Jpn. J. Clin. Ecol.（Vol.21 No.1 2012）10「第20回日本臨床環境医学会学術集会特集」
Original article（臨床環境21：10～23，2012）

シンポジウムInfluence of dinotefuran and clothianidin on a bee colony
Toshiro Yamada

Kazuko Yamada

Naoki Wada

Graduate School of Natural Science & Technology, Kanazawa University

Abstract
Recently it has become a serious problem that honeybees suddenly vanish in their colony, which is referred toas a colony collapse disorder（CCD）. We have made it clear by the field experiments for about four months what
effect neonicotinoid pesticides such as dinotefuran and clothianidin have on the occurrence of CCD. Eight colonies consisting of about ten-thousand honeybees in each colony were investigated under the practical beekeeping
conditions in our apiary. In this study foods containing dinotefuran of 1 ppm to 10 ppm or clothianidin of 0.4 ppm
to 4 ppm were fed into a beehive. Three levels of concentration were 10（high-conc.）, 50（middle-conc.）and 100（low-conc.）times lower than that in practical use. The changes of adult bees, brood and the pesticide intake ineach colony were directly examined. They suggest that each colony with the pesticide administered collapses tonothing after passing through a state of CCD, the high-concentration pesticides seem to work as an acute toxicity
and the low- and middle-concentration ones do as a chronic toxicity. CCD looks mysterious, but it is just one ofsituations where a colony dwindles to nothing. We have proposed a CCD occurrence mechanism based on our results. The NMR spectral analyses of dinotefuran and clothianidin in aqueous solution give the speculations thatboth are thermally stable under the heating condition of 50 ℃ ×24 hours and dinotefuran is radiationally stable2
under the ultraviolet-irradiation condition of 310 nm×50 W/m but clothianidin is unstable.
《Key words》dinotefuran, clothianidin, neonicotinoid pesticide, colony, collapse

Ⅰ．Introduction
A phenomenon referred to as a colony collapse disorder（CCD）

roa mite and Israel acute paralysis virus（IAPV）10～23）,
synergy-effect theory due to Nosema microsporescauses extremely serious problems

and systemic pesticide such as a neonicotinoid24, 25）,

for not only bee-keeping but also yielding agricultural

besides an environmental change-related stress one26, 27）,

products through honeybee pollination, and further-a beekeeping- related stress one due to transporta-

more sustaining ecosystem balance. The CCD dif-tion and hard work, a nutrition stress one due to hab-fers from the general bee-behavior such as swarming

itat loss28）, genetically modified（GM）crop one29, 30）, a

in that nearly all the adult bees rapidly vanish while

radiation one due to a cellular phone, a multiple

abandoning foods（honey, pollen）, brood and a queen.

causes one31～34）, etc. At present, any of them has not

Various theories on the cause of CCD have been

been yet demonstrated scientifically with CCD re-till now proposed, such as a pesticide theory due to

produced directly and experimentally. It is difficult to

1～4）

neonicotinoids

5～9）, a mite and plague one due to Var-

reproduce CCD in a laboratory where the behavior of

Reprint Requests to Toshiro Yamada, Graduate School of Natural Science & Technology, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192,
Japan

Influence of dinotefuran and clothianidin on a bee colony

11

bees limited in number is observed because CCD is arays are analyzed by the measurement of the proton

phenomenon seen in a colony of bees which are euso-

NMR spectrum in this study.

cial insects. In this work, colonies which consist ofenough honeybees（Apis mellifera）to behave as eu-

Ⅱ．Experimental and Evaluation Methodssocial insects are prepared under a natural environ-１．Experimental methods

ment in an apiary. Dinotefuran and Clothianidin are【Field experiment of pesticide administration】

widely used and well-known as a neonicotinoid pesti-

The pesticides were administered as foods of sug-cide in Japan mainly sprayed on rice field to extermi-ar syrup and pollen paste. Sugar syrup in a feeder

nate stinkbugs that mark with spots on grains of riceand pollen paste on the combs were fed to each colo-and degrade them. We cannot find any reports about

ny in a hive and they were exchanged for old ones

the influence of dinotefuran and clothianidin on aevery time.

honeybee colony through a long-term field experi-

content of 10% by Mitsui Chemicals Aglo, Inc. inment in an apiary. In order to elucidate the long-term

Tokyo（hereafter called “StarcklemateTM”）and DAN-

effect of these pesticides on a colony and the relation-

TOTSU® with clothianidin content of 16% by Sumi-

ship between these pesticides and CCD, field experi-

tomo Chemical Takeda Agro Company （hereafterments are carried out in an apiary based on ourcalled “DantotsuTM”）were used in this study. They

knowledge and experience in beekeeping.

are representative neonicotinoid pesticides in Japan.

STARKLE MATE® with dinotefuran

The concentrations of pesticide administered to

The solutions of commercial pesticides with a dilu-

each experimental run are diluted 10-, 50- andtion factor recommended for exterminating stink-

100-folds against the solution of commercial pesti-

bugs are as follows; a solution with a 1,000-fold dilu-

cide with a dilution factor recommended for extermi-

tion factor of a commercial concentration in sugar

nating stinkbugs in practical use. High-concentration

syrup for StarcklemateTM（dinotefuran of 100ppm inpesticides were administered only in the beginning ofsolution）and that with a 4,000-fold dilution factor forexperiment and after that they were never done in

DantotsuTM （clothianidin of 40 ppm in solution）.

this study to clarify the influence of acute toxicity of

Sugar syrup was made of an equal amount of sugar

the pesticides on a bee colony. Low and middle pes-

and water. The solution of pesticide administered toticides were administered every time to clarify theeach experimental run are diluted 10-, 50- andinfluence of chronic toxicity of them. In order to elu-

