EVALUATING IMPACT OF NEONICOTINOIDS
ON POLLINATORS
A PRELIMINARY PROJECT PROPOSAL
SUBMITTED TO DAC
MINISTRY OF AGRICULTURE
SUBMITTED BY
DR. R.K.THAKUR
MEMBER SECRETARY
COMMITTEE ON FINALIZATION OF PROTOCOLS
ALL INDIA COORDINATED RESEARCH PROJECT
(HONEY BEES AND POLLINATORS)
DEPARTMENT OF ENTOMOLOGY,
INDIAN AGRICULTURAL RESEARCH INSTITUTE
PUSA, NEW DELHI
1
EVALUATING IMPACT OF NEONICOTINOIDS ON POLLINATORS
In recent years, insecticides belong to a chemical class, ‘neonicotinoids’ have been the
fastest growing class of insecticides in modern crop protection, with widespread use against a
broad spectrum of sucking pests. These chemicals target the nAChR which are more abundant in
insects thus made them highly specific against insect than mammals. In India, these pesticides
are approved for use against sucking pests infesting a number of agricultural/ horticultural crops
including bee attractive crops like mustard, cucurbits, cotton, citrus and sunflower. Imidacloprid
and thiamethoxam are approved on mustard and imidacloprid, thiacloprid, acetamiprid,
thiamethoxam and clothianidin on cotton. Among these neonicotinoids, clothianidin,
imidacloprid and thiamethoxam were categorized as highly toxic and acetamiprid and thiacloprid
as moderately toxic to honey bees. Besides lethal effects, under laboratory and semi-field
conditions with artificial dosing these insecticides also cause sub-lethal effects on useful
beneficial fauna including honey bees like reduced feeding and locomotor activity in bumble
bees, decision making in honey bees, thus having the potential to endanger vital ecological
services i.e. pollination provided by these beneficial insects. Since bees contribute towards 35
per cent of global crop production hence are quite important for sustainable agriculture. Based on
these research findings and association of these insecticides in causing colony collapse disorder
of honey bees (CCD), in Europe, recently European Commission (EC) issued a two year
moratorium for the use of three neonicotinoids viz.clothianidin, imidacloprid and thiamethoxam
for pest management on bee attractive crops. At the same time there is paucity of scientific data
on the harmful effects of neonicotinoids on honey bees in India, therefore, this research proposal
is formulated to study the effects of neonicotinoids applied on mustard and cotton on honey bees
when applied for pest management under Indian conditions.
IMPACTS OF NEONICOTINOIDS INSECTICIDES ON APIS AND NON APIS
POLLINATORS
Under-estimation of the pivotal role of insect pollinators is a key constraint to the
sustainability of contemporary agricultural practices. In agricultural ecosystem, many
agricultural crops are dependent on insects for their pollination, and assisted pollination may
have to be done when natural pollination is insufficient in order to reduce potential yield loss.
About 80% of the world’s flowering plant species are dedicated on animal pollination, mostly by
insect (FA0, 2007).The worldwide economic value of the pollination services provided by insect
pollinators in 2005 was estimated about €153 billion. The United Nations also reported on the
economics of ecosystems and biodiversity that insect pollination was valued at £134 billion.
Many plants have evolved intricate relationships with many insect pollinators, without which
they would not reproduce and maintain their diversity. At the global level, the convention on biodiversity has identified the importance of pollinators with the establishment of the International
Initiative for the Conservation and Sustainable Use of Pollinators (International Pollinators
Initiative-IPI) in 2000, facilitated and coordinated by FAO.Many ecosystems, including agroecosystem, depends upon pollinator diversity to maintain their biodiversity.
The whole world is suffering from decreasing of pollinators (both in abundance and diversity
although there is a deficit of robust data for many regions); its deficit directly impactspollination
service; pollination depletion affects agro- ecosystems crop productivity and their sustainability.
Pollination is one of the most essential ecosystem services effectual for crop productivity and
human livelihoods.Conservation and management of pollinators is still ignored in the agriculture
development program by the policy makers. With the commercialization of agriculture,
2
especially in developing countries, burning challenges are emerging to sustain their crop
productivity.Amongst insects, undoubtedly honey bees are ultimate pollinators because they are
active the whole year for pollination; do not hibernate, works longer periods daily than other
pollinators, number of worker bees per colony is also higher than the other insects, dense hairs
on the corbicula for carrying abundant pollens and their average foraging rate and meticulous
handling of flowers are much advance than other pollinators. Bees are dominant pollinators in
tropical region because their food sources are totally dependent upon flower.Apisspp. have been
considered to be most effectual pollinator for many plant species but unfortunately they are
drastically declining due to CCD (colony collapse disorder) causing global concern for
pollination services.
India Government has already passed food security bill which require more food grain to
fulfilment of their commitment.India population proliferating day by day now rich about 125
coror and food requirement have positive correlation with population; therefore more food
require for sustenance. India is a developing country where cultivated land has been exploiting
for construction of new roads, industrialization, new colonization etc. therefore for fulfilment
food security bill commitment only one way is remaining: enhance the productivity. Pollinators
can play pivotal role to enhance the productivity which is expected to be a tool of 2nd green
revolution.
