the effect of imidacloprid combined with oxalic acid...
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The Effect of imidacloprid combined with oxalic acid mite treatment on Apis mellifera mortality
Courtney Wadley
MAY 19, 2016Advanced Scientific Research 2a
Nena Tippens
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley1
Abstract:
This study is a comprehensive collection of information on global bee colony decline and
the factors that contribute to it. Imidacloprid and oxalic acid have each individually been known
to contribute to Apis mellifera mortality and bee colony decline. While these factors have been
studied individually, little to no research has been conducted on how the combinations of these
and other factors affect mortality rates of Apis mellifera. Results suggested that the combination
of feeding Western honey bees imidacloprid and treating them with oxalic acid leads to
extremely significant and high mortality rates compared to other individual treatment groups.
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley2
Research Question
How does exposure to Varroa mite treatment and plant pesticide affect Apis mellifera
mortality?
Background Research
Introduction:
Agricultural Research in the United States has revealed that the global population of bees
(domestic and wild) is rapidly declining. This is troubling news, as bees serve as one of
agriculture’s most important pollinators. Honey bees for many years have been successful in
supporting human agriculture through pollination (Santos, et al., 2009). Some scientists believe
that the phenomenon is occurring because bees are being exposed to sub-lethal amounts of
pesticides, while others claim miticide (chemical treatment used to control pests in hives)
indirectly affect bee mortality. Many factors such as bee diets, pathogens, parasites, diseases,
global warming, and pesticide use were each thought to be the primary cause of this population
calamity, but recent studies suggest that bees’ interaction with multiple factors may be the
problem’s root (Pettis, et al., 2013).
Rapid decline in bee population negatively impacts the global economy. Because bees
provide such an essential ecological service, humans depend on food produced as a result of
pollination to make profit in the primary sector. There has been a six percent decline in vegetable
availability, and with many crop yields producing similar patterns, profits are also on a major
decline. Humans utilize many resources, including money, to harvest the pollinated crops. When
there is too high a demand and not enough supply, global food prices increase, making synthetic
and less healthy options objectively cheaper. This evidence proves the recent increase in bee
mortality is not beneficial to the primary sector of the global market (Potts, et al., 2010).
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley3
The recent decline in bees can be attributed to many factors. But while the factors have
been thought, in many cases, to be the cause of such a population and global crisis, these factors
have never been considered together as cause for global bee colony decline. This is a pressing
issue which needs to be studied with more intensity and focus on the relationships between
factors as opposed to individual effects. The two most commonly individually studied factors
seem to be the effect of plant pesticides on Apis mellifera and the effect of mite treatments on
Apis mellifera (sources). This is why experimenting to see the effect of mite pesticides and plant
pesticide treatments combined on bee mortality is so important to the future of global
environment health.
Apis mellifera:
The subject of this experiment is the Apis mellifera (or Western honey bee). Western
honey bees have existed for millions of years serving as pollinators (Hester, 2015). Apis
mellifera were brought to North America during the seventeenth century with the American
colonists. This is why Western honey bees are also known as European honey bees (Sammataro
and Avitabile, 1998). Western honey bees are classified as eusocial insects. Eusocial insects are
organisms which cooperate in a cohesive pattern and collectively share attributes of one
organism. Therefore, each colony functions as one cohesive organ system and performs tasks as
a superorganism. Eusocial insects, such as bees, work specifically as nest-bound superorganisms,
meaning the hive members depend on each other to maintain a hive’s (equilibrium) homeostasis.
Honey bees cannot survive individually without the support of a hive or colony (Bonoan, et al,
2014).
Apis mellifera hives maintain homeostasis and functions as superorganisms through a
caste system. The hive caste system dictates which occupants of the hives perform certain tasks.
According to the Ontario Beekeeper’s Association, the queen, workers, and drones make up the
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley4
three hive castes. The queen and worker bees are all female while the drone bees are male. The
queen bee decides whether a bee will be born a male drone or female worker through
fertilization. Fertilized eggs are born female workers while unfertilized eggs are born as drones
(2015).