100-folds against the solution of commercial pesti-

cidate the change in strength of a colony with eusoci-cide with a dilution factor recommended for extermi-

ality, this study will focus on the long-term behavior

nating stinkbugs in practical use. Now, we call thesuch as the change in the numbers of adult bees,

concentrations of 10-fold, 50-fold and 100-fold dilu-

brood and a queen in a colony, and the total intake oftion “high”, “middle” and “low”, respectively. A so-

pesticide leading to the collapse of a colony duringlution with a 10-fold dilution factor of a recommended

the administration of pesticide.

concentration of Starcle MateTM is hereafter called S-

Generally, pesticides sprayed on the fields are di-high（10 ppm of dinotefuran）in RUN-2 and a solution

luted in water outdoors and the aqueous solutions arewith a 10-fold dilution factor of a recommended con-heated and irradiated with ultraviolet rays by the ex-

centration of DantotsuTM is hereafter called D-high（4posure to sunlight. In order to clarify the photolytic

ppm of clothianidin） in RUN-5. Similarly, the solu-

and pyrolytic properties of dinotefuran and clothiani-tions with the middle and low concentrations fordin under the exposure to sunlight, their aqueous so-

Starcle MateTM and DantotsuTM are called S-middle（2lutions heated at 50 ºC and irradiated with ultraviolet

ppm of dinotefuran）（RUN-3）, S-low（1 ppm of di-

Jpn. J. Clin. Ecol.（Vol.21 No.1 2012）12

notefuran）（RUN-4）, D-middle （0.8 ppm of clo-

foods with pesticide in their hive if they prefer natu-

thianidin）（RUN-6）and D-low（0.4 ppm of clothiani-ral nectar, pollen and water in the surrounding fields.

din）（RUN-7）, respectively.

The details of each

In order to count the number of adult bees as cor-experimental run are tabulated in Table 1. Pollen

rectly as possible, experiments were conducted be-paste was prepared by kneading two parts of pollen

fore foraging bees went out of the hive in the earlywith one part of sugar syrup containing the pesticide,morning except on rainy days, fundamentally atwhere the pollen was prepared by mixing the pollen

weekly intervals（rarely, at two-weekly intervals）.

substitute “Feed-Bee ” with pure pollen at a ratio ofEight standard hives with the entrance to the east

1:1. Pictures of both sides of all the combs, a feeder,

were arranged on a hill from south to north. Eachthe inside, the outside of a hive, etc. were taken withwas composed of six combs and one feeder of sugara digital camera at weekly intervals（rarely at two-syrup. In order to prevent honeybees from swarm-

weekly intervals）. Pictures of circumstances arounding, a comb foundation was newly added in the hive of

the entrance of a hive were taken with a digital cam-each blank run（control）when necessary. We haveera with a half-hourly–interval timer monitoring the

conducted a series of experiments since July 18th inactivities of honeybees such as carrying the dead2010 for about four months when there were notbees out of a hive and the like.

many flowers in bloom and it is the less-swarming®

Experiments were conducted in an apiary, where

season of honeybees in Japan.

crop-dusting is controllable and honeybees could
freely visit flowers in the field and avoid taking theTable 1

Outline of foods (sugar syrup, pollen paste) on each experimental run

RUN No. Administered pesticide

A Dilution of
A Dilution of thecommercial product１） reference solution２）

Content ofpesticide３）0 ppm

B-1（Blank run） （control）

10,000-fold dilution

10 ppm

Ｓ1010000
50

Notation４）

Note５）RUN01

No pesticide

RUN02

StarckleTM（dinotefuran 10%）

RUN03

Starckle （dinotefuran 10%）50,000-fold dilution

50

2 ppm

Ｓ 50000

S-middle

RUN04

StarckleTM（dinotefuran 10%） 100,000-fold dilution

100

1 ppm

Ｓ100100000

S-low

RUN05

Dantotsu （clotianidin 16%）

40,000-fold dilution

10

4 ppm

D 40000

D-high

RUN06

DantotsuTM（clotianidin 16%）200,000-fold dilution

50

0.8 ppm

D50200000

D-middle

RUN07

Dantotsu （clotianidin 16%）0.4 ppm

100

RUN08

No pesticide

１）

TM

TM

TM

400,000-fold dilution

10

100

0 ppm

10

D

400000S-highD-low

B-2（Blank run） （control）

Dilution of commercial pesticide means that a commercial pesticide is diluted with sugar syrup up to a given dilution factor. For example,
in RUN02 a commercial StarckleTM containing dinotefuran of 10% is diluted with 10,000 parts of sugar syrup, where the solution in RUN02
contains dinotefuran of 10 ppm. The concentration of a pesticidal constituent included in a commercial pesticide are dinotefuran of 10% in
StarcklemateTM and clothianidin of 16% in DantotsuTM, respectively.
２）
Dilution of the reference solution represents a dilution factor diluting the reference solution which is recommended as a concentration of
extermination of stinkbugs, where the reference solution of StarcklemateTM and DantotsuTM have a 1,000-fold dilution of a commercial
product（dinotefuran of 100 ppm in solution）and a 4,000-fold dilution of one（clothianidin of 40 ppm in solution）, respectively.
３）
Content of pesticide represents the content of main constituent of pesticide administered to each run. For example, a 10 ppm of dinotefuran is administered to RUN02, which is included in a 10,000-fold diluted StarcklemateTM.
４）
B-1 and B-2 represent blank runs. X and Y in SXY and DXY represent the X-fold dilution of the reference solution and the Y-fold dilution of
the commercial product, respectively, and the S and D represent StarcklemateTM and DantotsuTM , respectively.
５）High conc.（concentration）, middle conc., and low conc. means a 10-fold dilution of the reference solution , a 50-fold one and a 100-fold one
in this paper, respectively.