Neonicotinoids embarked in Indian market during 1st decade of this century whereas it was
available in the developed country mid of 1990s. Neonicotinoids are synthetic chemical
insecticides their structure and mode of action is similar to Nicotine.European states have voted
in favour of a proposal to restrict the use of pesticides linked to serious harm in bees. Europe will
enforce the world's first continent-wide ban on widely used insecticides alleged to cause serious
harm to bees. Although the vote by the 27 EU member states on whether to suspend the insect
nerve agents was supported by 15 nations, but did not reach the required majority under voting
rules. The commission proposed the suspension after the EFSA concluded that three
neonicotinoids; Thiamethoxam, Clothianidin and Imidacloprid – posed an unacceptable risk to
bees. The three will be banned from use for two years on flowering crops such as corn, oilseed
rape and sunflowers, upon which bees feed. US Freedom of Information Act,calling for a ban on
neonicotinoid use as seed treatments because of their toxicity to birds, aquatic invertebrates, and
other wildlife.
OBJECTIVES OF THE PROJECT
• To quantify the impact of neonicotinoids on brooding behaviour and survival of
honeybees.
• To access the impact of neonicotinoids on foraging behaviour of honey bees.
• To ascertain the insecticidal residue in honey.
• To determine the extent of contamination in pollen and nectar.
• To study the toxicity of neonicotinoids to honey bee (A. mellifera) foragers and brood.
• To study the effects of contaminated nectar and pollen on honey bee foragers and brood
in semi field studies.
• Field studies on colony development.
USE OF NEONICOTINOIDS IN DIFFERENT CROPS
Active ingredient
Imidacloprid
Crop/ use
Cotton
Citrus
Pest
Sucking pest like Jassids, aphids,
mealybugs, thrips, white fly.
3
leaf miners, aphids and thrips.
Acetamiprid
Cotton
Citrus
Thiamethoxam
Cotton
Citrus
Thiacloprid
Cotton
Dinotefuram
Cotton
Citrus
Mustard
Imidaclopridand
thiamethoxam
Clothianidin
Cotton
Sucking pest like Jassids, aphids,
thrips, , white fly.leaf miners, aphids
and thrips.Aphids
Sucking pest like Jassids, aphids,
mealybugs, thrips, white fly.leaf
miners, aphids and thrips.
Aphis, thrips, white flies, boll worms,
stumborar.
Thrips, aphids, mealy bugs, white flies
Leaf minors
Sucking pest like Jassids, aphids,
mealybugs, thrips, white fly.
Jassids and White flies.
FLOWER AND SOWING TIME AT DIFFERENT PROPOSED CENTRES OF STUDY
Sr.No.
1.
Centre Name
Nagaland
Crop name
a. Mustard
b. Citrus
2.
3.
Solan
Bhubneshwar
a. Mustard
a. Mustard
4.
Ludhiana
a. Mustard
c. Cotton
5.
Andhra
Pradesh
a. Citrus
b. Cotton
6.
Pantnagar
a. Mustard
Flowering time
rd
December (3 week) to
February (1st week)
March (2nd week) to April
(2nd week)
October to February
November to January
Jan to February
Mid- July to mid
September
November, December,
January, February, June,
July
September, October,
November, December
Sowing time
September (2nd week)
October
(1st week)
September to December
October (Last week) to
November (First week)
Oct. 10-30
April-May 15
-
June, July, August



Five Acre
Mustard Crop
Sown on 26th
Sept 2014.
Five Acres
Mustard crop is to
be sown on 5th
October 2014.
Five Acres of
Mustard Crop is
to be sown on
4

15th October
2014.
Rest of the 10
Acres will be
sown later, to be
decided.
METHODOLOGY
• Sown crop have all the agronomical practices for proper vegetative growthbefore start of
the flower, the crop will be covered by net to restrict the pollinators activity.
• The desire strength of bee colonies will be placed in the confined area with known
number of foragers in different treatments.
• Thereafter, the crop will be sprayed at the label recommended timing and application rate
for the specified crop with molecules to be tested. Hive bees will be allowed to visit the
treated crops.
• Foraging behaviour of bees will be observed and growth and development of bee
colonies will be observed.
• The growth and development of brood in incubator will observed at optimum temperature
& humidity.
• The contaminated pollen grains collected and stored by foragers in the comb will be
isolated in vials and send for residue analysis to the concerned laboratory.
FIRST YEAR PLAN OF WORK
• Mustard and cotton crop will be sown in a measured area.
• The crop covered with net to confine the bees in the sprayed area.
• Three frames colonies (small) will be placed in each treatments
• The specific doses of insecticides will be applied in the field according to the label
• Protocols followed to observe larval developments of honey bees in laboratory
conditions.
• Semi field tests on larval development of honey bees will be carried out
• Tunnel test on honey bees will also be conducted
SECOND YEAR PLAN OF WORK
• The selected mustard and cotton crop will be sown in a measured area.
• Honey bee colonies with 3 frames prepared & placed in each treatments before blooming.
• The entire crop area covered with net to confine the bees in the sprayed area.
• Some of the experiments may be conducted in farmers field.
• The specific doses of insecticides will be applied in the field
• All the protocols followed to observe the larval developments of honey bees in laboratory
conditions.
• Semi field tests on larval development of honey bees will be carried out
• Tunnel test on honey bees will also be conducted.