The queen is at the top of the caste system function, and is essential to the survival of a
colony or hive. A queen can be recognized as being slightly larger in size compared to the
average worker bee which has short wings and a narrow abdomen. Queens live mainly to
produce eggs, therefore creating more bees, for the entire hive. Queens have an average life span
of two to four years and during that time laid an average of 365,000 to 547,500 eggs per year.
The queen is vital to hive survival, and because of that, the workers keep a queen well fed and
protected. Young worker attendants care for the queen, and constantly lick and groom her body;
thereby, distributing pheromones which are essential to hive survival. Queen bees become fertile
through a behavior known as the mating flights, in which the queen leaves the hive or colony as
a virgin queen, shortly thereafter, inseminated with thousands of drone sperm. If promiscuous
mating is not successful, the queen would only mate with a low number of drones, leaving the
health of the hive at risk due to the lack of brood (bee pupae) genetic diversity (Hester, 2015).
Worker bees are second in the caste hive system and also vital to hive health, with their
population making up the majority of a hive. Worker bees are infertile females and are a hive’s
smallest bees. These bees serve primarily to keep the cohesive hive organ system running. A
worker bee has several hive responsibilities which change during her life-span. During the first
few days the newly hatched worker bee keeps cells clean and warms brood cells. The next few
days she would proceed in the days after to feed older and then younger larvae. Next, she would
transport food and repair damages to the hive. Some worker bees then go on to attend to the
queen. The penultimate job for some workers is to become a guard bee, which is a bee that
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley5
protects/surrounds the queen and hive. The final job of a female worker bee is to forage for
resources. The collection of resources is important for hive survival, and is essential to colony
homeostasis (Hester, 2015).
Drones are at the bottom of the caste system. This type of bee possesses short legs, large
compound eyes, and large flight muscles. They do not perform any tasks throughout the hive and
nor do they feed themselves, because drones either barely or do not possess the physical
characteristics required to forage in the environment. These bees only serve the purpose of
fertilizing virgin queens during mating season. Drones mate by participating in the spring mating
flight, in which bees will fly near a hive and try to fertilize a virgin queen during flight. After
mating with a virgin queen, drones fall to the ground and die. These bees live towards the bottom
of the hive and are usually kicked out of the hive by female worker bees or are dead by winter
time each year (Hester, 2015).
During the winter time bees often face varying temperatures, which signals danger for the
incoming brood and the health of the hive. It is important to note the natural maintenance
practices of the hive. Broods in the hive need to maintain a steady temperature of 35-36o Celsius
at all times for positive growth and development. To keep this temperature constant, the worker
bees of the hive cluster around the brood cells and flex their thoraces (which are pressed against
the pupae) rapidly (Bujok, 2002). Honey bees must constantly regulate temperature because their
hive is immobile. This specific temperature range must be maintained because if temperatures
are not favorable brood mortality and a hindrance in development can occur. Unfavorable
temperatures can also lead to weak immune systems, causing increases in bee risk and
susceptibility to pathogens, pesticides, and disease (Bonoan, et al., 2014).
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Pesticides:
Many factors must come together in a hive so a colony can survive, but detrimental
agricultural practices such as intensive pesticide use has proven to hinder hive homeostasis. In
fact, scientists have pinpointed intensive pesticide use as the reason for such global bee
population decline. Researchers Chakrabarti, Rana, Bandopadhyay, Naik, Sarkar, and Basur
recently conducted a study investigating the effects of sub-lethal exposure to pesticide in an
agricultural landscape on the olfaction and overall health of the Indian (Western) honey bee. The
scientists asked the question, if native Apis ceranae are exposed to sub-lethal amounts (1.81
+ .04) of pesticide for a significant amount of time, then how does this affect the bees’ olfactory
system? The results found were that all honey bee samples produced similar results, there was a
decrease in Proboscis Extension Reflex (PER) in High Intensity Crop (HIC) honey bees; there
was a higher intensity of free (Calcium) Ca2+ in the brain of LIC (Low Intensity Crop) honey
bees, and multiple pesticides impact bee olfaction capacity. The results were presented in a
simple and easy to read format with an informative section which described how the results were
found. The authors came to the conclusion that bees being exposed to sub-lethal amounts of
pesticides in agricultural setting impairs the olfactory senses of bees, causing them to lose their
way back to the hive. Bees cannot survive without the support of a colony just as a colony cannot
function without workers (2015).