13

Influence of dinotefuran and clothianidin on a bee colony【Nuclear Magnetic Resonance（NMR）measure­mentof dinotefuran and clothianidin】

sons.
The consumption of foods（sugar syrup, pollen

Dinoterufan（standard）and clothianidin（99.5%）

paste）by honeybees and the number of dead bees

were purchased from Kanto chemical（Japan）and

were estimated from photos and visual measure-

Dr. Ehrenstorfer GmbH （Germany）, respectively.

ments at every experiment. The intake of pesticide

These pesticides were used without further purifica-

was calculated from the consumption of foods. The

tion. Samples were dissolved in D2O containing 0.3%

total intake of pesticide leading to the collapse of atrimethylsilyl propanoic acid as a standard. Decom-colony is converted into the pesticide solution with aposition by heating at 50℃ for 24 hours and ultravio-

concentration of a commercial product（STARKLE

let（UV）light irradiation for 30 min at 310 nm × 50

MATE®, DANTOTSU®）from the consumption of

W/m2 was investigated by 1H-NMR measurements,

sugar syrup or pollen paste.

where the amount of UV light irradiation is equiva-

３．Definition of normalized number

lent to that of about 6.5-days UV radiation from the

To compensate for a difference in initial population

sun in Tsukuba city. UV light may be somewhat de-among runs and that in seasonal fluctuation of bee

creased in intensity because it is irradiated on a sam-

population, a relative change in the number of adultple through a glass container. UV light irradiation

bees is newly defined by the following Equation（1）was performed on Funakoshi NTM-10 trans-illumi-

Normalized number of adult bees（n
/ Bj / nB0） （1）=（nij / ni0）

nator. NMR spectra were obtained by JEOL ECS400 spectrometer at room temperature.
２．Evaluation methods
The change in the numbers of adult bees and broodin each colony was directly examined through a long
period of days in this work because the change in theweight of a hive contained all the changes in theweight of honey, pollen and others in addition to honey bees and brood.
The numbers of adult bees and brood（capped

Where,
nij=	the number of adult bees in RUN i after theelapse of j days,
ni0=	the initial number of adult bees in RUN i atthe start of experiment,nBj=	the number of adult bees in blank run after
the elapse of j days,
nB0=	the initial number of adult bees in blank run at
the start of experiment,

brood and visible larvae）on a comb were counted

where the arithmetic mean number of RUN-1 andand summed up in a hive. The number of adult bees

RUN-8 was used as the number of adult bees in blankon a comb was directly counted on a photo when lessrun in Equation（1）.

than several hundreds; it was indirectly counted

A period of brood is considerably shorter than thatwhen more than several hundreds by use of the refer-of an adult bee and not always contemporary withence photos which were directly counted beforehand.

each other colony. Therefore, the change in the num-

The sum total on all combs in a hive was used as theber of brood was evaluated without normalization.

number of adult bees for each run. The number ofbrood was evaluated on a photo by the ratio of the

Ⅲ．Results and Discussion

area occupied with brood to the whole surface on one

１．Change in the number of adult bees

side of a comb. The sum total of the area ratios on all

Table 2 shows the change in the number of total

combs in a hive was expressed as the number ofadult bees in a hive with the elapsed days for eachbrood for each run in this study.

run. Figure 1 shows the change in the number of to-

These numbers were double-checked by two per-tal adult bees normalized by Equation（1）. The fol-Jpn. J. Clin. Ecol.（Vol.21 No.1 2012）14

Table 2

Change in number of total adult bees with elapsed days for each run

Start of the experiment after the adjustment on initial number of total adult bees
Date in2010

Elapseddays（Pesticide）
１）（Dilution ）RUN 1control

RUN 2
S-highRUN 3
S-middle

Blank 1

StarcklemateTM

StarcklemateTM

StarcklemateTM

１）

１）

１）

No pesticide

10,000-fold

50,000-fold

RUN 4
S-low

RUN 5
D-highRUN 6
D-middle

DantotsuTM
１）

DantotsuTM
１）

100,000-fold

40,000-fold

200,000-fold

RUN 7
D-low

RUN 8
control

DantotsuTM

Blank 2400,000-fold１） No pesticide

Average ofBlanksBlank 1 & 2No pesticide

July 18

0

8950

11700

12720

10400

12880

11600

13400

10560

9755

July 23

5

11700

5450

5240

7900

5100

7800

11900

11400

11550

July 301211850（3900）

7250

8750（1770）

8900

12100

11800

11825

August 8

21

11100

（2550）

1235

9500（1775）

4060

10100

12400

11750

August 13

26

11400

（1450）

940

8500（1530）

［70］

9900

11800

11600

August 21

34

8900

（861）

325

4750

（640）

［275］

6300

10700

9800

August 26

39

9800

（980

200

5150

（890）

［36］

4340

9400

9600

September 5

49

9650

（760）

［178］

4590

（830）

［0］

［1840］

6370

8010

September 11

55

10600

（666）

［110］

3550

（810）

［1180］

7450

9025

［0］

3740

（730）

［975］

6150

8650

1395

（895）

［150］

7680２）

9990

２）

9990

（285）

7680２）

9990

［（0）］

２）

9990

September 17

61

11150

（264）

September 24

6812300２）（470）

October 10

84

12300

２）

October 30

10412300２）126

２）

November 21

12300

（415）

0

（0）

（740）

［0］

7680

7680

This shows a dilution factor of a commercial product. ２）The numbers of adult bees on the elapsed of 68 days in RUN 1 & 8 were substituted for that after that.
（Note）Parentheses（ ）show a state that foods（sugar syrup, pollen paste）without a pesticide were fed into a colony after the elapse of 12 days instead of foods with a pesticide. Brackets［ ］show a state that a queen had been lost. The average between RUN 1 & 8 was used as the number in blank run in calculation of normalized number.
StarcklemateTM contains a dinotefuran content of 10% and DantotsuTM contains a clothianidin content of 16%. Less than ten heads are expressed as zero.