EXPERIMENTAL DESIGN PER CROPS
• Treatments
• Molecule-1 x 3 replications =3
• Molecule-2 x 3 replications =3
5
•
•
•
•
•
Molecule-3 x 3 replications
Molecule-4 x 3 replications
Molecule-5 x 3 replications
Molecule-6 x 3 replications
Total number of plots
=3
=3
=3
=3
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ANTICIPATED OUTCOMES
• The impact of various neonicotinoids used in mustard, Cotton and Citrus crop could be
quantified.
• Growth & development of bee brood with the exposure of contaminated pollen can be
measured.
• Impact of neonicotinoids on foraging behaviour of bees can be determined.
• Residual effect of neonicotinoids in honey can be measured.
• On the basis of data generated through the various scientific trails can be used to seek
legitimate action for the sake of sustainable agriculture.
PROTOCOLS IN BRIEF
Laboratory tests
Honey bees (Apismellifera) larval toxicity test, provides useful guidance for honeybee
testing, breeding honey bees, studying honey bee biology and understanding honey bee pests and
pathogens.
Oral and contact toxicity of test compounds (Neonicotinoids) to larvae will be assessed in the
laboratory. They would be exposed to different doses of the compound by way of feeding or
topical application. Mortality values are used to provide a regression line and LD50.
The experimental unit consists of an individual cell containing a larva. A minimum of twelve
larvae from each of three colonies will be allocated to each treatment level and to the control and
toxic reference chemical. For each test, the following treatments and control(s) will be used. For
each chemical five increasing test concentrations (each containing a minimum 12 larvae X 3
colonies = 36 larvae minimum per treatment) will be used. The treatment will be supplied in the
food provided to the larvae. The study will be conducted according to OECD guideline 237 and
draft OECD guideline (Honey Bee (Apismellifera) Larval Toxicity Test, Repeated Exposure )
The treatment will be given to 2 day old larvae and the data will be recorded on the survivability
of the brood on Day 3 after treatment followed by on Day 8 , Day 15 Day 21(emergence day).
The number of emerged bees and non-emerged bees (pupal mortality) will be counted on Day
21. Brood survival rate and emergence rate will be calculated in percentage by comparing the
number of bees emerged on Day 21 to the number of larvae on Day 2 when dosing starts.
Simultaneously data will recorded on control larvae.. The studies will be considered valid if
control mortality prior to pupation is less than 15% and emergence of control is >80%. Toxic
reference mortality should be greater than 50%.
Semi field tests on larval development of honey bees
Semi-field tests involving cages, tunnels or tents, will be used. The cage will be having
minimum area of 40 m2. Cages having a surface of 8x5 m2 and approximately 2.5 m high will be
erected in a field. On top of a black water permeable cloth, the floor of the cage will be lined
with white water-permeable synthetic foil to facilitate recovery of dead honeybees. Small healthy
queen-right colony per cage containing approximately 3000-5000 bees and at least three full
frames containing all brood stages will be used. Honeybee hives will be placed on poles at
approximately 1.5 m high. The high position of the hive made the orientation of the honeybees
6
easier. Inside the cages the plants will be connected to a drip irrigation system, so that watering
of the plants occurred with minimal bee disturbance and without wetting the residue.
The test chemicals will be applied according to the label requirements; if the label permits
applications during flowering then applications should beduring the daytime when bees are
foraging most actively.
Data on larval development of honeybees will be recorded as per the methodology for the
laboratory tests. Tunnels will be used for control, test chemical and toxic reference to ensure data
generated can be interpreted.
Residues in pollen/nectar collected from honey bees prior to high tier testing.
For nectar collection each Apismellifera L. forager will be observed in the treated area.
The foragers will be collected with the insect collecting net and taken out of the net carefully
avoiding any pressure on its abdomen. The bees will be caught carefully from the wings and a
gentle pressure was applied on the abdomen upwards. The oozed out nectar, around mouth parts,
will be sucked up in lambda capillary tube and will be discharged into the vial.
For pollen collection, the bees will be caught carefully from the head and portion of the thorax in
between two fingers. The pollens foraged will be removed with the help of a hair brush (no. 1) in
vial. In each sample 20 foragers will be taken. After adding suitable solvent nectar and pollen
samples, collected will be marked and weighed vials. Similarly, samples will be collected for
other intervals starting from 0 h after spray (just after spray), 24 and 48h of spray. The collected
samples will be stored in deep freezer at −4°C temperature till extraction and clean up.
The collected samples of nectar and pollen will be for residue analysis.
Tunnel test on honey bees
Tunnel test comprised as a part of semi-field studies. The crop area covered by each
tunnel tent will be at least 40 m² with one bee hive placed in each tunnel. For the present study,
the honey bees will be exposed to the treated crop and checked under comparable conditions for
the different treatment groups, control and toxic reference. As standard, bee flight, bee mortality
and the condition of the colonies will be assessed. All tunnels will have the same orientation for
common disposal. The tunnel nets will be stretched out and embedded alongside the tunnel, thus
creating a closed environment limiting foragers' flights. Mustard crop will be grown under
tunnels. The studies will be conducted following the guideline European and Mediterranean
Plant Protection Organization – No. 170 (2010).
Twenty four semi-field tunnels will be erected:
- One tunnel for each replication/ pesticide to be tested. For six chemicals 18 semi-field tunnels
will be erected (3 replicates each)
- Three semi-field tunnels for control
- Three semi-field tunnels for reference chemical
The tested pesticide will be Appliedaccording to the rate and timing identified on the label for
the specified crop. The hives will be placed in the central part of the tunnel.
