Imidacloprid is a commonly used systemic insecticide which can induce several sublethal effects. Previous research has not shown any increased mortality in bees that were fed with sublethal doses. However, there is very little research conducted with the focus on survival rate of honeybees in the field. The aim of this study is to assess the influence of imidacloprid on the survival rate of honeybees under field conditions. Honeybees from different colonies were administered a single dose of imidacloprid of 0, 0.07, 0.7, 7 or 70 ng per bee. From each concentration one group was kept in the laboratory to assess lethal effects and one group was returned to the hive to assess possible sublethal effects. The surviving bees were counted regularly during 4 weeks. Analysis has shown no difference in survival rate between treatments in the laboratory. There is a difference between the 70 ng treatment and the control group in the field (

Blacquiere, T.

English

NARCIS 

Survival rate of honeybee (Apis mellifera) workers after exposure to sublethal concentrations of imidacloprid

Wageningen Yield 

Adindah Visser & Tjeerd Blacquière
Bees@wur, Plant Research International, Wageningen University & Research, PO
Box 69, 6700 AB Wageningen, The Netherlands, E-mail: tjeerd.blacquiere@wur.nl
Imidacloprid is a commonly used systemic insecticide which can induce
several sublethal effects. Previous research has not shown any increased
mortality in bees that were fed with sublethal doses. However, there is
very little research conducted with the focus on survival rate of honey-
bees in the field. The aim of this study is to assess the influence of imi-
dacloprid on the survival rate of honeybees under field conditions.
Honeybees from different colonies were administered a single dose of
imidacloprid of 0, 0.07, 0.7, 7 or 70 ng per bee. From each concentration
one group was kept in the laboratory to assess lethal effects and one
group was returned to the hive to assess possible sublethal effects. The
surviving bees were counted regularly during 4 weeks. Analysis has
shown no difference in survival rate between treatments in the labora-
tory. There is a difference between the 70 ng treatment and the control
group in the field (P<0.0005), but because this treatment was conducted
on only two colonies, this result can not be generalized. Therefore,
increased mortality due to sublethal effects of imidacloprid is not
shown in this study.
Keywords: honeybee, Apis mellifera, sublethal effects, survival rate, imi-
dacloprid, systemic insecticides
The past decades show an obvious decline in honeybees. From 1947 to 2008 the
honeybee population in the United States decreased with 60% (vanEngelsdorp et
al. 2008). Also in Europe there is an obvious decline (Biesmeijer et al. 2006).
There does not seem to be a specific cause for the almost worldwide decline of
honeybees, but there are probably multiple factors that contribute to the weak-
ening and increased mortality of colonies. Possible causes are the introduction of
exotic parasites, such as varroa, that undermine the vitality of the colonies and
indirectly increase mortality (Martin 2001), decline of foraging resources
(Biesmeijer et al. 2006) and the use of pesticides in agriculture (Oldroyd 2007,
Bortolotti et al. 2003).
Survival rate of honeybee (Apis mellifera)
workers after exposure to sublethal
concentrations of imidacloprid
PROC. NETH. ENTOMOL. SOC. MEET. - VOLUME 21 - 2010 29
New pesticides are regularly developed to protect crops against parasites and
diseases. Before the introduction of a new product on the market, the lethal
effect on bees will be measured by determining the oral and contact LD50-values
in laboratory tests and during semi-field experiments (EPPO 1992). However,
this LD50-value only gives information about acute toxicity in comparison to
other pesticides, but does not say anything about possible adverse long-term
effects. It is known that low concentrations of pesticides do not induce acute
mortality, but may result in sublethal effects.
Products from a relatively new and currently commonly used group of insec-
ticides, neonicotinoids, are far more toxic than formerly used insecticides, so a
much lower concentration is needed to induce the same effect. Because the used
concentration is very low, these products are not instantaneously toxic to hon-
eybees in the field. Low concentrations can, however, cause sublethal effects
which are usually not seen in laboratory LD50-tests. A well-known insecticide
from the group of neonicotinoids is imidacloprid, commonly used as a seed
dressing in agricultural field crops or as drench in greenhouses.
Imidacloprid is a systemic insecticide and is therefore, after being absorbed
by the roots, translocated to all parts of the plant, including the nectar and
pollen. Since the introduction in 1994 it has been used as seed treatment for sev-
eral crops, like sunflower and maize. Several beekeepers in France noticed