Normalized number of adult bees in the hive１）

1.0
: Blank
RUN1・RUN8 ブランク

0.8

0.6

RUN2

: S-high

RUN3: S-middle

RUN4: S-low

RUN5: D-high

RUN6: D-middle

RUN7: D-low

0.4

0.2

0.0
0

20

40

60

80

100

120

Elapsed day
Figure 1

Normalized number of adult bees in the hive with the elapsed days

15

Influence of dinotefuran and clothianidin on a bee colony

lowing results can be obtained: After the administra-

２．Change in the number of broodtion of the pesticides（dinotefuran, clothianidin）, theTable 3 and Figure 2 show the change in the num-

number of adult bees rapidly dwindled and the colony

ber of total brood in a hive with the elapsed days forbecame extinct afterwards. A queen bee did not dis-each run. The following results can be obtained fromappear until adult bees became few. It is confirmed

them: The number of brood sharply decreased after

from photos that brood and foods existed at the point

the first pesticide administration while taking a peakof queen’s loss. Wax-moth larvae did not exist in a

in some cases about five weeks later. Taking a peak

hive while adult bees decreased in number to nothing

was caused by stimulation in egg-laying of a queen

and for a while after the complete collapse of a colony.

due to the sharp decrease in the number of brood.

In S-high（RUN-2）and D-high（RUN-5）, adult

This suggests that a pesticide has some effect onbees were killed on the instant just after the adminis-

egg-laying and hardly any effect on eggs and larvae.

tration of pesticide. The foods with a high-concentra-

The decrement in brood roughly suggests that thetion pesticide were fed to a colony only in the firsthigher concentration of pesticide leads to the morestage of experiment and they were replaced by foods

serious egg-laying impediment of a queen. At thewithout pesticide twelve days later. A great number

elapse of twelve days, the egg-laying capacity of aof dead bees occurred in and around the hive forqueen rapidly declines and is kept low afterwards, in-twelve days after the administration of pesticide. In

dependently of the pesticide concentration, though a

S-high, some dead bees were found three weeks later

high-concentration pesticide was stopped while foods

but afterwards became a few. In D-high, a few deadwithout pesticide being fed.

bees were found three weeks later and afterwards.

From the long-term observational results of brood,

The colony became extinct fifteen weeks later in S-

a colony with the pesticide administered collapses tohigh and eighteen weeks later in D-high. A queen

nothing after passing through a state of CCD as sup-existed until the number of adult bees dwindled down

ported in a new article titled “in situ replication of

to zero in S-high and D-high.

honeybee colony collapse disorder”

In S-middle（RUN-3）and D-middle（RUN-6）
, the

35）

due to neonic-

otinoid pesticide（imidacloprid）which was publishednumber of adult bees decreased to nothing sevenjust after submitting this article to this journal.

weeks later in D-middle and about nine weeks later

３．Total intake of pesticide leading to the col-in S-middle. A queen existed until the number oflapse of a colony

adult bees dwindled down to 1.4 percent of the initial

Table 4 and Figure 3 show the total intake of pesti-

number in S-middle and 0.6 percent in D-middle. A

cide. The following results can be obtained fromnumber of dead bees occurred only in the early peri-

them: In the case of S-high（RUN-2）and D-highod after administration but they almost never oc-（RUN-5）, a colony resulted in a collapse even whencurred in S-middle and D-middle afterwards.

a high-concentration pesticide was administered to a

In S-low（RUN-4）and D-low（RUN-7）, the num-colony only in the first stage of experiment and after-

ber of adult bees decreased to nothing twelve weeks

wards foods with high-concentration pesticide waslater in the same period of time. A queen existed

stopped and replaced by those without pesticide.

until the number of adult bees dwindled down to zeroThis suggests that a colony probably collapses due toin S-low and about 14 percent of the initial number inacute toxicity in high pesticide concentrations which

D-low. Dead bees almost never occurred after ad-

is one tenth the concentration to exterminate stink-ministration.

bugs in practical use. If the rough assumption ismade that five hundred honeybees a colony newlyJpn. J. Clin. Ecol.（Vol.21 No.1 2012）16

Table 3

Change in number of total brood in a hive with elapsed days for each run
Start of the experiment after the adjustment on the initial number of broods
RUN 1
control