Observations
 Mortality and behaviour will be recorded at least 2 days prior to treatment and after treatment
on days 0, 1,2,3,5 and 7.Foraging activity will be assessed by monitoring number of bees/m2 of
treated crop. The behaviour of the bees on the crop and around the hive will be recorded. Dead
bee traps and those dying in rest of the cage (on water permeable sheets placed along paths or
edges of crops) will be counted.
 The condition of the test colonies (brood, pollen and nectar area) will be recorded just before
moving the colonies in to the cages, shortly before application of pesticide and after exposure
on different days.
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DETAILS OF PROTOCOLS TO BE ADOPTED
A) TOXICITY STUDIES (To be conducted by PAU Ludhiana And Division of
Entomology IARI, New Delhi):
1. Objective
: To test the toxicity of various neonicotinoids and a standard reference
insecticide against honey bees
: Apismellifera (workers)
: Serial concentrations of various neonicotinoids on cotton mustard
and citrus to be tested If residual toxicity is to be tested this should be
according to an accepted internation guideline, e.g. OPPTS Guideline
850.3030
2. Test Insect
3. Treatment details
Imidacloprid 17.8 SL
Thiamethoxam 25 WG
Thiamethoxam(For white flies)
Thiacloprid 21.7 SC
Thiacloprid 21.7 SC(For white flies)
Acetamiprid 20 SP
Clothianidin 50 WDG
Dinotefuran 20 SG
Thiamethoxam 25 WG
Imidacloprid 17.8 SL
1. Test method
:
A. COTTON
2.0 ppm, 0.2 ppm, 0.1 ppm,
0.05 ppm, 0.025 ppm, 0.012 ppm
2500 ppm, 250 ppm, 125 ppm,
62.5 ppm, 31.25 ppm, 15.625 ppm
5000 ppm, 500 ppm, 250 ppm,
125 ppm, 62.5 ppm, 31.25 ppm
24.955 ppm, 2.495 ppm, 1.247 ppm,
0.623 ppm, 0.311 ppm, 0.155 ppm
10.85 ppm, 1.085 ppm, 0.542 ppm,
0.271 ppm, 0.135 ppm, 0.067 ppm
1 ppm, 0.1 ppm, 0.05 ppm,
0.025ppm, 0.012 ppm, 0.006 ppm
1.5ppm, 0.15 ppm, 0.075 ppm,
0.037 ppm, 0.018 ppm, 0.009 ppm
2.5 ppm, 0.25 ppm, 0.125 ppm,
0.025 ppm, 0.031 ppm, 0.015 ppm
B. MUSTARD
1.25 ppm, 0.125 ppm, 0.062 ppm,
0.031 ppm, 0.015 ppm, 0.007 ppm
C. CITRUS
0.89 ppm, 0.089 ppm, 0.044 ppm,
0.022 ppm, 0.011 ppm, 0.005 ppm
Indirect contact Toxicity (ICT)
Leaves are to be sprayed at the standard application volume per unit area for the cropwith
pure water (Control) or with water suspensions of the insecticides to be tested. They are allowed
to dry in the shade for at least three hrs. After that, honey bees (10 bees/cage) are allowed to
walk freely on the cage bottom covered with leaves for 3 hrs. For each replication 2 or 3 cages
are to be used and replicate the test 2 or 3 times. Discard the replication with control mortality
>10%. Honey bees are considered dead when they remain totally motionless during a 10 seconds
observation period. Remove the leaves and honey bees are allowed to feed on sugar candy
throughout the trial and Note down the mortalities after 3 hrs, 6hrs, 12 hrs, 24hrs & 72 hrs. The
8
log concentration – mortality regression will be estimated by probit analysis using the MSTAT
Program based on calculations given by finney (1971)
FIELD TRIAL PROTOCOL (For Residue / Dissipation study)
1. Objective
:
To evaluate the residues and persistence (Dissipation) of
Neonicotinoids and a standard reference insecticide in Mustard.
Mustard
2. Crop
:
3. Treatment details :
Sr. No. Treatments
1.
Thiamethoxam 25
WG
4.
5.
6.
7.
8.
Design
Replications
Spray schedule
Spray Volume
Residue Analysis :
Dosage (g / ha)
T1 12.5-25
T2 25-50
Dosage of formulation / ha
T1 50-100
T2 100-200
:
RBD
:
3
:
Mustard : Single spray in according to the label
:
500 litres / ha.
Residues of neonicotinoids from the above mentioned
treatment to be determined in the leaves at 1,3,7, days after
spray or till the residues reaches BDL (0.01 mg/kg) as per
standardized methodology. In case of flowers, samples are
to be collected at 50 % flowering stage of the crop.
Analysis will be done by HPLC and LCMS.
FIELD TRIAL PROTOCOL (For Residue / Dissipation study)
1. Objective
:
2. Crop
:
3. Treatment details :
To evaluate the residues and persistence (Dissipation) of
neonicotinoids and a standard reference insecticide in Citrus.
Citrus
Sr. No. Treatments
Dosage (g / ha)
1.
Imidacloprid 17.8 SL T1 10 ml
T2 20
4.
5.
6.
7.
8.
Design
Replications
Spray schedule
Spray Volume
Residue Analysis :
Dosage of formulation / ha
T1 50 ml
T2 100
:
RBD
:
3
:
Citrus : Spray at 50% flowering of the crop
:
500 litres / ha.
