 



22) 


abstract (paper in 

Italian) 

In Italian 

(information taken 

from Blacquiere et 

al 2012). 

Reference 

Nucifora and 

Vasquez 

(1999) 

Bielza et al 

(2001) 

Colombo and 

Buonocore 

(1997) 

Imidacloprid 

(non-heated 

greenhouses 

‘ragusa cultivated 

with tomato) 

Semi-field 15g a.i./ha 6 weeks (i) Pollination (i) Significant (p



Bombus terrestris L. (Greenhouses)/fie 

ld exposure/adults 

Imidacloprid 

(Merit 0.5 G, granular 

formulation) 



duration 

(bumble bee 

hives introduced 

for pollination) 

Semi-field 

(pollination 

cages)/field 


[Tall fescue, 

(Festuca 

Arundinacea) with 

25-50% flowering 

(foliar spray) 

0.4483 kg 

a.i./ha 

+ 

1.5 cm of 

irrigation from 

lawn sprinklers 





rate 

(ii) Fruit 

setting/develop 

ment 

(iii) Life span of 

colony 

(iv) Broth 

consumption 

(v) Colony 

parameters at 

end of study 

30d Weight (g) 

(i) Colony (with 

hive) 

(ii) Workers 

(iii) Queen 

No. in colony 

(i) Workers 

(ii) Brood 

reduction in pollination 

following both hive 

introductions 

(ii) ‘Inferior’ flower 

pollinations 

(iii) No effects observed 

(few larvae found around 

hives when checks made) 

(iv) 1st hive: c. 1/3 of 

control consumption 

2nd hive: c. 4/5 of control 

consumption 

(v) 1st hive: no. small 

larvae, weight of workers, 

weight of nest all lower 

than controls (nest weight 

c. 54% of control). 2nd 

hive: less clear 

differences 


for any endpoint 

22) 

applications were 

also made to all 

test plots including 

controls. 

Reference 

(2005) 

Gels et al 

(2002)



Imidacloprid 

(Merit 75 WP, spray 

formulation) 


Imidacloprid 

(Merit 75 WP, spray 


duration 

white 

clover cover] 

Semi-field 

(pollination 

cages)/field 


[Tall fescue, 

(Festuca 



white 

clover cover] 

Semi-field 

(pollination 

0.336 kg 

a.i./ha 

+ 

1.5 cm of 

irrigation from 

lawn sprinklers 

0.336 kg 

a.i./ha 



chambers 

(iii) Honey pots 

Defensive 

response 

(i) Time to initial 

response (s) 

(ii) Duration of 

response (s) 

(iii) No. of bees 

responding 



hive) 

(ii) Workers 

(iii) Queen 

No. in colony 

(i) Workers 

(ii) Brood 

chambers 


Defensive 

response 


response (s) 


response (s) 


responding 




for any endpoint 

Significant treatment 

related decrease for all 



22) 

Significant effects 

seen only in non- 

Reference 

Gels et al 

(2002) 

Gels et al 

(2002)



formulation) 


Imidacloprid 

(Confidor LS 200) 



duration 

cages)/fieldexpos 

ure/adults 

[Tall fescue, 

(Festuca 



white 

clover cover] 

Semi-field (tent)/ 

contact and 

oral/adults 

+ 

No irrigation 



hive) 

(ii) Workers 

(iii) Queen 

No. in colony 

(i) Workers 

(ii) Brood 

chambers 


Defensive 

response 


response (s) 


response (s) 


responding 

150g a.i./ha 28d Effects of single 

drench 

application on 

tomatoes 

endpoints except weight 

of queen 

49% mortality, 58% in 

controls 



22) 

irrigated sprayed 

plots 

Table 21: Overview of studies of the effects of sublethal and chronic exposure to clothianidin and thiamethoxam on non-Apis bees 



Clothianidin 

(99.75% TG) 

Megachile rotundata 


duration 

Lab / Oral 

exposure/ larvae 

low (6 ppb), 

intermediate 

(30 ppb), or 

high (300 ppb) 

in pollen 

provisions 

Total 

developme 

nt period 




Development 


weight 

No lethal effects. 

No major sublethal effects 

on larval development. 

23) 

Reference 

Sechser et al 

(2002) 

Reference 

(1) Abbott et al 

(2008)

Clothianidin 

(99.75% TG) 


Thiamethoxam 

(Actara 25% WG) 


Thiamethoxam 

(Actara WG 25) 


Semi-field (flight 

cage) /Oral via 

pollen (chronic 

exposure)/colony 




Lab/ contact and 

oral/adults 

6 and 36 ppb 

(in pollen) 

From the 

MFRC to 

several 

dilutions: 

100 (MFRC), 

10, 1, 0.5, 

0.2, 0.1 ppm 

and 10 ppb (in 

sugar water) 

0.1 ppm (1/1000 

MFRC) only for 

foraging 

behaviour 

(i) 10g a.i./ha 

(ii) 10g a.i./ha 

(50ppm in 

70% sugar 

solution) 

> 80 days Pollen 

consumption, 

progeny weight, 

number of 

males, queens 

and workers, 

foraging 

Up to 11 

weeks 

(chronic 

exposure) 

behavior. 



foraging 

behavior 

No colony effects were 

observed. 

The foraging ability of the 

workers bees tested on 

artificial flowers did not 

differ among treatments 

(but sample size small). 


100% mortality at 0.5 and 

1ppm at 1 and 3 weeks of 

exposure, respectively, 

0.2, 0.1 ppm and 10 ppb 

killed 83, 25 and 6% of the 

workers, respectively. 

Chronic 

LC50 = 0.12 ppm 

Total reproductive failure 

at100, 10, 1 and 0.5 ppm. 

Significant effect at 0.1ppm 

with number of drones only 

14% of control. 