RUN 2
S-highRUN 3
S-midle

RUN 4
S-low

RUN 5
D-highRUN 6
D-middle

RUN 7
D-low

RUN 8
Control

Average ofBlanks（Pesticide）

Blank 1

StarcklemateTM

StarcklemateTM

StarcklemateTM

DantotsuTM

DantotsuTM

DantotsuTM

Blank 2

Blank 1
&2

（Dilution１））

No pesticide

10,000-fold１）

50,000-fold１）

100,000-fold１） 40,000-fold１） 200,000-fold１） 400,000-fold１） No pesticide

Date
in 2010

ElapsedDays

July 18

0

5.3

7.05

7.2

3.9

7.6

1.5

2

6.96

6.13

July 23

5

5.25

4.45

3.95

4.05

2.6

1.4

2.1

4.45

4.85

July 30

12

3.7

（0.8）

1.4

1.35

（0.25）

0.05

0.05

3.5

3.6

August 8

21

2.5

（0.4）

0

0.05

（0.2）

0.15

0.3

3.45

2.975

August 13

26

2

（0.4）

0

0.1

（0.05）

［0.2］

0.35

2.95

2.475

August 21

34

2.55

（0.4）

0.07

0.6

（0.3）

［0.006］

0.8

2.8

2.675

August 26

39

2.5（0.15）

0.1

0.25

（0.4）

［0］

0.235

2.05

2.275

September 5

49

1.4

（0.05）［0.039］0.036（0.05）［0］［0.049］2.1

1.75

September 11

55

1.5

（0.065）［0.065］0.008（0.098）

［0］

2.6

2.05

September 17

61

1.9

（0.042）［0.042］0.005（0.099）

［0］

3

2.45

２）

September 24

68

3.85

（0.016）

0（0.045）［0］

4.4

4.125２）October 10843.85２）（0.06）

0（0.141）［0］

4.4

4.125２）October 30

1043.85２）（0）

4.4

4.125２）（0.026）２）

１）

Dilution shows a dilution factor of a commercial product.
The numbers of brood on the elapsed of 68 days in RUN 1 & 8 were substituted for that after that.
（Note）Parentheses（ ）show a state that foods（sugar syrup, pollen paste）without a pesticide were fed into a colony after the elapse of 12 days instead of foods with a pesticide.
Brackets [ ] show a state that a queen had been lost. Where StarcklemateTM contains a dinotefuran content of 10% and DantotsuTM contains a clothianidin content of 16%.

8.0

Number of brood in the hive

RUN-1&-8: Blank
系列10
RUN-2:
S-high3

7.0

RUN-3: S-middle
系列4
6.0

RUN-4: S-low
RUN-5: D-high
系列6

5.0

RUN-6: D-middle
系列7
RUN-7: D-low
系列8

4.0

3.0

2.0

1.0

0.0
0

10

20

30

40

50

60

70

80

90

100

Elapsed day
Figure 2

Change in the number of brood expressed by the number of combs occupied
by brood in the hive with the elapsed days

17

Influence of dinotefuran and clothianidin on a bee colony

develop from larvae into imagoes while influenced by（RUN-4）and similarly little difference of that be-

foods with the pesticide fed and stored in the hive,

tween D-middle （RUN-6） and D-low （RUN-7）.

the lethal dose of dinotefuran can be estimated to beThis suggests that the pesticide may be little-metab-

0.1072 µg/bee for S-high（RUN-2）, 0.2434 µg/bee forolized and accumulated in the body tissues of bees

S-middle （RUN-3） and 0.1903 µg/bee for S-low

and then a colony probably collapses due to the（RUN-4）, respectively. Similarly, the lethal dose ofchronic toxicity when the accumulated pesticide pass

clothianidin can be estimated to be 0.0360 µg/bee fora certain threshold.

D-high （RUN-5）, 0.1150 µg/bee for D-middle

On closer investigation, the total intake of pesti-（RUN-6）and 0.0706 µg/bee for D-low（RUN-7）,

cide leading to the collapse in S-middle or S-low is36）

reported that the LD50

about 150 percent of that in S-high. Similarly, the to-values of dinotefuran and clothianidin are 0.0750 µg/

tal intake of pesticide in D-middle or D-low is about

bee and 0.0218 µg/bee, respectively. The report by

150 percent of that in D-high. From the above it can

respectively. Iwasa et al.

Iwasa et al.

36）roughly supports the results of thisbe suggested that the total intake of pesticide leading
to the collapse in the low or the middle（chronic tox-

work.

icity）is about 150 percent of that in the high（acuteOn the other hand, a colony probably collapses due
to chronic toxicity in the middle and low pesticide

toxicity）.

concentrations as already concerned about possible

The total intake of dinotefuran（StarcklemateTM）

chronic problems caused by long-term pesticide ex-leading to the collapse of a colony is almost four times

posure in nectar . Because there is little difference

as much as that of clothianidin（DantotsuTM）in theof the total pesticide intake leading to the collapse ofconcentration of commercial product, independent ofa colony between S-middle （RUN-3） and S-low

the pesticide concentration; that is, S-high/D-high ≒4）

Table 4

Total intake of pesticide for each run calculated from the intake of foods
RUN 1
Control

RUN 2
S-highRUN 3
S-middle

RUN 4
S-low

from sugar syrup [g]

0

63.3

99.8

95

63.2

98.8

93.2

0

from pollen paste [g]

0

5

5.4

4.7

5.2

5.2

4.8

0

from both foods [g]

0

68.3

105.2

99.7

63.4

104

98

0

from sugar syrup [mg]

0

63.3

99.8

95

15.8

24.7

23.3

0

from pollen paste [mg]

0

5

5.4

4.7

1.3

1.3

1.2

0

from both foods [mg]

0

68.3

105.2

99.7

17.1

26

24.5

0

from sugar syrup [mg]

0

6.33

9.98

9.5

2.53

3.95

3.72

0

from pollen paste [mg]

0

0.5

0.54

0.47

0.2

0.2

0.19

0

from both foods [mg]

0

6.83

10.52

9.97

2.73

4.15

3.91

0

Fiducial concentration Total intake of pesticide

Reference solutiona）[g]

Commercial productb）[mg]

Active ingredientc）[mg]

no pesiticidea）dinotefuran

RUN 5
RUN 6
RUN 7
D-high D-middle D-low

clotianidin

RUN 8Controlno pesticide

Total intake of pesticide solution converted into the reference solution with a concentration to exterminate stinkbugs
Total intake of pesticide solution with the concentration which is converted into the concentration of commercial productc）
Total intake of pesticide converted into the amount of an active ingredient which is dinotefuran for RUN-2, -3 and -4 or clotianidin for RUN5, -6 and -7（Note）The total intake of pesticide which was converted into the pesticide solution with a concentration of a commercial product（StarcklemateTM, DantotsuTM）from the comsumption of sugar syrup or pollen paste.
Where StarcklemateTM contains a dinotefuran content of 10% and DantotsuTM contains a clotianidin content of 16%.
b）

Jpn. J. Clin. Ecol.（Vol.21 No.1 2012）18

Figure 3

Total intake of pesticide with a converted concentration into that of commercial
product for each run

4; S-middle/D-middle ≒ 4; S-low/D-low ≒ 4.1. The
ratio between the dilution factor to make the solution
to exterminate stinkbugs of clothianidin and that offollowing speculations:
1）Dinotefuran and clothianidin is not decomposed
at 50 ºC.

dinotefuran is 4000:1000=4:1. Considering that each2）Dinotefuran is ultraviolet-stable because of lackof them has the same insecticidal activity against aof chromophore under the conditions of radiation

seems to have almost theintensity（RI）=50 W/m2, wavelength（WL）=310 nm

same insecticidal activity against a honeybee as Dan-and radiation time（RT）=0.5 hrs equivalent to about

totsu .