Residues of neonicotinoids from the above mentioned
treatment to be determined in leaves at 1,3,7, days after
spray or till the residues reaches BDL (0.01 mg/kg)as per
standardized methodology. In case of flowers, samples are
9
to be collected at 50 % flowering stage of the crop.
Analysis will be done by HPLC and LCMS.
FIELD TRIAL PROTOCOL (For Residue / Dissipation study)
1. Objective
:
2. Crop
:
3. Treatment details :
To evaluate the residues and persistence (Dissipation) of
Neonicotinoids and a standard reference insecticide in Cotton.
Cotton
Sr. No.
Treatments
Dosage (g / ha)
2.
-
3.
Untreated control
(Water Spray)
Imidacloprid 17.8 SL
Dosage of formulation /
ha
-
4.
Thiacloprid 21.7% SC
T1 20-25 ml
T2 40-50
T1 24-30
T2 48-60
T1 100-125 ml
T2 200-250
T1 100-125
T2 200-250
T1 120-144
T2 240-288
T1 10
T2 20
T1 500-600
T2 1000-1200
T1 50
T2 100
T1 20
T2 40
T1 25
T2 50
T1 100
T2 200
T1 100
T2 200
T1 50
T2 100
T1 25-30
T2 50-60
T1 15-20
T2 30-40
T1 200
T2 400
T1 125-150
T2 250-300
T1 30-40
T2 60-80
T1 20-25
T2 40-50
T1 40-50
T2 80-100
For white flies
5.
Acetamiprid 20 SP
(For Aphids and Jassids)
For white flies
6.
Thiamethoxam 25 WG
For white flies
7.
Dinotefuran 20 SG
8.
Clothianidin 50 WDG
(For Jassids)
For white flies
4.
5.
6.
7.
8.
Design
Replications
Spray schedule
Spray Volume
Residue Analysis :
:
RBD
:
3
:
Cotton : Single spray after 65-70 days after sowing
:
500 litres / ha.
Residues of neonicotinoids from the above mentioned
treatments to be determined in leaves at 1,3 and 7 days after
spray or till the residues reaches BDL (0.01 mg/kg) as per
standardized methodology. In case of flowers, samples are
10
to be collected at 50 % flowering stage of the crop.
Analysis will be done by HPLC and LCMS.
B) SEMIFIELD TESTS (To be conducted by AICRP Centres):
Title: Evaluation of Impact of Neonicotinoids to the Honeybee Apismellifera L. and Apiscerana
under semi-field conditions (tunnel test)
Objective
The objective of this study is to determine the impact of Neonicotinoids in a realistic application
scenario on the honeybees Apismellifera L., Apiscerana, Bumble bees and Stingless bees under
semi-field condition (tunnel test) following the guideline European and Mediterranean Plant
Protection Organization – No. 170 (2010).
Principle
To evaluate mortality, flight activity, foraging activity, colony strength and behavior of small
healthy honeybee and other pollinators colonies after being confined in a tunnel to foraging on a
crop treated according to a pre-flowering GAP,
Design and layout of the Test
One test item treatment as per label recommendation, one toxic reference and one water control
treatment, each with three replicate tunnels and one bee colony per tunnel.
Details of bee colonies
Small healthy bee colonies procured from local bee keepers, disease free with no history of
medical treatment within the last four weeks are selected. The size of the colony will be chosen
based on available crop area per tunnel. For a tunnel of minimum 40 m2, each colony should
consist of the equivalent of approximately 3 full frames of brood cells in all stages, 1-2 food
comb with honey and pollen and approximately 3000-5000 worker bees. All hives will be
equipped with a dead bee trap at the entrance. The colony should be prepared as a “split” with
young workers shortly (2-3 days) before placing in the tunnels to minimize the number of
established foragers and placed in the tunnels at the start of flowering when there is sufficient
attractive crop. Soon after introduction into the tunnels any established foragers will fly into the
mesh sides of the tunnels and result in large numbers of dead foragers over the first few days
after introduction which in unrelated to treatment. Colonies should be prepared at least 3-5 km
from the test site to ensure re-orientation. During the whole testing, colony will be supplied with
water. Colonies will be exposed in the tunnel for a period of at least 14 days, or as long as the
flowering condition of the crop allows and then removed to a site where no pesticide exposure
will occur, where all colonies will be set up together in one location. The colonies are required
to be established as per flowering pattern of crops.
Test Conditions
Tunnels with a minimal size of 40 m2 will be used. The minimum height of the tunnel should be
2.5 m, to guarantee and unhindered flight of the bees. The covering gauze will have a maximal
mesh size of 3mm. The test crops will be mustard, cotton and citrus.
11
Test Product
The test product and toxic reference will be prepared freshly (within 2 hrs of application)
according to the GAP, in water and stored cool before use.
Application of test item
The application for the toxic reference and the control will be performed a few days after the bee
colonies have been set up in the tunnel, when the relocation-related background mortality after
the transfer has ceased. Treatment of toxic standard and water control will be done during full
bee flight (normally late morning). The application rate for each treatment will be recorded
based on nozzle output rate and time for application. Application will be made to the tunnels in
the order, control and then test product. The toxic reference will be applied at flowering.
During of the study
The assessment of the number of dead bees will be carried out approximately at the same time of
a day in the morning. Flight activity assessment will be done during times of high flight activity
of the bees. The total observation period of the colony development will be for at least 28 days.
