MELISSA - Report http://www.dafne.at/dafne_plus_homepage/index.php?section=dafneplus&content=result&come_from=&&search_fields[offer_number]=100472&search_fields[title_ger]=&search_fields[research_objective]=&search_fields[beauftragungsjahr]=&search_fields[antragsteller]=&search_fields[projektleiter]=&project_id=2909 Summary The number of reports about honey bee colony losses or damages from many countries has increased over the last years. The potential causes are numerous and could differ case by case. According to the current knowledge, a single factor is rarely responsible. In fact, in many cases more likely a combination of etiological factors is involved, e.g. colony management and good apicultural practice, environmental and anthropogenic elements as well as honey bee pests and parasites. In spring 2008 severe honey bee losses occurred in Germany (Rhine valley), in Italy and Slovenia during and after sowing of clothianidin coated maize seed with pneumatic seed drills. Further investigations in Germany proved the causal connection between the use of this seed dressing insecticide and the reported damages in honey bee colonies. In order to assess the possible relevance of this problem to Austria, the project “Investigations in the incidence of bee losses in corn and oilseed rape growing areas of Austria and possible correlations with bee diseases and the use of insecticidal plant protection products” (acronym: “MELISSA”) was carried out in the years 2009 – 2011 on behalf of the Federal Ministry of Agriculture, Forestry, Environment and Water Management and the Austrian federal provinces. The aim of the project was to identify possible correlations between the incidence of honey bee losses in production areas of maize and oilseed rape and bee diseases or the use of plant protection products on the basis of field data. Summing up, the results of the MELISSA-project give evidence that in Austria regional clustered bee damages had occurred in the years 2009 – 2011, which were frequently associated with the use of maize and oilseed pumpkin seeds coated with insecticides, as proved by residue analysis. The strong local component and the accumulation in areas with small-scale structured agriculture indicated special environmental conditions resulting in an increased exposition of honey bees to the identified insecticidal plant protection substances in the affected areas. Regulatory measures to prevent honey bee losses due to the exposure of bees to insecticidal seed dressing substances have significantly improved the situation. However, repeatedly observed incidences of honey bee mortality in defined regions suggest their systematic correlation with local factors contributing to increased exposure of bees. In addition to considering environmental factors, all measures to mitigate risks have to be implemented invariably and with discipline.

1. Investigations on the incidence of bee losses in corn and
oilseed rape growing areas of Austria and possible
correlations with bee diseases and the use of insecticidal
plant protection products
(Acronym: MELISSA)
Order for project: Federal Ministry of Agriculture, Forestry, the Environment and Water Management (BMLFUW)
Funding: BMLFUW, Federal provinces of Austria, AGES, Consortium of seed and plant protection companies
Duration: 1.3.2009 – 31.1.2012
Details and full report: www.dafne.at
Project Nr. 100472
Project manager: DI Leopold Girsch
Involved AGES-Divisions and Departments:
Division for Food Security: Inst. for Seed and Propagating Material, Phytosanitary Service and Apiculture;
Inst. for Sustainable Plant Production; Inst. for Plant Protection Products;
Division for Food Safety;
Department Data, Statistics & Risk Assessment
www.ages.at Austrian Agency for Health and Food Safety (AGES)

2. MELISSA
Objectives
• documentation of incidences of honey bee losses in production
areas of maize and oilseed rape
• analysis of possible causes (honey bee pathogens and
parasites, plant protection products)
• evaluation of results with respect to measures taken to prevent
honey bee losses
• development of decision guidances for authorities, beekeepers
and farmers for the implementation of measures to prevent
honey bee losses by pathogens, parasites and plant protection
products
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation:
www.ages.at 2
Robert Womastek; SCFCAH - PPP Legislation Meeting, 27-28 Sept 2012

3. Studies conducted
• diagnosis for pathogens and parasites in honey bees
Varroa destructor, Nosema spp., 7 honey bee viruses
• analysis for residues
- insecticidal seed coating products
- other active substances of plant protection products
sampled matrices:
bees, beebread, extracted honey
plants
seed
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation:
www.ages.at 3
Robert Womastek; SCFCAH - PPP Legislation Meeting, 27-28 Sept 2012

4. Symptoms in honey bees and
bee colonies
- increased numbers of dead bees inside and outside the hives
- flightless bees with symptoms of trembling, cramping,
disorientation, paralysis, abnormal wing movements,
hyperexcitation, disordered locomotory activity
o at time of maize sowing (simultaneously or from next day onwards)
o ca. 10 – 14 days after beginning of maize sowing
- flightless crawling bees or groups of bees on the soil or in the
grass in front of beehives
o from the beginning of maize sowing
o in many cases also considerably later in the season (May until beginning of June)
- intensity and duration of symptoms
o one to several days – in some cases considerably longer
o in some particular cases massive damages occured when bees showed a
multiple contamination by insecticidal seed coating substances and other
insecticides
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation:
www.ages.at 4
Robert Womastek; SCFCAH - PPP Legislation Meeting, 27-28 Sept 2012

