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Field trial: mortality of bee brood by the addition of stearin to beeswax Reybroeck W. ILVO

Field trial: effect of the addition of a mixture of stearic

and palmitic acid (called stearin) to beeswax on the development of the worker bee brood

Final report: June 30, 2017

Dr. Wim Reybroeck

ILVO -T&V, Melle, Belgium


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Introduction In 2016, beekeepers alerted problems related to the poor development and dying-off of the bee brood after the insertion of new wax foundations which had been produced on an industri-al scale. Following analysis, it appeared that the abnormal beeswax contained much higher levels of stearic and palmitic acid, which pointed to the addition of stearin. Not only was the addition of 20 to 30% stearin signalled in Belgium, but also in Germany and the Netherlands. There are few literature data available on the effects of stearin on the bee brood. As such, the Federal Public Service for Public Health, Food Chain Safety and Environment asked the Flan-ders Research Institute for Agriculture, Fisheries and Food (ILVO) in Melle (BE) to carry out this study. Objectives To examine whether the addition of stearin to beeswax leads to mortality of part of the worker bee brood ('shot brood'). If mortality is identified, to determine the extent of the mortality for various percentages of stea-rin. Material and methods Reference beeswax Beeswax from Cameroon was used as a reference: 'Selected cast beeswax', Dadant Blatt, 41x26.3 cm, Lot 460, Best before 13/02/2020 (from a Belgian producer of wax foundations) = ('Ref'). The wax contains very low levels of pesticides (analysis result is known) and complies with the melting point, acid and ester values as specified for pure beeswax (1). Stearin The stearin used is a mixture of the (predominant) saturated fatty acids palmitic acid C16 and stearic acid C18 (CAS No. 67701-03-5) obtained from crude palm oil (from a Belgian producer of fatty acids) with a composition as shown in Table 1. Table 1. Composition of the stearin used in the field tests (producer's analysis).

Stearin

Origin:

palm oil

Palmitic

acid

C16:0

Stearic

acid

C18:0

Others

(C12,C14,

C15, C17,

C18:1, C20)

Acid value

Saponification

value

(mg KOH/g)

Melting point

43.5% 54.2% 2.3% 206.1 206.1 Ca. 56 °C

Cast wax foundations with added stearin The reference beeswax was re-melted in a Pyrex beaker from which newly cast wax founda-tions ('A') where made in the laboratory in a wax foundation mould with water cooling 'Normal 350 x 200' (Graze, Weinstadt, DE). The cast wax foundations (‘A’) have the same composition as the purchased wax foundations.

Stearin was added to the wax in increasing weight concentrations, namely 15, 20, 25, 30, 35

(1) As established in Regulation (EU) No. 231/2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No. 1333/2008 of the European Parliament and of the Council (for acid value and saponification value), and in accordance with the relevant literature.


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and 40%. Each time, wax foundations were cast from the various wax compositions, in the same way as (‘A’). The cast wax foundations obtained were: 15% (‘B’), 20% (‘C’), 25% (‘D’), 30% (‘E’), 35% (‘F’) and 40% stearin (‘G’). Samples (‘A’), (‘B’), (‘D’) and (‘F’) of the beeswax mixed with stearin were analysed by the la-boratory Ceralyse (Celle, DE), which is specialised in analysing beeswax, for acid and ester value, saponification number, and the hydrocarbons content (see below 'Results and discus-sion'). A gas chromatographic fingerprint was also carried out on these samples by Ceralyse (see below 'Results and discussion'). Wax foundation sample examined in parallel (‘H’): Industrially produced beeswax foundations from a producer of wax foundations. The re-sults for sample H are not included in this report. Apiary and bee colonies The apiary is situated at Brusselsesteenweg 370, Melle (BE). Four Dadant Blatt bee hives were used, populated with the European honey bee (Apis mellifera). Two queens (Carnica

Troiseck) were bred by Jacques Levrau and fertilised in Kreverhille in 2016, one queen (F1 - Carnica Troiseck) was bred by André Decaluwe and fertilised in 2016 at his apiary while the last queen is a young queen (2017), a daughter of a queen of Willy Geirnaert and fertilised in Melle. Experimental design Purchased wax foundations made of good beeswax were used (‘Ref’). These were cut to the size of Dadant Blatt super frames, with interior dimensions of 13x41 cm. Four openings of 8x8 cm (= 64 cm2) were applied to these wax foundations, into which a piece of the cast wax foun-dation to be tested could fit (Figure 1). The wax foundation with the openings and the pieces of cast wax foundation were held in place by a suitably positioned and fused small thread. For each test frame, a piece of cast reference wax was applied as a kind of internal check.