unusual honeybee mortality after foraging on sunflowers, and blamed the use of
the new systemic pesticides. Because of this accusation, several studies focussed
on the sublethal effects of imidacloprid on honeybees, such as impaired olfacto-
ry memory (Decourtye et al. 2004) and learning performance (Ramirez-Romero
et al. 2005), and decreased mobility of bees (Medrzycki et al. 2003). Such effects
are not likely to be detected in the current registration procedure.
Although research has not yet given a complete answer to the question
wether imidacloprid is responsible for increased honeybee colony losses, most
researchers agree that the registration procedure must be adapted to the new gen-
eration of pesticides. Because sublethal effects are not assessed in laboratory
tests but are likely to lead to mortality under field conditions, it is important to
develop a method to measure mortality due to sublethal effects and integrate
such a method in the registration procedure for new insecticides. A possible
method is to focus on the survival rate of honeybees in the field, after exposure
to imidacloprid. The aim of this study is to assess the influence of imidacloprid
on the survival rate of honeybees under field conditions.
MATERIAL AND METHODS
Apiary
For this study four honeybee (Apis mellifera mellifera) colonies were used (A, B,
C and D). The colonies, held in 10-frame hives, were installed at the research site
SURVIVAL OF HONEYBEES EXPOSED TO SUBLETHAL IMIDACLOPRID
30
of Wageningen University and Research centre with 10 m distance between the
hives. Four days before the start of the experiments, each hive was provided
with a Muenstertrap (Illies et al. 2002), to collect dead bees that were removed
from the colony by undertaker bees. The colonies had not been treated against
parasites or diseases for at least 6 weeks before the start of the study, nor during
the study. The colonies were fed with extra sugar dye ad libitum.
Animals
From each colony four groups of 100 bees were collected. From two colonies each
100 extra bees were collected (colonies C and D). The bees were held in a flight
cage for at least 1 h, then they were put in small metal cages in groups of 10 bees
per cage. Each group was fed with a certain concentration of imidacloprid. After
feeding, the bees were sedated with carbon dioxide to ease handling. From each
group 50 bees were transferred to small cardboard cages (10 bees per cage) in a cli-
mate room (25 ± 2 °C and 80% relative humidity) and 50 bees were marked with
acryl paint on their thoraxes and returned to the colony. The experiment started
with one colony every week, for 4 weeks, on: 28 April, and 5 , 11 and 20 May 2009.
Experimental feeding
Admire® (70% imidacloprid) was administered to four replicate groups of 100
bees by ingestion at four concentrations: 0.07, 0.7, 7 and 70 ng imidacloprid per
bee per group. Each concentration was administerd as a single dose (10 μl per
bee). The concentration of 70 ng per bee was administered to bees of only two
colonies. The feeding solution for control groups was 50% sucrose, the same
solution was used for dissolving Admire® to various concentrations for the
experimental groups. The bees were provided clean sucrose solution ad libitum
after at least 30 min after they consumed the entire experimental solution.
Observations
The remaining marked bees in the colonies were visually counted at several
times after the treatment for 4 weeks. Dead bees in the Muenstertrap and in the
cages in the climate room were counted as well. Dead bees in the cages in the cli-
mate room were removed after each observation. The bees were counted three
times a week for the first 2 weeks after starting the treatments, and two times a
week for the last 2 weeks.
Statistical analysis
The Cox proportional hazard model was used to compare data (i.e., hazard rate)
from treated groups with control groups. Dead bees that were found in the
Muenstertrap were recorded as censored, whereas bees that were found alive in
the Muenstertrap were recorded as failures. Bees that died during handling or
that escaped from the cages in the climate room, were recorded as censored. Data
for laboratory and field observations were analysed separately, treatment and
colony were entered as covariates.
ADINDAH VISSER & TJEERD BLACQUIÈRE
31
RESULTS
After feeding the experimental solution, sublethal effects such as decreased
mobility, trembling and black-out were observed in the 7 and 70 ng treatments.
The survival probability for bees in the laboratory is shown in Fig. 1A. Colony
A was not analysed for the laboratory observations. Due to a problem with leak-
ing feeding tubes in the cardboard cages, too many bees had to be entered as cen-
sored events. The results for the remainder colonies showed no significant dif-
ferences between treatment and control groups.
Significant differences between the control group and treatments were