Research reveals the pesticides which most affects honey bees negatively are
neonicotinoids. Neonicotinoids are commercial insecticides which are used to kill or remove
unwanted pests from plants. Neonicotinoids were introduced into commercial agriculture
production in the 1990’s, and are now the most used pesticides/insecticides in the world. These
insecticides are utilized for agriculture in over one hundred and twenty different countries to
control over one hundred and forty different pests such as moths, caterpillars and other insects
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley7
harmful to crop health. Neonicotinoids are so widely used that they were valued at one point five
billion dollars in the 2008 stock market. The pesticide works by remaining present in plant
tissue, killing insects that consume the plants, but are thought to have no toxic effect on
mammals. Numerous studies, including conclusions found in the study conducted by
Chakrabarti, Rana, Bandopadhyay, Naik, Sarkar, and Basur, proves even sub-lethal exposure to
these pesticides are toxic to the honey bee. Many countries and organizations such as the
European Union have taken this information into account and have banned use of the three most
controversial pesticides. These pesticides include Imidacloprid, Clothianidin, and Thiamethoxam
(Lundin, et al., 2015). Honey bee risk is when Apis mellifera have higher chances of being killed
or hurt, and hindering bee population growth and sustainability. Neonicotinoids can hinder
cognition of honey bees, affecting how they forage and increasing Apis Mellifera mortality risk
(Tan, et al., 2014).
One research study confirmed claims that pesticides can even increase chance of
infection due to pathogens and parasites. In Crop Pollination Exposes Bees to Pesticides Which
Alters Their Susceptibility to the Gut Pathogen Nosema ceranae, scientists tested the hypothesis
that if Apis melifera experience consumption of honey bee diets, parasites, diseases and
pesticides in an agricultural setting, then the interaction the bees have with parasites will have
stronger negative effects on managed honey bee colonies. The background provided to support
the hypothesis includes information on research concerning the sub-lethal effects of pesticides on
bees, surveys on the different colony reserves and building material used, and background on
Nosema (the investigated pathogen thought to impair olfactory senses within Apis mellifera).
The results showed that all pollen collected contained pesticides, insecticides and fungicides. The
amount of pesticides found was discovered to be similarly large across all hives. In addition to
the pesticides present, researchers also found 147 out of all 630 bees were infected with Nosema.
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley8
Nosema impairs the olfactory senses of honey bees and causes them to become lost on their way
back to the hive from foraging. The researchers came to the conclusion that pesticides used by
farmers have significant effects on a bee’s susceptibility to parasite and Nosema infection (Pettis,
et. al, 2013).
Imidacloprid:
As stated previously, Imidacloprid has been banned from commercial use in certain
countries and organization membership provinces. This is because Imidacloprid is a type of
neonicotinoid pesticide. The metabolites of Imidacloprid aggravate nicotinic acetylcholine
receptors and negatively impact the pollinating behavior of Apis mellifera. Use of Imidacloprid
on crops statistically decreases pollination service quality by 6%-20%. This means that 6%-20%
of crops do not get completely pollinated by the bees. This decrease in performance also
negatively affects bees’ abilities to perform their duties such as enter the hive, forage, and even
return to the hive. A bees’ ability to return to the hive is crucial for bee and hive survival (Tan, et
al., 2014).
Imidacloprid is the most commonly used and researched pesticide (Lundin, et al., 2015).
In one research study, Imidacloprid Alters Foraging and Decreases Bee Avoidance of Predators,
Ken Tan and his fellow researchers hypothesize that if neonicotinoids (harmful insecticides) alter
honey bee foraging behavior, then neonicotinoids would also impair a bee’s ability to sense
danger and avoid predators. To support their hypothesis, Tan provided an extensive background
on how Imidacloprid (a neonicotinoid) decreases foraging activity of bees and how
neonicotinoids affect bee behavior around the world. Neonicotinoids have varying effects on
Apis mellifera around the world depending on region and indigenous species. The results of this
experiment showed there is a huge effect of pesticide concentration on the number of bees which
returned to feeders after foraging, and that increasing pesticide concentration reduces the average
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley9
amount of nectar collected, and that exposing bees to heavy concentrations of Imidacloprid
causes bees to not avoid dangerous predators. After gathering all of the results, it was concluded
that neonicotinoid pesticides impair honey bee cognitive responses and also concluded that
significant concentrations of neonicotinoids can impair a bee’s ability to sense and avoid danger.