6.5-days UV radiation amount from the sun in Tsu-

stinkbug, Starcklemate

TM

TM

When converting the food consumption into the

kuba city. This is somewhat different from the under-amount of active ingredient which is pure dinotefuran
or clothianidin, the ratios of total intake of pesticides

water photolysis testing results of dinotefuran with axenon arc lamp under the conditions of RI = 400-

in high, middle and low concentration are 2.50 for S-

416 W/m2, WL=300-800 nm and RT=3.8 hrs37）equiv-

high/D-high, 2.53 for S-middle/D-middle and 2.55 foralent to about 400-days UV radiation amount from

S-low/D-low, respectively. From the above, the in-the sun in Tsukuba city. The difference may comesecticidal activity of clothianidin is about 2.5 times asfrom the amount of UV light irradiation. As a pesti-

strong as that of dinotefuran, while slightly increas-cide is expected to be photo-decomposed as soon as

ing with decrease in pesticide concentration.

possible after sprayed, about 400-days UV radiation

４．Photolytic and pyrolytic properties of dinotefu-

amount from the sun seems to be too much in com-

ran and clothianidin on the assumption thatparison with the half-life of 180 days regulated byan aqueous solution of pesticide is exposed

law38）.

to sunlight3）Clothianidin is decomposed by ultraviolet rays

Figures 4 and 5 show the measured results of theunder the same conditions as dinotefuran because it

proton NMR spectra for dinotefuran and clothianidin,

has a thiazole ring absorbing ultraviolet rays. This isrespectively. These NMR spectral analyses give theapproximately similar to the underwater photolysis

Influence of dinotefuran and clothianidin on a bee colony

Figure 4

NMR spectra of dinotefuran in D2O（A）without any treatment,（B）after
heating at 50 ℃ for 24 hours, and（C）after UV light irradiation for 30 min.

Figure 5

NMR spectra of clothianidin in D2O（A）without any treatment,（B）after heating
at 50℃ for 24 hours, and（C）after UV light irradiation for 30 min. The increased
and decreased signals were shown in the figure as up and downward arrows.

19

Jpn. J. Clin. Ecol.（Vol.21 No.1 2012）20

testing results of clothianidin with a xenon arc lamp
under the conditions of RI=18 W/m2, WL=360-480

ed in water is stable under the sunlight and the toxic-

nm and RT=40-42 minpresumed to collapse as bellow:

39）

equivalent to about 3-days

ity does not change for a long time, a colony can be

UV radiation amount from the sun in Tsukuba city.

Foraging bees are killed instantly on the spot

The decomposition products by ultraviolet rays seem

where a pesticide is directly sprayed. The death of

to be extremely diverse because the skeleton of ni-

many foraging bees leads to the conversion of house

troguanidine is biodegradable under the anaerobic

bees into foraging bees, and as a result a lack of housecondition. The specification and toxicity of the de-bees and imbalance of colony composition. When for-composition products are unexamined.

aging bees take water, nectar or pollen containing

５．Rational mechanism of CCD occurrence

pesticide in high concentrations, they are killed in-

Dinotefuran and clothianidin can lead to the col-stantly near the sprayed spot. Judging from the fact

lapse of a bee colony, judging from the following ex-

that about 5 ppm of clothianidin was detected in theperimental findings in this study: 1）Dinotefuran andwater near the rice paddy40）, the above assumption

clothianidin are probably little-metabolized and most-seems to be plausible. When in middle concentra-

ly accumulated chronically in the body tissues of bees

tions, some are killed instantly near the sprayed spot

and work as an chronic toxicity in low and middle

and others come back to their hive and then soon die.

concentrations.

2）A high-concentration pesticide

In this case many dead bees are found near their hive.

seems to work as an acute toxicity just by one doseOn the other hand, the sprayed pesticide is diluted

judging from the total pesticide intake till the collapse

with water in a rice paddy or rain, or the toxicity ofof a colony, which is less than that of low or middle

nectar has been diluted by new nectar flowing out inconcentration pesticide, and the state of dead bees.

a flower. In such cases foraging bees are scarcely

3）
As a period of brood is very short, the low-concen-

killed on the spot. And ingesting water, nectar andtration pesticide does not much affect the brood butpollen with a pesticide in low concentrations, forag-

does a queen having a long lifetime and results in theing bees carry the low toxic ones back to their hive.

inhibition of her egg-laying. 4）Both dinotefuran and

The low toxic ones are ingested by house bees, broodclothianidin are thermally stable. And dinotefuran isand the queen, or stored in combs as honey and bee

stable under ultraviolet irradiation but clothianidin isbread, and then the pesticide accumulated in the body

unstable （quite susceptible to deterioration fromof bees passes a certain threshold of toxicity. Whenultraviolet light）. 5）
Starcklemate （dinotefuran）seems

the brood taking the pesticide become foraging bees,

to have almost the same insecticidal activity against athey cannot come back to their hive because of being

honeybee as Dantotsu （clothianidin）when they are

disoriented or becoming exhausted due to chronic

prepared to have the same insecticidal activity against

toxicity as suggested in the recent article on homing

a stinkbug.

failure in foraging honeybees41）. The egg-laying ca-

TMTM

We can infer the following plausible mechanism ofpacity of a queen declines through ingesting the low