Figure 1. Scale diagram of a Dadant Blatt super frame with 4 pieces of wax foundation to be tested (1 reference wax foundation and 3 different situations).


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Each test frame was hung separately in a bee colony in the super, to enable the wax honey-comb to be built up by the worker bees. After 2 to 3 days, the queen was located in the bee colony in question, and enclosed on the test frame with built-up honeycomb, by placing a small frame with a flat queen excluder on both sides of the test frame. The queen confinement cage was placed in the middle of the super. That way, the queen can only lay eggs in the cells of the frame in question. After 2 days, the queen was removed from the frame and released into the brood area below, under a queen excluder, so that there could be no disruption by the subse-quent relaying of eggs in cells by the queen. The egg-filled test frame was checked each time to see whether an egg had been laid in each cell, and was photographed each time. After these checks, the test frame was replaced in the colony for the further development of the worker bee brood, which was also monitored. The final check (and photo-recordings) was al-ways carried out at the moment that all the larvae had pupated, and the brood was sealed. In this phase, a distinction can clearly be made between open cells in which the egg/larva has died, and the sealed cell with a living pupa. To this end, the detailed photographs of each situ-ation were printed and the open and closed cells were manually counted. As such, the survival percentage could be calculated. Given that a bee honeycomb is built up on both sides of the wax foundation (middle wall), results were obtained for each test frame for both the left and right side, in other words results for around 210 cells for each side. This approach makes it possible to rule out as many external influence factors as possible (queen, food, etc.) on the survival of the brood. The position within the frame itself may have an influence; for this reason, the positions of the pieces of wax foundation were changed when the test was repeated: each situation was at least tested in duplo. At the same time, the paral-lel situation was tested in a second bee hive (with a different queen). In addition, the pieces of wax foundation were mounted in another position: position 1 to position 3; position 2 to position 4; position 3 to position 1; position 4 to position 2. Tests were carried out over the period April 27 - June 20, 2017 with various climatological con-ditions. For the first tests, it was very cold with little nectar flow, the second period was very warm with significant nectar flow of the acacia (Robinia), while at the start of the third test peri-od, the temperatures and the nectar flow were normal, but towards the end it was once again very warm with significant nectar and honeydew flow (Castanea). Notwithstanding the fact that in all cases, eggs had been laid in practically every cell of all test frames, it was subsequently identified in several cases that the eggs in the cells against the side of the frame were no longer cared for in the cold weather period in the test frame 1 in col-ony 4 (weakest colony). In this test frame, for position 1 and 4, only the half of the cells, laid towards the middle, were counted (indicated in blue in Table 2). On the other hand, it was observed in the warm period that cells with eggs were quickly filled with nectar and half-ripe honey in strong colonies. The counts of the positions in question were cancelled (indicated in yellow in Table 2). It is noteworthy that this never occurred in positions 2 and 3, except in test frame 4 which was completely cancelled. It was also observed that this occurred less quickly for the reference wax (‘A’). In Table 2, an overview is shown of the different test frames and the tested wax foundations.


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Table 2. Overview of the different test frames, the position of the tested wax foundations and the date on which the queen was placed in confinement.

Test frame

Side Wax foundation

Starting date Position 1 Position 2 Position 3 Position 4

1 left A B C D

30/04/2017 right D C B A

2 left A E F G

30/04/2017 right G F E A

3 left A H D H

30/04/2017 right H D H A

1bis left C D A B

25/05/2017 right B A D C

2bis left F G A E

25/05/2017 right E A G F

5 left C D A B

05/06/2017 right B A D C

Legend: Blue: results based on a count of the half (directed towards the middle) of the inserted piece of bees-wax due to incomplete care of the brood (cold); Yellow: results cancelled due to the presence of nectar/half-ripe honey.