5. Observed symptoms in the apiaries
(1)
• increased bee mortality
Residue analysis in bees: Residue analysis in bees:
*Clothianidin 0.0021 mg/kg; *Clothianidin 0,0033 mg/kg;
*Thiamethoxam 0,0088 mg/kg *Imidacloprid 0,0273 mg/kg
*LOD = 0,0002 mg/kg; *LOQ = 0,001 mg/kg
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 5

6. Observed symptoms in the apiaries
(2)
• flightless crawling bees from the beginning of maize sowing and in many
cases also considerably later in the season
Residue analysis:
Residue analysis: bees: negative;
bees: negative bee bread positive: *Imidacloprid <
LOQ **Thiacloprid < LOQ
*LOD = 0,0002 mg/kg; *LOQ = 0,001 mg/kg; ** LOQ: 0,01 mg/kg
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 6

7. Observed symptoms in the apiaries
(3)
• large quantities of dead bees in cases of multiple contamination by insecticidal
seed coating substances and other insecticides
Insecticides detected in dead bees by residue analysis: *Clothianidin, *Fipronil,
*Fipronil-sulfone, **Chlorpyrifos
*LOD = 0,0002 mg/kg; *LOQ = 0,001 mg/kg; ** LOQ: 0,01 mg/kg
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 7

8. Bee yards with suspected bee poisonig –
results of residue analysis year 2009
Insecticidal seed coating materials: Clothianidin, Thiamethoxam,
Imidacloprid, Fipronil and Fipronil-sulfone
< LOQ
≥ LOQ
negative
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 8

9. Bee yards with suspected bee poisonig –
results of residue analysis year 2010
Insecticidal seed coating materials: Clothianidin, Thiamethoxam,
Imidacloprid, Fipronil and Fipronil-sulfone
< LOQ
≥ LOQ
negative
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 9

10. Bee yards with suspected bee poisonig –
results of residue analysis year 2011
Insecticidal seed coating materials: Clothianidin,
Thiamethoxam, Imidacloprid, Fipronil and Fipronil-sulfone
< LOQ
≥ LOQ
negative
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 10

11. Results of residue analysis for
„other plant protection substances“
Bees and Beebread
– Several different active substances were detected
– insecticides
– herbicides
– fungicides
– unapproved substances according to VO (EG) Nr.
1107/2009 were detected in single cases (e.g.
phosalone, chlorfenvinphos,paraoxon-methyl,
diphenylamin)
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 11

12. Colony losses during hibernation
period 2009/2010 (1)
4 beekeepers supposed losses to be connected with the uptake of potentially
contaminated pollen during late summer and autumn of the previous year
a) Diagnostic results for pathogens and parasites
In beekeeping operations 1 – 3 Varroosis could be identified as the most likely cause for
colony losses (Varroa-infestation rate bee samples: 17-39 %; brood samples: 24-56 %)
In beekeeping operation 4 severe failures to comply with good apicultural practice were
revealed (establishment of very weak mating nucs late in the year, no protective measures
against shrews, dysentery and N. ceranae as cofactors), therefore no residue analysis was
performed
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 12

13. Colony losses during hibernation
period 2009/2010 (2)
b) Residue analysis
• No residues from insecticidal seed coating materials or
other active substances of plant protection products were
detected in
– bee bread samples (pooled by apiaries) from 3
beekeeping operations
– dead bees (pooled sample) from one beekeeping
operation
• Conclusion: In these 4 cases there are no indications for
a contamination of hive products by the investigated
analytes
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 13

14. Colony losses during hibernation
period 2010/2011
6 beekeepers supposed losses to be connected with the uptake of potentially
contaminated pollen during late summer and autumn of the previous year
a) Residue analysis of bee bread from dead or very weak colonies
• Case 1: Clothianidin < LOQ
• Case 2: Imidacloprid < LOQ
• Cases 3 - 5: negative
• Case 6: no residue analysis was performed (severe case of varroosis: 24 % infestation
rate in remaining brood cells)
b) Diagnostic results for pathogens and parasites
In cases 3 - 6 pests and parasites were most likely the causes for colony winter losses.
Conclusions: an exposure to insecticide seed coating material was confirmed for 2
cases of winter losses. Because Imidacloprid is used both as a seed coating and a
spray treatment the source for this contaminant remained unclear.
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 14