Recording of Meteorological data
During the whole testing, data on temperature, relative humidity and rainfall will be collected on
daily basis and wind speed will be recorded during application inside the tunnel.
Mortality of honeybees
It will be assessed daily on linen sheets spread out in front of the hive and along central paths
running the length and across the tunnel. Additionally, dead bees present in the traps will be
counted. Assessment will done daily
The number of dead bees should be recorded separately as adult worker bees, larvae, pupae and
drones (males).
Mortality assessments in dead bee traps will be conducted throughout the whole studies; all other
mortality assessments only as long as the hives are set up in the tunnels.
Flight activity
Flight activity will be recorded 3 times each day (at peak foraging times, e.g. morning, early
afternoon, late afternoon) in a 1 m wide transect along the length of each side of the tunnel for
the 14 days the colonies are within the tunnels. This will also depend upon the crop under study.
At each assessment time the number of bees that are both foraging on flowering plants and flying
around the crop will be counted during walking along the transect in a set time period. e.g. 2
mins per transect. Flight activity assessment will be conducted as long as the colonies are set up
in the tunnels.
Condition of the colonies
The condition of the colonies will be assessed on the day before introduction into the tunnels and
on days 7 (± 1 day), 14 (± 1 days), 21 (± 1 day) and 28 (± 1 day) (the last 2 assessment whilst the
colonies are at the monitoring site)
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In order to record effects of the test item, the following parameters will be assessed:




Strength of the colony (number of combs covered with bees),
Presence of a healthy queen,
Comb area with pollen and nectar (in sq. inch),
Comb areas containing eggs, larvae and capped cells (square inches)
The estimation of the areas containing brood and food will be done in square inches each side of
each frame containing, pollen, nectar/honey, eggs, larvae, capped cell or are empty. This needs to
be done by a person (preferentially a beekeeper or bee researcher) experienced and trained with
this specific method.
Brood and colony condition assessments will be done throughout the whole study.
Evaluation of the test results
The evaluation of the results will be done by comparing the results using appropriate statistical
tools in the test item treatment to the water treated control and treatment and furthermore by
comparing the pre and post application data regarding






Mortality in the dead bee traps and on the linen sheets (number of dead adult worker
bees, pupae and larvae)
Flight activity in the crop
Condition of the colonies
Strength of the colonies
Brood development
Average brood area per hive
C) FIELD TESTS (To be conducted by AICRP Centres):
Title: Evaluation of potential effects of application of Neonicotinoids to the honey bee
Apismellifera L. and Apiscerana under field conditions.
The following study is designed to determine the effects of spray application of [active
ingredient] as the formulation X applied to crop on honey bee (Apismellifera L.) colonies in the
field when applied according to the GAP/label. During the exposure phase, the honeybee
colonies will be monitored at the fields with assessment for immediate post-exposure effects
(mortality and behaviour). The study will be conducted as field study (open field) under local
practical conditions in a representative crop-growing area. This study will be conducted in
accordance with the guidelines of the European and Mediterranean Plant Protection Organization
No. 170 (3) (OEPP/EPPO, 2010).
The exposure phase will be carried out using two experimental groups; one group comprised
three plots each with 6 honeybee colonies exposed to active substance treated crop. The other
group comprised three plots each with 6 honeybee colonies exposed to untreated (control) crop.
Application
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The test product and toxic reference will be prepared freshly (if practically feasible within 2 hrs
before application) according to the GAP, in water and stored cool before use. The application
will be made with calibrated equipment and the applied rate will be determined measured by
determining the volume of the test item before and after application with an application tolerance
of ±10%.
Test Location and Design
The study will be conducted at eight AICRP centres. There will be six different plots of 1-2 ha,
three of which as control and three as treatment replicates. The six different sites will be
separated by a least 2-3 km from each other to avoid bee foraging on the other plots where the
same crop as used in the study is grown. Moreover, the study fields will be located away from
other crops in flower and significant areas of weeds. The pesticide use history of all fields used
in this study will be documented for at least the two previous cropping seasons before the start of
study.
Agronomic records for the plots and their environment (including the presence of any weeds
around the test plots and location of other crops) and meteorological data for the experimental
period will be recorded.
Dosing
The test product will be applied in a use scenario according to the label recommendations and
Good Agricultural Practice.
Data Collection
Raw data will be collected in pre-approved formats during the entire study.
Honey bees effects assessments
All effects assessments will take place on the test plots or as long as the crop is still in bloom.
Hives are to be kept at test plots, and the colonies will be assessed for a further 28 days after
moving them altogether to an untreated area where no pesticide exposure is likely.
Test Bee Hives
Six normally developed queen-right bee colonies will be used per treatment and control i.e. a
total of 72 colonies. The colonies will be as similar to one another as possible. The hives will
contain 8-9 frames including at least 10,000 bees, 3-4 frames of brood and 2 frames of stores.
The colonies will be moved to the test sites at the onset of bloom of the crop. The hives will be
placed on the field edge such that bees will fly over the plot. If necessary, colonies will be
supplied with water throughout the test.
Exposure and Monitoring phase
On day after the setup of the hives, the activity of the bees at the hive will be assessed from
sunrise to sunset to determine when the bees are actively foraging. This will be assessed by
counting the number of bees leaving the hives for 30 seconds at half an hour intervals until
activity had started (at least 1 bee/ 30secs/ 2hives) and then once per hour.