Results and discussion

a) Results of analyses of wax samples of reference wax, and from wax mixed with stearin (Ceralyse, Celle, DE)

The results of the analysis at Ceralyse of acid value, ester value, saponification value and total hydrocarbon content in samples (‘A’), (‘B’), (‘D’) and (‘F’) are shown in Table 3. Table 3. Ceralyse analysis results

Acid value Ester value Saponification

value (mg KOH/g)

Total hydrocarbons

Stearin added

Normal values (1)

17-24 70-80 87-104 13-13.5 %

(African origin) ---

Sample (‘A’) 18.8 75.4 94.2 13.9 % 0 %

(100 % ‘Ref’) = (‘A’)

Sample (‘B’) 47.3 65.0 112.3 11.5 % 15 %

(85 % ‘Ref’)

Sample (‘D’) 65.7 57.3 123.0 10.4 % 25 %

(75 % ‘Ref’)

Sample (‘F’) 84.7 49.8 134.5 8.8 % 35 %

(65 % ‘Ref’)


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From the results, it appears that the addition of stearin results in an increase in acid value and in the saponification number. On the other hand, both the ester value and the percentage of hydrocarbons decline. In the results, Ceralyse observed that the hydrocarbons content is slightly higher than normal for beeswax of Cameroonian origin (‘A’). This can be attributed to the addition of a very small quantity of hydrocarbons (such as paraffin) or to the fact that the beeswax largely consists of old honeycombs, meaning that this content can increase slightly. From literature, it appears that such small deviations do not have any impact on the development of the bee brood. A gas chromatographic fingerprint was also carried out at Ceralyse. In the chromatograms, the peaks for palmitic and stearic acid are clearly present in the samples (‘B’), (‘D’) and (‘F’). The chromatograms of the reference wax (‘A’) and wax sample (‘D’) are shown in Annex 1.

b) Results of brood mortality All the inserted wax foundations were well built-up by the bees, and then completely laid with eggs by the queens. Dying-off of the brood occurred at the start of the larval stage. The counts were made at the moment the surviving brood had pupated (sealed cells), which made the counting easier. The results of the counts are shown in Table 4. In addition, survival was also calculated with regards to the survivals obtained with the reference wax (Cameroon) on the same side as the test frame. No dying-off during the pupa stage was observed: the pupae de-veloped into imago (adult bees). Table 4. Results of the survival of the worker bee brood in beeswax of various composition.

Frame Side Situa-tion

Survival (%) Average

survival (%)

Survival w.r.t. refer-

ence (%)

Average survival

w.r.t. refer-ence (%)

Code

1

L A

66.3 67.5

100 100.0 Ref Cameroon

R 68.8 100

L B

29.8 34.5

45.0 51.0 15% stearin

R 39.2 57.0

L C

29.8 31.7

45.0 46.9 20% stearin

R 33.5 48.7

L D

32.7 27.8

49.3 41.3 25% stearin

R 22.9 33.3

2

L A

85.9 88.8

100.0 100.0 Ref Cameroon

R 91.7 100.0

L E

40.5 35.7

47.2 40.4 30% stearin

R 30.9 33.7

L F

26.9 29.3

31.3 33.0 35% stearin

R 31.7 34.6

L G

35.8 30.4

41.6 34.4 40% stearin

R 25.0 27.3


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Frame Side Situa-tion

Survival (%) Average

Survival (%)

Survival w.r.t. refer-

ence (%)

Average survival

w.r.t. refer-ence (%)