15. Suspected bee poisoning incidents
during bloom of maize or later in
the year 2011
Reports from 5 beekeeping operations (5 bee yards)
Reasons suspected by beekeepers:
• case 1: “spray applikations in seed maize or grape-vine“;
residue analysis bees: Imidacloprid 0,0016 mg/kg
• case 2: “plant protection activity“:
residue analysis bees: Imidacloprid < LOQ (LOQ = 0,001 mg/kg)
• cases 3, 4: “contaminated pollen from maize, oilseed-pumpkin, grape-vine“;
residue analysis negative
• case 5: “unidentified plant protection activity“; residue analysis not possible
due to insufficient sample size
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 15

16. Clothianidin – three-years-comparison of
residue results for samples of bees and bee bread
MELISSA-Vergiftungsverdacht
LOD Rückstandsanalysen auf Clothianidin
= 0,0002 mg/kg; LOQ = 0,001 mg/kg
Matrix: Bienen und Bienenbrot
Legend:
CLO >= BG CLO < BG CLO n.n. BG = LOQ
100% 0 0 n.n. = not detected
90% 7
15
2 15
80%
2
11 7 9
70% 19
28
60% 9
18 42
29
50% 1 3 1 2 0 1 1
40% 2
30% 24 42
7 3 27 2
20%
2 7 Bees + Bee bread:
10% 0
4
5 9
1 significant decline
2 2
0% 0 0 0 0 2 0 0 0 0 0 0 0 0 in percentage of
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
positive samples
B K NOE OOE ST T W V
from 2009 - 2011
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 16

17. Thiamethoxam – three-years-comparison of
residue results for samples of bees and bee
bread
MELISSA-Vergiftungsverdacht
LODRückstandsanalysen auf Thiamethoxam
= 0,0002 mg/kg; LOQ = 0,001 mg/kg
Matrix: Bienen und Bienenbrot
Legend:
THI >= BG THI < BG THI n.n. BG = LOQ
100% 0 n.n. = not detected
90%
80%
70%
44
60% 16 30
5 25 44
36
50% 1 20 3 1 48 31 4 1 1
40%
30%
12
20%
0
2 2 Bees + Bee bread:
10% 7 2
1
3
0
2 3
1
10
5 significant decline in
0% 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0
percentage of positive
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
samples from 2009 -
B K NOE OOE ST T W V
2011
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 17

18. Fipronil + Fipronil-sulfone – three-years-
comparison of residue results for samples of
bees and bee bread
MELISSA-Vergiftungsverdacht MELISSA-Vergiftungsverdacht
Fipronil
Rückstandsanalysen auf Fipronil
Matrix: Bienen und Bienenbrot
Rückstandsanalysen auf Fipronil-sulfon
Fipronil-sulfone
Matrix: Bienen und Bienenbrot
FIP >= BG FIP < BG FIP n.n. FIP-S >= BG FIP-S < BG FIP-S n.n.
100% 100%
90% 90%
80% 80%
70% 70%
60% 3 60%
3
28 59 28 62 50
50% 1 20 3 1 6 49 20 38 32 36 50 1 1 50% 1 20 3 1 6 49 20 39 32 37 1 1
40% 40%
30% 30%
0 0
20% 20%
1 1
10% 10%
0 4 0
2 1 1 3 0 2 2
0% 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 0 1 1 2 0
1
0% 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
B K NOE OOE ST T W V B K NOE OOE ST T W V
LOD = 0,0002 mg/kg; LOQ = 0,001 mg/kg
Bees: significant decline
in percentage of positive
samples
Legend: BG = LOQ; n.n. = not detected
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 18

19. Imidacloprid – three-years-comparison of
residue results for samples of bees and bee
bread
MELISSA-Vergiftungsverdacht
LOD = 0,0002 mg/kg; LOQImidacloprid
Rückstandsanalysen auf = 0,001 mg/kg
Matrix: Bienen und Bienenbrot
IMI >= BG IMI < BG IMI n.n. Legend:
100% 0
BG = LOQ
n.n. = not detected
90%
80%
1
70%
14
60% 25
33
46 19 63 48
50% 1 3 1 6 30 40 4 1 1
40%
30%
1
20%
3
3
Bees + Bee bread:
10%
3 4 5
significant increase
2 1 1
1
0% 0 0 0 0 0 0 0 1 0 0 0 2 2 0 0 0 0 0 0 0 0 in percentage of
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
2009
2010
2011
positive samples
B K NOE OOE ST T W V from 2009 - 2011
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 19