found in the colonies in the field. The P-values and hazard ratio’s are shown in
Table 1, Fig. 1B shows the survival probability for the bees inside the colonies
in the field. Over all colonies, a significant difference was only found between
the 70 ng treatment and the control group (P<0.0005) with a hazard ratio of
3.069. Marked bees from all treatments except 70 ng were seen carrying pollen
loads.
SURVIVAL OF HONEYBEES EXPOSED TO SUBLETHAL IMIDACLOPRID
32
Figure 1. Survival rate for bees in (A) the laboratory, and (B) the field. Black, control;
red, 0.07 ng; green, 0.7 ng; dark blue, 7 ng; light blue, 70 ng.
Table 1. P-values and hazard ratio’s for significant differences between treatments.
Colony Treatment P HR
B 7 ng / control 0.012 0.532
C 70 ng / control 0.006 1.945
D 7 ng / control 0.039 2.033
D 70 ng / control <0.0005 6.191
ABCD 70 ng / control <0.0005 3.069
DISCUSSION
The LD50 value for imidacloprid can vary between 3.7 and >81 ng per bee
(Schmuck et al. 2001, Nauen et al. 2001). Most researchers, however, assume 4-40
ng per bee to be general. In this experiment bees in the laboratory did not die
with 70 ng. To be sure that these results were not imputed to the used product,
an LD50 test following EPPO guideline no. 170 was conducted with the same
concentrations and the used product and a product from a newer batch. In this
test no difference in death rate was observed between the two used products.
The oral 48-h LD50 in this study was estimated to be >70 ng per bee.
A significantly lower survival rate was found in the treatment of 70 ng per
bee. This mortality was not observed in the laboratory and is therefore
ascribed to sublethal effects. However, this treatment is conducted on only
two colonies and the result can therefore not be generalized. Although research
has shown that leaf guttation drops from corn plants germinated from imida-
cloprid-coated seeds can contain such concentrations or even more, bees did
not seem to be attracted to drink from field-collected guttation drops
(Girolami et al., 2009).
All colonies were infested with varroa. In colony A many bees with
deformed wing virus have been observed. If the colonies were weakened by par-
asites and other pathogens, they might show a deviant interaction with imida-
cloprid.
Imidacloprid was administered only once and the bees were returned to the
hives at the end of the day. If bees collect contaminated nectar and pollen, they
would be chronically exposed to imidacloprid. According to Suchail et al. (2004)
the half-life of imidacloprid in the honeybee ranges between 4.5-5 h, which indi-
cates that most of the effects could be expected during the first few hours after
consumption. These effects cannot be seen in this study, because most bees were
in the hive at least till the next morning. If bees consume contaminated nectar
or pollen in the field, they possibly suffer directly from sublethal effects
(Bortolotti et al. 2003). 
From this study it cannot be concluded that a single dose contamination of
food induces a higher bee mortality in the field. Even though bee mortality due
to sublethal effects cannot be demonstrated with this study concept, it is never-
theless very important to develop a practical method to assess both lethal and
sublethal effects of pesticides, and integrate this method in the registration pro-
cedure for pesticides. 
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SURVIVAL OF HONEYBEES EXPOSED TO SUBLETHAL IMIDACLOPRID
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A survey of honey bee colony losses in the U.S., fall

Effects of Cry1Ab protoxin, deltamethrin and imidacloprid on the foraging activity and the learning performances of the honeybee Apis mellifera, a comparative approach.

Effects of imidacloprid in sub-lethal doses on honey bee behaviour. Laboratory tests.

Effects of sub-lethal doses on the homing rate and foraging activity of honey bees.

Guideline on test methods for evaluating the side-effects of plant protection products on honeybees.

Metabolism of imidacloprid in Apis mellifera.

Parallel decline in pollinators and insect-pollinated plants in Britain and the Netherlands.

Risk posed to honeybees (Apis mellifera L, Hymenoptera) by an imidacloprid seed dressing of sunflowers.

The influence of different bee traps on undertaking behaviour of the honey bee (Apis mellifera) and development of a new trap.

The role of Varroa and viral pathogens in the collapse of honeybee colonies: a modelling approach.

Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae).

Translocation of neonicotinoid insecticides from coated seeds to seedling guttation drops: a novel way of intoxication for bees.

What’s killing American honey bees?

Name not available

Survival rate of honeybee (Apis mellifera) workers after exposure to sublethal concentrations of imidacloprid

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Wageningen Yield

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