Code

3

L A

90.9 89.6


R 88.2 100.0

L H

Commercial wax R

L D

47.1 49.5

51.8 55.3 25% stearin

R 51.9 58.9

L H

Commercial wax R

1bis

L D

25.6 18.7

32.2 26.3 25% stearin

R 11.7 20.4

L A

79.7 68.5


R 57.4 100.0

2bis

L F

24.1 22.0

27.3 25.1 35% stearin

R 19.9 22.9

L G

33.6 33.1

38.0 37.8 40% stearin

R 32.6 37.5

L A

88.4 87.6


R 86.8 100.0

L E

33.0 31.8

37.3 36.2 30% stearin

R 30.5 35.1

5

L D

38.8 42.5

46.0 48.9 25% stearin

R 46.1 51.8

L A

84.3 86.7


R 89.0 100.0

The results of the replications per situation are summarised in Table 5. Survival was also cal-culated with regards to the survivals obtained with the reference wax on the same side as the test frame. These results are shown in Table 6. Discussion In all test frames, the survival of the worker bee brood was lower in wax with added stearin than survival in the reference wax, regardless of the weather, nectar/honeydew flow, queen bee, or the bee population. In most cases, the dying-off of the worker bee brood was very lim-ited in the reference wax. The dying-off of the brood was sometimes observed in the cells where the frame thread had fused. The addition of 15% stearin to beeswax results in significant mortality of the worker bee brood in the cells built up on wax foundations made of this kind of wax. Higher additions (up to 40%) result in an increase in mortality. Related to the mortality of worker bee brood in the reference wax, the average mortality in the wax with added stearin was at least 49.0% (with 15% stea-rin). The highest average mortality amounted to 71.0% (with 35% stearin).


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Table 5. Summary of the results of the survival of the worker bee brood in beeswax of various composi-tion (per situation).

Survival (%) for each type of wax

A B C D E F G H

66.3 29.8 29.8 32.7 40.5 26.9 35.8

68.8 39.2 33.5 22.9 30.9 31.7 25.0

85.9

47.1 33.0 24.1 33.6

91.7

51.9 30.5 19.9 32.6

90.9

25.6

88.2

11.7

79.7

38.8

57.4

46.1

88.4 86.8 84.3

89.0

Average survival (%) for each type of wax

81.5 34.5 31.7 34.6 33.7 25.7 31.7

Table 6. Summary of the results of the survival of the worker bee brood in beeswax of various composi-tion (per situation) related to the survival in reference wax.

Survival (%) with regards to the survivals obtained with the reference wax for each type of wax

A B C D E F G H

100.0 45.0 45.0 49.3 47.2 31.3 41.6

100.0 57.0 48.7 33.3 33.7 34.6 27.3

100.0

51.8 37.3 27.3 38.0

100.0

58.9 35.1 22.9 37.5

100.0

32.2

100.0

20.4

100.0

46.0

100.0

51.8

100.0 100.0

100.0

100.0

Average survival (%) with regards to the survivals obtained with the reference wax for each type of wax

100.0 51.0 46.9 43.0 38.3 29.0 36.1


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Conclusion The results show that beeswax with added stearin (in the tested percentages from 15% on-wards) is not suitable for use as a raw material for the production of wax foundations for use in apiculture. Remarks regarding the results Attention is drawn to the fact that the test was carried out with very pure reference wax, and in healthy bee colonies. In many practical cases, beeswax still contains a certain percentage of added hydrocarbons (for example paraffin) and a higher content of pesticide residues which may have an additional (mutually reinforcing) negative influence on the development of the bee brood. Other types of stearin (for example of a different origin and with a different ratio of palmitic ac-id/stearic acid) can generate a different result. The study only provides a snapshot of the effect of stearin over a very short timeframe of the life cycle of a bee hive. With significant brood losses, the chances of survival of a bee colony are seriously curtailed.


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Annex 1. Chromatograms

Fig. 2. Chromatogram of the reference wax (‘A’).


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Fig. 3. Chromatogram of wax sample D (with 25% stearin). The first two major peaks are pal-mitic and stearic acid, respectively.


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Annex 2. Photographs

Photo 1: Start phase test frame 2bis, left side.

Photo 2: Half built-up test frame 5, left side.

Photo 3: Queen confinement cage (test frame surrounded by frames with queen excluder).


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Photo 4. Detail of wax honeycomb (‘D’) on test frame 1bis, left side, fully laid with eggs.

Photo 5. Detail of end phase for wax honeycomb (‘A’) (reference) on test frame 2bis, right side as used during the counting of open and closed cells.


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Photo 6: End phase test frame 2bis, left side.

Photo 7: End phase test frame 2bis, left side, with template.