20. Exposure of bee yards (%) to insecticidal seed coating
materials1 in the years 2009 – 2011 according to results of
residue analysis in bees or bee bread
Anteil der Bienenstände mit bzw. ohne
Rückstandsnachweis für insektizide Saatgutbeizmittel in
1 Clothianidin, Thiamethoxam, Imidacloprid, Fipronil and
Bienen oder Bienenbrot
Fipronil-sulfone: LOD = 0,0002 untersucht, bzw. 7 Proben nur aufmg/kg
* 2011 wurden keine Monitoringstände mg/kg; LOQ = 0,001
"sonstige Pflanzenschutzmittel" untersucht
100% 7
2
90%
5 27
80% 8
70% 66
Bienenstände
13 n.u.** Legend:
Bee yards
11
60%
neg. BG = LOQ
50% 7 neg = negative
< BG
40% 20 28 n.u. = not analysed
30% 55 ≥ BG
2
20%
43 7 *7 samples just analysed for
10% 4 „other active substances of
0% plant protection products;
2009 2010 2011 2009 2010 2011*
** in 2011, no monitoring
Suspected poisoning
Vergiftungsverdacht Monitoring
Monitoring beeyards were investigated;
Significant downtrend of exposed bee yards during years of study.
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 20

21. Residue analysis of extracted honey
In 2009 9 samples and in 2010 62 samples were analysed for Clothianidin, Thiamethoxam,
Imidacloprid: all negative (LOD = 0,0006 mg/kg; LOQ = 0,002 mg/kg)
2011: 13 samples analysed: all were in compliance with the MRL-regulations and fully marketable
according to the Austrian legislative framework.
Analytes n.d. > LOQ < LOQ Samples (n=)
*Clothianidin1 11 0 2
*Thiamethoxam2 12 0 1
*Imidacloprid1 11 1 1 13
*Fipronil2, *Fipronil-sulfone1 13 0 0
Thiacloprid2 5 4 4
Amitraz3 12 1 0
Legend: n.d. = not detectable; LOQ = level of quantification; LOD = level of detection
1 LOQ = 0,0002 mg/kg; LOD = 0,00006 mg/kg
2 LOQ = 0,0005 mg/kg; LOD = 0,00015 mg/kg
3 LOQ = 0,01 mg/kg
*for these substances LOD and LOQ, respectively, were lower by a factor of 10 in 2011 compared to the years 2009 and 2010.
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 21

22. Wind conditions at time of maize sowing 2011
„no sowing of insecticidal coated maize and oil pumpkin seed with pneumatic seed
drills at a windspeed > 5 m/s (18 km/h)“
Wind-peak values (wsp)
25
20
Windspeed (m/sec)
15
Hours
10
5
Day
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 22

23. Wind conditions in April at time of maize sowing
years: 2009, 2010, 2011
„no sowing of insecticidal coated maize and oil pumpkin seed with pneumatic seed
drills at a windspeed > 5 m/s (18 km/h)“
Neusiedl/See 2009 - 2011
Location Neusiedl/See
Wind-Spitzenwerte 12 Uhr
Wind-peak values (wsp) at 12 (a.m.)
wsp-12h-2009 wsp-12h-2010 wsp-12h-2011
200
20
Windspeed (m/sec)
Windgeschwindigkeit
150
(Zehntelmeter/sec)
15
100
10
50
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Tag
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 23

24. Heubach-values of abrasion tests from
enforcement (marketing) control samples
of insecticidal coated seed batches in 2010
and 2011
0,80
2010 2011
Grenzwert Abrieb
0,75 g / 100.000 permissible abrasion value: 0.75 g/100.000 kernels
Maximum Korn
0,70
0,60 Average = 0.35 = 0,35 g / 100.000 Korn
Mittelwert g/100.000 kernels Average = 0.17 g/100.000 / 100.000 Korn
Mittelwert = 0,17 g kernels
0,50
0,40
0,30
0,20
0,10
0,00
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 24

25. Summary
• Results of the MELISSA-project give evidence that in Austria regional clustered bee
damages had occurred in the years 2009 – 2011, which were frequently associated with
the use of maize and oilseed pumpkin seeds coated with insecticides.
• Regulatory measures to prevent honey bee losses due to the exposure of bees to
insecticidal seed dressing substances have significantly improved the situation. However,
repeatedly observed incidences of honey bee mortality in defined regions suggest their
systematic correlation with local factors contributing to increased exposure of bees.
• Seed dressing quality and seed drill equipment still need further improvement and
sowing of insecticidal dressed seed with pneumatic seed drills has to be avoided under
windy conditions
• Insecticidal seed dressings should be used solely based on a given risk in plant
production with therefore reasoned indication.
• An adjustment in the approval procedure is required for risk assessment and approval of
insecticidal substances concerning the specific characteristics and possible routes of
exposure of bee toxic seed dressing substances.
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 25

26. For further Details, please contact
Dr. Rudolf Moosbeckhofer
AGES, Institute for Seed and Propagation Material, Phytosanitary
Service and Apiculture
Department for Apiculture and Bee Protection
rudolf.moosbeckofer@ages.at
Rudolf Moosbeckhofer, Leopold Girsch - AGES Division Food Security; Presentation: Robert Womastek; SCFCAH - PPP
Legislation Meeting, 27-28 Sept 2012 26