Neonicotinoids are proving to be a key factor in global bee decline (Tan, et. al, 2014).
Miticide:
Another variable considered in the study is miticide. Miticide is man-made pest control
used heavily around the 1980s and still used today to remove mites from bee hives. In 1984,
Tracheal mites and Varroa mite infestations massively decreased United States bee population,
prompting the invention of miticide. These chemicals served as anthropogenic (man-made) hive
maintenance for years, and are supposed to not harm bees while killing off the mites. However,
evidence has shown that it is difficult to create miticide completely safe for bees yet still be
effective. Even with the advanced technology of miticide, Apis mellifera continue to die off in
colonies at alarming rates (Burley, 2007).
Bee mortality has shown a rapid increase in recent years, and rates have increased in
many regions of the world. Bee mortality rates are the average numbers of bee or colony deaths
per year. The United States has lost fifty nine percent of its bees from 1947 to 2005 and
meanwhile, Europe has lost twenty five percent of its colonies from 1985 to 2005. This
troublesome news proves that mortality rates are rapidly increasing year by year. There are
various consequences associated with this massive bee genocide: significant decrease in crop
yield (amount of food produced), decrease in obtainable profit from crops, and drastic negative
environmental impacts. When speaking specifically of harsh environmental impacts, a few
effects concerning bees are very important to understand. Western honey bees are a primary
agricultural pollinator. The crops they pollinate account for one third of all of the food humans
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley10
consume. Without bees, there would be a significant increase in demand for pollinator intensive
crops such as almonds, oil seed rape, watermelon, apples, broccoli, and many others. This
decline causes a decrease not only in biodiversity but also food supply. Pollination is the major
ecological service they contribute to human beings (Burley, 2007).
Nest Homeostasis:
Nest homeostasis is largely affected by the environmental conditions and factors of a
region/area. Homeostasis of a nest is defined as the monitoring and maintenance of internal
environmental conditions and temperatures. Hive homeostasis contributes to large brood rearing,
stable rearing conditions, forager warming, and the seasonal population of the hive. To maintain
hive homeostasis, proper worker bee characteristic (strong thorax muscles, strong wings)
development is of the upmost importance because they will often have to respond to extreme
differences in temperature throughout the year. Compounding problems limits homeostasis of the
hive. It does this by placing physical strain on the growth and development of worker bees, but
regardless of bee architecture, worker bees are still able to properly maintain a hive because of
physiological flexibility. (Winston, 1991).
Bees maintain homeostasis of the hive through temperature control. Honey bees try to
improve internal homeostasis by covering any openings with propolis. Propolis is a sticky sap
which acts as a sealant and provides proper insulation for the hive. During the winter, some
workers will even protect the hive with a curtain or cover of propolis. Comb design and structure
also contributes to hive homeostasis. This is because the hive’s brood chambers are covered with
protective temperature buffering wax comb layers. These layers are also protected by other
worker bees when they flex and contract thorax muscles in clusters around the hive between
combs. This action is often most necessary during winter due to extreme cold temperatures.
Honey bees store their energy during winter and use it through ‘hibernation’ periods to warm the
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley11
hive and maintain cell brood temperature. Workers will even consume stored honey to generate
the massive amounts of heat needed to maintain the hive. Extreme heat is not usually a problem,
with most tropical hives being located under shady areas. To produce heat during the winter
clusters, bees gathered together during the heating process, will often break apart to allow these
bees to feed for continuation of proper hive and brood warming. Even with significant honey
reserves, a hive can die during winter because bees were not allowed to occasionally leave the
cluster to feed. There is almost no brood rearing during this time period because of the constant
temperature fluctuations in the hive (Winston, 1991).