CCD occurrence as an example from the findings

toxic ones but a queen remains until the collapse.

mentioned above: Figure 6 shows the schematic dia-

The imbalance of colony composition also causes thegram of CCD occurrence mechanism due to neonic-

decrease in the egg-laying activity of a queen and fi-

otinoid pesticides. Considering the fact that the con-nally leads to a collapse of the colony with a queen

centration of pesticide sprayed on fields is at least ten

remaining. Even if a colony appear to be vigorous

times higher than that in this study, and under the

before wintering after it has been influenced by low

assumption that a low-concentration pesticide dilut-

toxicity in autumn, it probably fails in wintering due

Influence of dinotefuran and clothianidin on a bee colony

Figure 621

Schematic diagram of CCD occurrence mechanisms due to neonicotinoid pesticides

to chronic toxicity. Even when a colony does not col-tion becomes low, the low-concentration pesticide

lapse and looks active, a neonicotinoid pesticide

carried by foraging bees continues to affect a colonycauses an egg-laying impediment of a queen and a

for a long time and finally leads to a collapse of a col-decrease in immune strength of bees leading to the

ony or the failure in wintering. Even if a colony does

infestation of mites in a colony.

not collapse and looks active, it causes an egg-laying
impediment of a queen and a decrease in immune

Ⅳ．Conclusion
A colony rapidly dwindled after the administration

strength of bees leading to the infestation of mites ina colony.

of dinotefuran or clothianidin and finally became extinct after taking on an aspect of CCD. That is, aqueen bee did not disappear until adult bees became
few and brood and foods existed in the colony at the
point in time when a queen disappeared. Wax-mothlarvae did not exist for some time after the extinction

Acknowledgment
The authors have received valuable advices and informative
collaboration from Mr. Seita Fujiwara, Dr. Yasuhiro Yamada
and people involved in bee-keeping. And NMR analyses were
performed with the support of the Advanced Science Research Center of Kanazawa University. This study was sup-of colony. This means that the CCD is just one of

ported from a fund for research on bees granted by Yamada

situations where a colony dwindles away to nothing

Apiary.

although it may look mysterious. These strongly
suggest that the neonicotinoid pesticides such as dinotefuran and clothianidin can most probably causes
CCD whose mechanism is proposed as follows: In
supposing that a pesticide is sprayed and diluted inwater of a rice paddy or an orchard and its concentra-

References

１） “Honey Bee Die-Off Alarms Beekeepers, Crop Growers
and Researchers”, Penn State Live of The University’s Official News Source in Pennsylvania State University, January 29 in 2007２） vanEngelsdorp D, Evans J.D.: Colony Collapse Disorder:Jpn. J. Clin. Ecol.（Vol.21 No.1 2012）22

A Descriptive Study. PLoS ONE 4: e6481, 2009
３） Maini S, Medrzycki P, Porrini C.: The puzzle of honey bee
losses: a brief review. B. Insectol. 63: 153-160, 2010

bee monitoring project: a long term study to understandperiodically high winter losses of honey bee colonies.
Apidologie, 41: 332-352, 2010

４） Johnson R.: Honey Bee Colony Collapse Disorder. Con-

19）Le Conte Y, Ellis M, Ritter W.: Varroa mites and honeygressional Research Service 7-5700, www.crs.gov,

bee health: can Varroa explain part of the colony losses ?

RL33938, January 7, 2010

Apidologie 41: 353-363, 2010５） Hileman B.: Why are the bees dying ? Chem. Eng. News
85: 56-61, 2007
６） Johnson R.M, Pollock H.S, Berenbaum M.R.: Synergistic
Interactions Between In-Hive Miticides in Apis mellifera. J. Econ. Entomol. 102: 474-479, 2009７） Girolami V, Mazzon Squartini A.: Translocation of Neonicotinoid Insecticides From Coated Seeds to Seeding Gut-

20）Bacandritsos N, Granato A, Budge G, et al.: Sudden
deaths and colony population decline in Greek honey beecolonies. J. Invertebr. Pathol. 105: 335-340, 201021）Soroker V, Hettzroni A, Yakobson B, et al.: Evaluation ofcolony losses in Israel in relation to the incidence of
pathogens and pests. Apidologie 42: 192-199, 201122）Pohorecka K, Bober A, Skubida M, Zdanska D.: Epizootic

tation Drops: A Novel Way of Intoxication for Bees. J.

Status of Apiaries with Massive Losses of Bee Colonies

Econ. Entomol. 102: 1808-1815, 2009（2008-2009）. J. Apic. Sci. 55: 137-150, 2011８） Mullin C. A, Frazier M, Frazier J.L, Ashcraft S, Simonds23）Di Prisco G, Pennacchio F, Caprio E, et al.: Varroa de-R, vanEngelsdorp D, Pettis J.S.: High Levels of Miticides

structor is an effective vector of Israeli acute paralysis

and Agrochemicals in North American Apiaries: Implica-

virus in the honeybee, Apis mellifera. J. Gen. Virol. 92:

tions for Honey Bee Health. PLoS ONE 5: e9754, 2010
９） Johnson R.M, Ellis M.D, Mullin C.A, et al.: Pesticides and151-155, 201124）Alaux C, Brunet J.-L, Dussaubat C, et al.: Interactions

honey bee toxicity–USA. Apidologie 41: 312-331, 2010

between Nosema microspores and a neonicotinoid weak-10）Minkel J.R.: Mysterious Honeybee Disappearance

en honeybees（Apis mellifera）. Environ. Microbiol. 12:

Linked to Rare Virus. Sci. News（Scientific American）,
September 7, 2007

774-782, 200925）Aufauvre J, Biron D.G, Vidau C, et al.: Parasite-insecti-11）Vanengelsdorp D, Underwood R, Caron D, Hayes J.: An

cide interactions: a case study of Nosema ceranae andestimate of managed colony losses in the winter of 2006-

fipronil synergy on honeybee. www. nature. com / scien-

2007: A report commissioned by the apiary inspectors ofAmerica. Am. Bee J. 147: 599-603, 200712）Cox-Foster D.L, Conlan S, Holmes E.C, et al.: A metagenomic survey of microbes in honey bee colony collapse
disorder. Science 318: 283-287, 200713）Boecking O, Genersch E.: Varroasis-the ongoing crisis inbee keeping. J. Verbrauch Lebensm 3: 221-228, 200814）Teixeira E.W, Chen Y.P, Message D, Pettis J, Evans J.D.:
Virus infections in Brazilian honey bees. J. Invertebr.
Pathol. 99: 117-119, 2008

tific reports 2: 326, 201226）Sahba A.: The mysterious deaths of the honeybees. CNN
Money, March 29, 2007, retrieved on April 4 in 200727）Le Conte Y, Navajas M.: Climate change: impact on honey bee populations and diseases. Rev. Sci. Tech. OIE 27:
499-510, 2008
28）Naug D.: Nutritional stress due to habitat loss may explain recent honeybee colony collapses. Biological Conservation 142: 2369-2372, 200929）Hopwood L.: GE and bee Colony Collapse Disorder -- sci-

15）Highfield A.C, El Nagar A, Mackinder L.C.M, et al.: De-ence needed!, Letter from a Chair of Sierra Club Genetic

formed Wing Virus Implicated in Overwintering Honey-

Engineering Committee to Senator Tomas Harkin on

bee Colony Losses. Appl. Environ. Microbiol. 75: 7212-

March 21 in 2007, retrieved on March 23 in 2007

7220, 200916）Schafer M.O, Ritter W, Pettis J.S, Neumann P.: WinterLosses of Honeybee Colonies（Hymenoptera: Apidae）:

30）Latsch G.: Collapsing Colonies: Are GM Crops Killing
Bees? International –Spiegel Online –News” on March 22
in 2007, retrieved on February 24 in 2008

The Role of Infestations With Aethina tumida（Coleop-31）vanEngelsdorp D, Evans J.D, Saegerman C, Mullin C, ettera: Nitidulidae）and Varroa destructor（Parasitiformes:al.: Colony Collapse Disorder: A Descriptive Study. PLoS

Varroidae）. J. Econ. Entomol. 103: 10-16, 201017）Berthoud H, Imdorf A, Haueter M, Radloff S, Neumann
P.: Virus infections and winter losses of honey bee colonies（Apis mellifera）. J. Apicult. Res. 49: 60-65, 201018）Genersch E, von der Ohe W, Kaatz H, et al.: The German

ONE 4: e6481, 200932）Ratnieks F.L.W, Carreck N.L.: Clarity on Honey Bee Collapse? Science 327: 152, 201033）Ellis J.D, Evans J.D, Pettis J.: Colony losses, managed
colony population decline, and Colony Collapse DisorderInfluence of dinotefuran and clothianidin on a bee colonyin the United States. J. Apicult. Res. 49: 134-136, 201034）Medrzycki P, Sgolastra F, Bortolotti L, et al.: Influence of

2338）Japan Food Safety Commission Report on Toxicity Assessment of Pesticides – A Review of the Criteria forbrood rearing temperature on honey bee development

Registration of Pesticide Residues in Soil –: 3, 2005

and susceptibility to poisoning by pesticides. J. Apicult.

39）Japan Food Safety Commission Report on Toxicity As-

Res. 49: 52-59, 201035）Lu C, Warchol K. M, Callahan R.A.: In situ replication ofhoney bee colony collapse disorder. B. Insectol. 65: 99106, 201236）Iwasa T, Motoyama N, Ambrose J.T.: Mechanism for thedifferential toxicity of neonicotinoid insecticides in thehoney bee, Apis mellifera. Crop Protection 23: 371-378,
200437）Japan Food Safety Commission Report on Toxicity Assessment of Pesticides – Dinotefuran –: 23-24, 2005

sessment of Pesticides – Clothianidin – 3rd Ed.: 13-14,
200840）Kakuta H, Gen M, Kamimoto Y, Horikawa Y.: Honey bee
exposure to clothianidin: analysis of agroxhemicals using
surface enhanced Raman spectroscopy. Res. Bull. Obihiro Univ. 32: 31-36, 201141）Henry M, Beguin M, Requier F, et al.: A Common Pesticide Decreases Foraging Success and Survival in Honey
Bees. Science Express, 29 March 2012, Science 1215039:
1-4, 2012

要約
蜂崩壊症候群（CCD）と呼ばれる現象は養蜂や農業のみならず、生態系の危機へ繫がる深刻な問題である。病原体説や農薬説など様々な CCD 原因説が提案されているが、決定的な結論は出ていない。これまで CCD 原因解明のために、限定された条件下での実験や CCD 発生後の巣箱内の病原体の分析等が行われてきたが、CCD 発生過程の長期現場実験は殆ど行われていない。欧米ではネオニコチノイド系農薬を状況証拠から使用禁止した国も多いが、日本では科学的根拠が確定されていないため禁止に至っていない。そこで、日本で広く使われているジノテフランとクロチアニジンの長期投与実験を行い、その間の蜂数や蜂児数の変化および農薬摂取量を追跡し、蜂群が CCD の状態を経由して消滅に至ることを初めて明らかにした。また、太陽光下での蜜蜂の農薬摂取を想定して、これらの分解特性を調べた。NMR スペクトル解析により、熱的には両農薬とも安定であり、紫外線に対してはジノテフランは安定であるもののクロチア
ニジンは不安定であることが判った。
《キーワード》ジノテフラン、クロチアニジン、ネオニコチノイド系農薬、蜂群、崩壊