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These data will be used to identify the most appropriate times for subsequent assessment of bee
activity at the hive and in the crop.
Subsequent assessments will be aimed at collecting bee activity and behaviour observations 3
times each day but the exact timing will be determined by suitable weather (e.g. assessments will
not be made during periods of rainfall).
If practically feasible, all following assessments should be done simultaneously or at least within
a as short as possible time frame for all replicates, in order to make sure that environment
conditions during the individual assessments are well comparable.
Mortality in Front of the Bee Hives and in the Field
When the hives are set up at the test plots, dead bee traps will be fitted to each hive and waterpermeable sheets of 1.5 m width and about 3 m length spread out in front of the hives.
At three places within the crop water permeable sheets (each area: approximately 15 m2) will be
placed between the rows of the crop on which any dead bees found will be counted. Each day
the number of dead bees will be recorded (the bees in the dead bee trap and on the sheets in front
of the hives were combined).
Assessments of number of dead bees will be made daily until the end of the exposure period. In
the first days after set up of the hives at the test plots, increased mortality figures are to be
expected in both treatments and control colonies, caused by the transport and relocation stress to
which the bee colonies were exposed before. This can be differentiated from potential treatmentrelated mortality by comparison with the control colonies.
Activity and Behaviour of the Bees in the Field
Bee activity and behaviour will be recorded at the hive and in the field each day from the setup
of the bee hives in the field. Activity at the hive will be assessed by counting the number of bees
leaving the hive for 30 seconds three times per day (timing based on the assessments on the day
before spray application).
The observations in the field will be undertaken by walking along the rows for a total of 10-30
minutes per plot (depending on the attractiveness of the crop (highly attractive crop 10 mins, less
attractive crop 30 mins) three times per day (timing based on the intitial activity assessment at
the hive. Each transect will be at different areas of the plots. The numbers and behaviour of the
foraging bees on and number of bees flying over the transect will be recorded. Assessments of
activity at the hive and in the field will be made 3 times on each day until the end of the exposure
period at the test plots.
Colony Condition and Development of the Brood
The condition of all colonies will be recorded and the development of the bee brood checked 2±2
days before setup of the colonies at the test fields, every 7±1 days until the end of the entire
study exposure period (i.e. until the end of the additional 28 day additional observation period
when the hives have been removed from the test fields).
The following parameters were assessed:

Weight of each colony
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



Strength of the colony (number of combs covered with bees and % area of each combs
covered with bees)
Presence of a healthy queen (presence of eggs, presence of queen cells)
Visual assessment of the pollen storage area and area with nectar (square inch)
Visual assessment of the area containing eggs, larvae and capped cells (square inch)
The measurements will be done for all combs per hive. Afterwards the mean values were
calculated for each hive and assessment date. These assessments need to be done by and
experienced and specifically trained person, preferentially a bee keeper or bee scientist. One
and the same person should do the estimates for all study hives.
Visual inspections of the colonies were undertaken for signs of bee disease (Nosemaapis,
Acarapsiswoodi, viruses, European foulbrood, as well as any other locally relevant diseases)
at each assessment. Varroaassessments were undertaken by placing a sticky sheet on the
floor of each colony and counting the Varroamite fall. The sheets were replaced with clean
sticky sheets at the time of colony inspections outlined above and counted after return to the
laboratory.
D) RESIDUE STUDIES (To be conducted by Division of Agro. Chemicals, IARI, New
Delhi):
Experiments to be conducted (i) under controlled conditions in tunnel (ii) under field conditions
The treatments would be according to the recommended uses of neonicotinoids. The
recommended uses of neonicotinoid formulations in cotton, mustard and citrus are as follows.
Crop
Pesticide
Formulation Dose
Pests controlled
Acetamaprid
20% SP
10 g ai, 500-600 L per ha Aphids, Jassids
Cotton
Whiteflies
Clothianidin
50% WDG
15-20 g ai, 500L per ha
Jassids
White fly
Dinotefuran
20% SG
30-40 g ai, 500L per ha
White Fly,
Jassids, Aphids
&Thrips
Imidacloprid
70% WG
10 g ai, 500-600 L per ha Jassids, Aphids,
Thrips
48% FS
300 – 540 g aiper 100kg Aphids,Whitefly,
seed
Jassids, Thrips
70% WS
350 – 700g ai per 100kg Aphids, Jassids,
seed
Whitefly, Thrips,
30.5% M/M
21-26.25 g ai, 500 –
Aphid,
SC
750L per ha
JassidThrips
17.8% SL
20 – 25g ai, 500 - 700 L
Aphid, Jassid,
per ha
Whitefly, Thrips
Acephate 50%
518g ai, 1 kg or L
Aphid ,Jassids,
+ Imidacloprid
formulation 500 L per ha Thrips, White
1.8% SP
flies, Bollworms
Thiacloprid
21.7% SC
24 – 30g ai, 500 L per ha Aphid, Thrips,
Jassid
Thiamethoxam 30% FS
3 g ai/kg seed
Aphid, Jassids,
whiteflies,
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Mustard
Citrus
70% WS
300
25% WG
25 g ai, 500-750 L per ha
Thiamethoxam
12.6%+Lambd
a cyhalothrin
9.5%ZC:
Imidacloprid
200g ai, 500 L per ha
70% WS
490g ai per 100kg seed
Thiamethoxam
25% WG
Imidacloprid
17.8% SL
Thiamethoxam
25% WG
12.5-25.0 g ai, 5001000L per ha
10g ai, vol depending on
size of tree
25g ai, 1000 L per ha
Aphid, Thrips
whiteflies,
Jassids
Jassid,
Thrips,whiteflies,
Aphid
Jassids, Aphids,
Thrips and
Bollworm
Mustard
sawfly &
painted bug
Aphid
Leaf miner,
psylla
Psylla
Sample Collection for residue studies:
Folliar sprayed pesticides
Pesticides will be applied according to the label relevant for the specified crop
Materials to be sampled : leaves, flowers, nectar and pollen
Sampling intervals : 24 hours, 3 days, 7 days, 15 days, 30 days, 45 days after application
depending on the flowering period of the crop.