In conclusion, there are various factors at play when it comes to bee colony decline. Most
scientists in the past have believed it to be attributed to an individual factor when in truth, it may
have been a combination of factors all along. Each factor discussed in detail has proven to have
significant impacts on the behavior, but especially the mortality of Apis mellifera. The wide
disparity between studies involving factor combinations versus individual factor study without
evidence for a pinpoint source of bee colony decline suggests there might be some knowledge
left to gain. While individually the factors of Imidacloprid and miticide use have been thought to
negatively impact the mortality rate of Apis mellifera, the notion of combining these factors to
see if they have somewhat of a “synergistic” effect on Apis mellifera mortality seems to be a
concept worth exploring. Global hive homeostasis trends may truly be dependent on more factors
than just the two being studied, but in terms of research progression, the next step clearly is to
take combinations of factors into account. If the study conducted shows any amount of statistical
significance, it may be high time to start looking into more and more factor combinations. This
study focuses on looking outside the box in research methodology to find additional causes of
bee colony decline.
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley12
Hypothesis
If Apis mellifera are exposed to combinations of miticide to treat Varroa mites and
neonicotinoids, then hive mortality rates will be higher than mortality rates of those treated
individually with only miticide or only pesticide.
Justification
The purpose of this study is to expand understanding on the impact of how inadvertent
chemical combinations affect bee mortality and colony health. The combination of
neonicotinoids, a systematic pesticide widely used on food crops, with commonly used Varroa
mite treatments have individually proven in the past to be indicators of bee colony decline. Bee
colony decline is a plague which has spread throughout the globe, killing off entire populations
of bees. There have been several indicators hypothesized to be the cause of this massive bee
colony decline, but so far research has not produced conclusive results. Little to no research
however, has been conducted on how these indicators might work in tandem to cause bee colony
decline or increase bee mortality. The mite treatment and plant pesticide used in the experiment
are used most frequently on crops and hives around the world. Therefore, this study is vital to
expanding the understanding of how these indicators in combination affect bee death not just in
Western honeybees, but also bee species around the globe which are most exposed to these
commonly used plant pesticides and mite treatments.
Materials:
1 hive nucleus 1 Thermo scientific incubator (in lab)
1 Corning Stirrer/Hot Plate (In lab) 1 Teflon magnet coated stir bar (in lab)
1 Micro pipette 1 scientific balance (in lab)
Mason jars (for solutions) 140 cotton balls
Ziploc containers (cages) 2lbs Oxalic acid
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley13
2lbs Imidacloprid Screen Wire
Estimated Budget
1 hive nucleus - $150.00
16 16oz. Mason Jars (for solutions) - $57.60
20 Ziploc containers (cages) - $10.99
250g Oxalic acid - $80.70
100 mg Imidacloprid - $60.50
Cotton ball jumbo bag - $1.50
Phifer 48in by 25ft charcoal fiberglass screen - $34.86
Estimated total: $396.15
Methods: Step by Step
1. An insulated location to conduct the experiment was secured. Conducted the experiment
at the Georgia Tech ecology lab in Atlanta, Georgia.
2. In the ecology lab, there were three different solutions made for experimental use. First,
two liters of 1:1 sucrose solution were made by measuring out on a scientific balance
1000 grams of sugar and mixing it with 800 ml of water in a two liter glass bottle. The
solution was then placed on a hot plate and mixed together by a Teflon magnet coated stir
bar. Next, the Imidacloprid solution was made by placing .0125grams in a 500milliliter
bottle and mixing it with the previously prepared sucrose solution poured into the 500
milliliter bottle. .0125 g is the amount of imidacloprid bees are on average exposed to in
the field. Finally, the Oxalic acid solution was made by mixing 35g of Oxalic acid
powder in a 500ml glass bottle with sucrose solution.
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley14
3. After the solutions were made, frames were collected with a majority of capped brood
and placed in a Thermo-incubator. The incubator temperature was set to thirty-five
degrees Celsius and the brood were left to emerge from their caps overnight.
4. Twenty bee cages were then constructed to hold Apis mellifera for experimentation.
a. Twenty Ziploc containers were purchased. Then, a square three inch by three inch
hole was cut in each container lid and pre-cut screen wire was duck taped to the
lid top.
b. Twenty cages were created and each cage was labeled by group. The four groups
were Control (1:1 Sucrose Solution), Imidacloprid w/Oxalic acid, Imidacloprid
Only, and Oxalic acid w/ 1:1 Sucrose Solution. There were five cages designated
to and labeled for each experimental group.