EC50 = 35 ppb 

(i) 100% mortality 



(i) 7d 

(i) 7d 

(i) 12d 

(i) Contact 

toxicity glass 

plate 

(ii) Oral activity 

(iii) contact and 

residual activity 

(ii) 100% mortality 

(iii) 95% mortality 

(2) Franklin et al 

(2004) 





of sublethal 


the bumblebee 




Mommaerts et 

al (2010) 

Sechser et al 

(2002)

Thiamethoxam 

Bombus terrestris L. 

Thiamethoxam 


Semi-field 

(Greenhouses)/fie 

ld exposure/adults 

(bumble bee 

hives introduced 

for pollination) 

Semi-field (tent)/ 

contact and 

(iii) 40g a.i./ha 

(4x maximum 

rate) 

2 x 100g 

a.i./ha 7d apart 

(drip irrgation) 

OR 

Single 

application at 

200g a.i./ha 

(drip irrigation) 

(i) 40g a.i./ha 

on tomatoes 

6 weeks (i) Pollination 

rate 

(i) Hives 

placed in 

(ii) Fruit 

setting/develop 

ment 

(iii) Life span of 

colony 

(iv) Broth 

consumption 

(v) Colony 

parameters at 

end of study 

(i) Effects of 

foliar application 

(delaying release by 2d 

reduced mortality to 65%) 

(i) Significant (pdouble 

application>single 

application 

2nd hive: less clear 

differences 

(i) 92% mortality at 3 

weeks following 



Other phytosanitary 

applications were 

also made to all 

test plots including 

controls. 

Alarcon et al 

(2005) 

Sechser et al 

(2002)

Bombus terrestris oral/adults 

Thiamethoxam 



Semi-field 

(tunnel)/ contact 

and oral/adults 

(ii) 100g a.i./ha 

(iii) 40g a.i./ha 

(iv) 150g 

a.i./ha 

(i) 10g a.i./ha 

(ii) 10g a.i./ha 

(50ppm in 

70% sugar 

tent either 

immediatel 

y spray dry 

or 1wk later 

(ii) 28d 

observation 

, Hives 

placed in 

tent either 

immediatel 

y spray dry 

or 14d later 

(iii) 24d 

observation 

. Sprayed 

1, 2, 7 or 

14d before 

exposure 

(iv) 28d 

(i) 28d 

(ii) 35d 

(Phacelia) 

(ii) Effects of 


(tomatoes) 

(iii) Effects of 


(tomatoes) 

(iv) Effects of 

single drench 

application on 

tomatoes 

(i) Effects of 

single 

application via 

irrigation (Trial 

1) 

(ii) Effects of 

single 

application via 

immediate introduction. 

68% mortality if 

introduced after 1wk. 

Control mortality 50% 

(ii) 93% mortality at 3 

weeks following 

immediate introduction. 

94% mortality if 

introduced after 14d. 

(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 

(i) No negative effects on 

hives observed 

(ii) No negative effects on 

hives observed 



Sechser et al 

(2002)

Thiamethoxam 



Semi-field 

(tunnel)/ contact 

and oral/colony 

solution) 

150 and 161g 

a.i./ha (via 

irrigation 

system – 

single drip 

application) 

Hives 

opened 13- 

36d after 

application 

irrigation (Trial 

2) 

Impact on 

broods 

No negative impact 

deteceted. 



Sechser and 

Freuler (2003)

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Walorczyk S, and Gnusowski B, 2009. Development and validation of a multi-residue 

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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, 


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, 


10. (varroacid* or miticid*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, ti, 


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, 




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, 


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, 


21. (sublethal* or sub-lethal*).mp. [mp=ab, bc, bo, bt, cb, cc, ds, ge, gn, mc, mi, mq, or, ps, sq, st, 


22. 19 or 20 or 21 

23. 17 or 18 



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, 


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, 


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, 


33. 27 or 28 or 29 or 30 or 31 or 32 





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, 


38. 15 or 37 


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, 


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 



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] 




49. "interact*".m_titl. 



52. 36 use mesz 

53. 51 use mesz 

15 th 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 



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 


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 


*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 



(c) 2012 The Thomson Corp 

File 434:SciSearch(R) Cited Ref Sci 1974-1989/Dec 


Set Items Description 

S1 1690058 AGROCHEMICAL? OR PESTICID? OR CHEMICAL()CONTROL? OR HERBIC- 

ID? 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 



S21 486 S11 AND S19 

S22 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 (unique items) – ITEMS PRINTED FROM DATABASES NOT 

PREVIOUSLY SEARCHED 

S31 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 FOULBROOD? OR BACILLUS? OR LEISSOCOCCUS? OR PATHOGEN? OR 

D- 

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 



SYSTEM:OS - DIALOG OneSearch 

File 155:MEDLINE(R) 1950-2012/Aug 08 


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 


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 


*File 156: Toxfile has been reloaded with the 2012 MeSH Thesaurus. 

File 10:AGRICOLA 70-2012/Jul 


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 

THORA- 



COBOMBUS OR CERATINA OR ZADONTOMERUS OR FLORILEGUS OR HABROP- 

ODA OR ACENTRON OR AGAPOSTEMON OR AUGOCHLORELLA OR 

AUGOCHLORO- 

PSIS OR BEES OR BOREOCOELIOXYS OR COELIOXYS OR COLLETES OR CO- 

LLETIDAE OR DIALICTUS OR DIEUNOMIA OR EOXENOGLOSSA OR EUMELIS- 

SODES 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 PARAUGOC- 

HLOROPSIS 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 SU- 

B-CHRONIC? OR MULTIPLE()EXPOSUR? OR REPEATED()EXPOSUR? OR INC- 

REMENT?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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