Seed treatment with neonicotinoids in mustard and cotton
Pesticides will be applied according to the label relevant for the specified crop
Materials to be sampled : leaves/plant, flowers, nectar and pollen
Sampling intervals: 15 days after application (only plant), at initiation of flowering (0-5%), 50%
flowering, 75% flowering depending on the flowering period of the crop.
The collected samples will be properly packed as follows:
1. Plant/flowers : in polythene bags
2. Pollen and nectar : in screw capped vials and then sealed
3. Honey : In glass bottles of appropriate size
4. Wax materials and dead honeybees : Plastic boxes
The containers to be properly tagged giving information about place, crop, treatment and
sampling interval.
The containers containing samples to packed in dry ice in a bigger PUF box and sent by courier
to Division of Agricultural Chemicals, IARI, New Delhi
Pesticide Residue Analysis
The samples received from different centres will be taken out of the dry ice, allowed to warm up
to room temperature. Appropriate quantity of material will be weighed and processed for residue
analysis. The materials, that could not be processed, will be stored in a deep freezer.
Samples will be processed and analysed for parent molecule as well as their toxicologically
significant metabolites. The methods will be standardised and validated before being used for
sample analysis. The tentative protocols of the methods are given below.
Sample processing
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Plant samples : Plant samples will be processed by QuEChERS method. Plant samples
(leaves/flower) 100 g will be homogenized in high volume homogenizer. 10 g of representative
homogenized plant material will be taken in 50 mL centrifuge tube and 10 mL of acetonitrile,
anhydrous Magnesium sulfate and sodium chloride will be added and then subject to rotospin at
50 rpm for 5 minutes. The material will be again homogenized by using a low volume
homogenizer at 12,000 rpm for 2 minutes. The contents will be centrifuged at 5000 rpm for 10
minutes at 5⁰C. 1 mL aliquot of the supernatant will be taken in a micro centrifuge tube,
anhydrous Magnesium sulfate and PSAwill be added and vortexed for 2 minutes and centrifuged
at 500 0 rpm for 5 minutes. 0.5 mL supernatant will be filtered through 0.45µM Simlicity®
Filtration system and analysed.
Honey/Necture/Pollen : Sample will be extracted and cleaned up using Solid phase extraction
method using suitable using cartridges or modified QuEChERS method.
Wax, dead honey bees – Modified QuEChERScleanupmethod : About 20 dead honey bee will be
homogenized and around 2.5 g of representative homogenized material will be taken in 50 mL
centrifuge tube and processed similar to plant samples.
Estimation
LC-MS-MS instrument will be used for residue analysis. The methods will be standardised for
the analysis of acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid and
thiamethoxamand their toxic metabolites.
The toxicological information and metabolites included in residue definition is given below:
Pesticide
ADI LD50 Honeybee
Residue definition (codex)
0.07
Acute
oral
toxicity:LD
50
~
14.53
sum of acetamiprid and its
Acetamiprid
microg./bee
desmethyl (IM-2-1)
LD50 8.85 microg. a.s./bee (EXP 60707 A
metabolite
tested formulation) (acetamiprid 20 %)
Acute contact toxicity: LD50 ~ 8.09
microg./bee (acetamiprid)
LD50 9.26 microg. a.s./bee (EXP 60707 A
tested formulation) (acetamiprid 20 %)
0.1
acute contact basis (LD50 > 0.0439 µg/bee
Clothianidin
Clothianidin
Acute oral toxicity:LD 50: 0.00379ug/bee
Dinotefuran
0.2
Acute contact toxicity: LD50 : 0.0275ug/bee
Acute oral toxicity:LD 50: 0.023ug/bee
Acute contact toxicity: LD50 : 0.047ug/bee
Imidacloprid
0.06
Thiacloprid
0.01
Thiamethoxam
0.08
Acute oral toxicity:LD 50: 5 ng/bee
Acute contact toxicity: LD50 : 14-24 ng/bee
14.6
LD50 oral = 17.3 μg/bee
LD50 contact = 38.8 μg/bee
Acute oral LD50: 0.005 μga.s./bee
Acute contact LD50 : 0.024 μga.s./bee
Sum of dinotefuran, 1methyl-3-(tetrahydro3furylmethyl) urea (UF) and
1-methyl-3-(tetrahydro3furylmethyl)guanidiumdih
ydrogen (DN)
imidacloprid and its
metabolites containing the
6-chloropyridinyl moiety
Thiacloprid
sum of thiamethoxam,
CGA322704, CGA 265307
and MU3
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