5. The next day, the newly emerged bees were taken from their frames and placed in cages.
Ten bees were placed in each cage, meaning there were fifty bees designated to each
experiment group.
6. Each cage was then treated with its designated group treatment
a. Control Group – Cotton balls were drenched with 1:1 Sucrose solution, put on a
microgram (mg) weigh boat, and placed in each control group cage to orally feed
the Apis mellifera.
b. Imidacloprid w/Oxalic acid Group – Cotton balls were drenched in Imidacloprid
solution and placed in each Imidacloprid/Oxalic acid cage on an mg weigh boat.
Then oxalic acid was applied by using a micro-pipette to place one twenty micro-
liter drop on each bee in the cages.
c. Imidacloprid Only - Cotton balls were drenched in Imidacloprid solution and
placed in each Imidacloprid only cage on an mg weigh boat.
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley15
d. Oxalic acid w/ 1:1 Sucrose Solution - Cotton balls were drenched with 1:1
Sucrose solution, put on a microgram (mg) weigh boat, and placed in each Oxalic
acid w/ 1:1 Sucrose Solution group cage to orally feed the Apis mellifera. Then
oxalic acid was applied by freezing the cages for ninety seconds and afterwards
using a micro-pipette to place one twenty micro-liter drop on each bee in the
cages.
7. After administering the different treatments, the bee cages were placed back in the thirty-
five degree Celsius incubator and left over night.
8. After twenty-four hours, each cage was removed out of the incubator and a death count
was taken. Results and observations were then recorded and analyzed.
Results:
The project measured the effect of imidacloprid pesticide combined with oxalic acid mite
treatment on Apis mellifera mortality through an unpaired t-test and averages. By looking at
figure one, it is obvious to see the differences in mortality using oxalic acid vs. not using oxalic
acid. The control group were bees which had no oxalic acid administered. The graph shows that
no apparent deaths were recorded after the twenty-four hour experiment period. Results were
also recorded for Apis mellifera which were fed imidacloprid only. The graph shows that feeding
Apis mellifera imidacloprid only produces small amounts of mortality. On average those bees
which were fed imidacloprid only had low mortality rates. Results were also recorded for Apis
mellifera which were fed imidacloprid solution and treated with oxalic acid. The graph shows
very high mortality rates for this category, and has the highest mortality rates out of all the
experimental groups. The last group results were recorded for was oxalic acid treatment with
bees fed imidacloprid solution. This group also showed high mortality rates after a twenty-four
hour period, but proved to have an average mortality rate ten percent less than that of the
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley16
imidacloprid w/oxalic acid group. A Chi Square test was used to measure the significance of
these results combined. A Chi Square test was used because it compares the expected to
observed results of two or more variable groups. This test had more than two variable groups and
means, therefore requiring the use of a Chi Square test to measure significance. The Chi Square
test found a P-value of 5.9 x 10−13. The P-value rejects the null hypothesis the number of bees
that survived are equally distributed. This result is strongly significant, and suggests that the
combination of imidacloprid pesticide and oxalic acid mite treatment greatly increases average
Apis mellifera mortality.
Figure one displays the collective results of Apis mellifera death after twenty-four hours.
By looking at figure one and Apis mellifera death raw data one can infer that control had no bees
die during after each trial. It is also safe to infer that any combination using oxalic acid, whether
it be with sugar water or imidacloprid, results in high amounts of bee deaths after each trial.
From these results it is clear to see that the combination of imidacloprid with oxalic acid and the
administration of oxalic acid and sugar water (Appendix C).
Figure 1: Apis mellifera Death after 24hrs
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley17
From figure two, it is clear to see that Apis mellifera not treated and only fed sucrose
solution have on average no mortality and an extremely high percent survival rate after a twenty
four hour period.
Figure 2: Control Group Average Death & Survival
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley18
From figure three, it is clear to see that Apis mellifera treated with imidacloprid only have a
relatively low average mortality rate and moderate average survival rate after a twenty four hour
period. There exists in the graph on average a twenty percent survival rate and eighty percent
death rate.
Figure 3: Imidacloprid Only Group Death Average Death & Survival
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley19
From figure four, it is clear to see that Apis mellifera treated with oxalic acid and fed 1:1
Sucrose solution have a relatively high average mortality rate and moderately low average
survival rate after a twenty four hour period.
Figure 4: Oxalic Acid w/ 1:1 Sucrose solution Group Average Death & Survival
From figure five, it is clear to see that Apis mellifera fed Imidacloprid and treated with
oxalic acid have an extremely high average mortality rate and an extremely low average survival
rate after a twenty four hour period.
Figure 5: Oxalic Acid w/Imidacloprid Average Death & Survival
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley20
From these various averages, it can be determined that on average the combination of
imidacloprid and oxalic acid is ten percent more potent than the mortality rates of Apis mellifera
just being treated with oxalic acid and fed the 1:1 sucrose solution. It can also be determined that
the mortality rate average of bees treated with oxalic acid and fed with imidacloprid is seventy-
two percent higher than the average mortality rate of bees just fed imidacloprid only.
International data referenced in the lit review suggests that imidacloprid on average resulted in
the most bee deaths, due to imidacloprid’s ability to interfere with Apis mellifera olfactory
senses (Chakrabarti, et. al, 2015). That study focuses solely on one factor (or perceived cause) of
the recent trend in bee mortality increase, and its results contradict the findings of this study.
Thus, suggesting that research with combinations of factors needs to be more thoroughly
researched when it comes to finding the ultimate cause of global Apis mellifera mortality
increase.
Discussion:
The purpose of this study is to expand understanding on the impact of how inadvertent
chemical combinations affect bee mortality and colony health. The combination of
neonicotinoids, a systematic pesticide widely used on food crops, with commonly used Varroa
mite treatments have individually proven in the past to be indicators of bee colony decline.
Researchers have studied these factors individually multiple times, often resulting in numerous
single-cause studies, but currently there is little to no research on how combinations of these
factors affect Apis mellifera mortality. Bees come into contact with many chemicals, pesticides,
and particulates in the field, making it easy for them to “pick-up” two or more possibly
synergistic factors. More research on combinations of strong-correlation factors regarding Apis
mellifera should be conducted, because there are many individual factors that could possibly
have a negative synergistic effect on bees.
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From the results of this project, it is clear that the combination of feeding Apis mellifera
imidacloprid pesticide solution produces significantly high mortality rates for bees compared to
other experimental groups. According to the Graph Pad Software Inc. unpaired T-test, the results
of this group compared to that of other experimental group mortality rate showed a statistical
significance of .9286. This is extremely significant, and indicates that the combination of
neonicotinoid pesticides such as imidacloprid with the mite treatment of oxalic acid is very
potent to bees. The high mortality rates of even just that of bees treated with oxalic acid and fed
1:1 Sucrose solution (average of 82% mortality) indicates also that the use of oxalic acid to treat
mites might be hazardous to the health of bees. This is important to consider for any commercial
beekeepers or even hobby beekeepers in the United States especially, because oxalic acid
globally is presumed to be safe for usage and is slowly starting to gain tentative approval for use
by beekeepers. The results presented suggest overall, it is not safe to use oxalic acid dribble to
treat mites and treating crops with imidacloprid most likely will enhance the negative effects of
utilizing it.
This project is the first project to try to find a correlation between combinations of
pesticide and miticide honey bee exposure and globally decreasing Apis mellifera population.
Because of this, there is little to no information to compare it to. However, there is other data
about the effects of these factors individually correlating to global bee decline. In China, the data
for imidacloprid showed that testing at the experiment’s highest dose the result significance was
P = .012, respectively . This shows low significance in terms of p-value testing. Data in India
studying only the effects of intensive pesticides found olfactory senses were impaired
significantly by p<.01 using the 1 tail Mann Whitney U Test (Chakrabarti, et al., 2015). Those
results are of generally low significance. The results of the experiments in China and India
indicate why it is becoming more and more relevant to study factor combinations. Studying
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley22
factors individually does not provide enough statistically significant evidence to pinpoint one
factor as the main cause of the recent trends in global Apis mellifera decline.
There are three major improvements which should be made to this project. One major
improvement suggestion is to possibly wait a longer period of time before applying treatment to
the Apis mellifera. It would possibly be best to wait a day or so before administration of these
chemicals and pesticides because new-born bees have soft cuticles, which absorb liquids and
other chemicals relatively quickly compared to those bees of 2-3 days old with hardened cuticles.
Another improvement suggestion is to possibly lower the dose of oxalic acid administered to the
Apis mellifera. This might improve the experiment because bees are exposed to different dosages
in different countries, including India, often with lower dosage standards than the United States
gives for commercial usage of the product. A final improvement on experiment suggestion is to
handle the bees more carefully. While putting the new bees in their cages some exhibited
symptoms of diarrhea and vomit. This was probably due to the fact that many bees had to
quickly be placed in the cages, possibly causing discomfort and panic. This may affect results
being that they do not experience that process in their natural environment.
There are multiple ways to expand and extend this project. One future experiment to
conduct would be the effect of cuticle formation on Apis mellifera toxin susceptibility. That
experiment could be used to either support or deny the notion that soft cuticles make bees more
susceptible to absorption of harmful substances. The project could also indicate what time of
year is best to treat Apis mellifera as a commercial or hobby beekeeper. Another future
experiment would possibly explore honey bee diet, and look to see the effect of honey type on
the overall health of bees. This project could explore the various contents of multiple honey
types around the world, and even examine what content is in the honey of the country with the
largest population of bees. A final future experiment suggestion is a honey bee psychological
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley23
study which focuses on how honey bee foragers choose the flowers they pollinate. This will help
scientists and the entire beekeeping community learn which flowers honey bees are most
attracted to and possibly need to plant more of. There are various future experiments which can
extend further upon the effect of imidacloprid pesticide combined with oxalic acid mite
treatment on Apis mellifera mortality.
The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley24
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Appendix
Appendix: Page 24 Table of Contents
Appendix A: Page 25 Pictures of Materials
Appendix B: Page 26-27 Pictures of Experiment
Appendix C: Page 28 Apis mellifera Death after 24 Hours Raw Data
Appendix D: Page 29 Apis mellifera Death after 24 Hours Graph
Appendix E: Page 30 Apis mellifera Average Death after 24 Hours Raw Data
Appendix F: Page 31-32 Apis mellifera Average Death after 24 Hours Graphs
Appendix G: Page 33 Acknowledgements
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Appendix A
a. Bee Cages b. Honey Bee Brood Comb
c. Sucrose Solution d. Oxalic Acid
e. Imidacloprid f. Incubator Chamber
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Appendix B
A. Bee Cages with Hive Nucleus B. Capped Brood Cells
C. Bee Cage with Cotton Ball D. Glass Solution Jars
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E. Making Sucrose Solution F. Dissolving Sucrose into water
G. Measuring Out Imidacloprid H. Georgia Tech Urban Beekeeping Rooftop
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Appendix C
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Appendix D
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Appendix E
Apis mellifera Death after 24hrs
Treatment Type Percent Survival Percent DeathControl (1:1 Sucrose Solution) 100% 0%Imidacloprid w/Oxalic Acid 8% 92%Imidacloprid Only 80% 20%Oxalic Acid w/Sucrose Solution 18% 82%
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Appendix F
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The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley35
Appendix F:
Acknowledgements
First and foremost, I would like to thank my wonderful, amazing, talented mentor for
helping me through this process. I certainly could not have asked for a better person to not only
advocate for my project, but also be an advocate for me. When I started this project I did not
think I would ever apply for a grant, acquire membership into the Metro Atlanta Beekeeper’s
Association or even the Georgia Beekeeper’s Association, attend an amazing seminar on
beekeeping, use a smoker, and even have the ability to conduct my experiment in a professional
ecology lab. To me you truly are the world’s greatest mentors and one of the world’s greatest bee
lovers, and I could not have asked for a better advocate or a better person to help me on this
journey.
Second, I would like to thank the Georgia Beekeeper’s Association. Without your help, support,
and grant for my research, this project may not have been possible. It is truly inspiring to me to
have a group of people who believe in me and support my ambitious endeavors. I will be forever
grateful
Finally, I would love to thank Dr. Jennifer Leavey and the Georgia Tech Ecology lab for
allowing me to conduct my experiment in their facilities. It is a young researcher’s dream come
true. I can only imagine what would have happened if I had gone another direction without a
proper lab, so thank you for supporting such an ambitious project and an even more ambitious
young lady.
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