A new formulation of oxalic acid for Varroa destructor control applied in Apis mellifera colonies in the presence of brood

Abstract

An organic product based on oxalic acid was evaluated for use in Varroa control under spring/summer climatic conditions in Argentina. The formulation consists of four strips made of cellulose impregnated with a solution based on oxalid acid. Forty-eight beehives were used to assess the product efficacy. Residues of the product were also tested in honey, bees, and wax. Each trial had respective control groups without oxalic treatment. At the beginning of the experiment, four strips of the formulation were applied to the colonies belonging to the treated group. Falling mites were counted after 7, 14, 21, 28, 35, and 42 days. After the last count, the strips were removed and colonies received two flumethrin strips for 45 days. Falling mites were counted throughout this period. Average efficacy of the organic product was 93.1 % with low variability. This product is an organic treatment designed for Varroa control during brood presence and represents a good alternative to the synthetic treatments.

Full text

A new formulation of oxalic acid for Varroa destructor
control applied in Apis mellifera colonies in the presence
of brood
Matías MAGGI
1,2
,Elian TOURN
3,4,5
,Pedro NEGRI
1,2
,Nicolás SZAWARSKI
1
,
Alfredo MARCONI
3,4,5
,Liliana GALLEZ
6
,Sandra MEDICI
1,2
,Sergio RUFFINENGO
7
,
Constanza BRASESCO
1
,Leonardo De FEUDIS
1
,Silvina QUINTANA
8
,Diana SAMMATARO
9
,
Martin EGUARAS
1,2
1
CIAS, Centro de Investigación en Abejas Sociales (ex Laboratorio de Artrópodos), Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Mar del Plata, Funes 3350, 7600, Mar del Plata, Argentina
2
CONICET, Consejo Nacional de Investigaciones Científicas y Técnicas, Rivadavia1917, C1033AJ Buenos Aires,
Argentina
3
Departamento de Agronomía, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina
4
Ministerio de Agricultura, Ganadería y Pesca de la Nación, EEA INTA Bordenave, Bahía Blanca, Buenos Aires,
Argentina
5
Cooperativa de Trabajo Apícola Pampero Ltda., Bahía Blanca, Buenos Aires, Argentina
6
LabEA, Depto. Agronomía, Universidad Nacional del Sur, 8000, Bahía Blanca, Argentina
7
Cátedra de Apicultura, Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, 7620, Balcarce, Argentina
8
Laboratorio de Biología Molecular, Fares Taie Instituto de Análisis, Mar del Plata, Argentina
9
USDA ARS Carl Hayden Honey Bee Research Center, Tucson, AZ, USA
Received 14 July 2014 –Revised 4 August 2015 –Accepted 26 October 2015
Abstract –An organic product based on oxalic acid was evaluated for use in Varroa control under spring/summer
climatic conditions in Argentina. The formulation consists of four strips made of cellulose impregnated with a
solution based on oxalid acid. Forty-eight beehives were used toassess the product efficacy. Residues of the product
were also tested in honey, bees, and wax. Each trial had respective control groups without oxalic treatment. At the
beginning of the experiment, four strips of the formulation were applied to the colonies belonging to the treated
group. Falling mites were counted after 7, 14, 21, 28, 35, and 42 days. After the last count, the strips were removed
and colonies received two flumethrin strips for 45 days. Falling mites were counted throughout this period. Average
efficacy of the organic product was 93.1 % with low variability. This product is an organic treatment designed for
Varr oa control during brood presence and represents a good alternative to the synthetic treatments.
Varroa destructor / control / oxalic acid / bee brood / Argentina
1. INTRODUCTION
Varroa destructor (Acari: Varroidae), an obli-
gate ectoparasitic mite of the honey bee, Apis
mellifera (Hymenoptera: Apidae), feeds on the
hemolymph of adult bees, larvae, and pupae in
capped brood cells. In Argentina, severe
V. d e s t r u c t o r infestation of A. mellifera results
in honey bee colony death within 1 to 2 years of
initial infestation (Eguaras and Ruffinengo 2006).
In recent years, resistance to acaricides has be-
come a major problem in the control of mite
populations. Increased tolerance to the most wide-
ly used synthetic active ingredients has been
Corresponding author: M. Maggi,
biomaggi@gmail.com
Manuscript editor: Yves Le Conte
Apidologie Original article
*INRA, DIB and Springer-Verlag France, 2015
DOI: 10.1007/s13592-015-0405-7

observed (Maggi et al. 2010a,2011). Because of
the resistance to acaricides, there is a renewed
interest in Argentina for substances of natural
origin, such as essential oils and their components
or organic acids, especially formic acid and oxalic
acid (Eguaras et al. 2001,2003; Maggi et al.
2010b; Ruffinengo et al. 2014). This situation
related to Varr oa resistance has been observed
around the world (Sammataro et al. 2005).
Oxalic acid (OA) is widely used for controlling
V. destructor because of its high efficacy (>90 %)
and low risk of hive contamination (Gregorc and
Planinc 2001,2002; Nanetti et al. 2003; Marinelli
et al. 2006; Rademacher and Harz 2006;
Bacandritsos et al. 2007). OA is applied to colo-
nies by spraying or trickling a solution of OA and
sugar-water over the bees or by evaporating crys-
tals with heat (Rademacher and Harz 2006).
Hovewer, most tests have been carried out during
a broodless period, and have reported more than
95 % efficacy (Higes et al. 1999; Charriere and
Imdorf 2002). So, mite mortality after treatment
with oxalic acid seems to be directly influenced by
the presence of brood because oxalic acid does not
kill the mites in sealed brood cells (Imdorf et al.
2003). Several reports agree on the use of oxalic
acid when no brood is present in winter (Barbero
et al. 1997). Its efficacy when brood is present is
around 60 % (Charrière 1997; Rademacher and
Harz 2006). Thus, the usefulness of the acid
seems limited in warm climates with a long brood
rearing period.
Taking into account that oxalic acid is a suit-
able compound for the control of V. d e s t r u c t o r in
broodfree colonies during the autumn and winter
period within the concept of Integrated Va r ro a
Control, the major challenge to scientists is to
develop a newformulation that can be used during
permanent brood presence in colonies of
A. mellifera . The main goal of this article was to
evaluate the efficacy of a new formulation based
on oxalic acid for when brood was present.
2. MATERIAL AND METHODS
Location of study. Summer field trials were carried out
during March and April 2010 in the experimental apiary
located near Bahía Blanca city (Buenos Aires,
Argentina) and during January and February 2011 in
the experimental apiary of The Charrúas Government
(Downtown of Charrúas, Entre Ríos, Argentina). The
average temperature recorded during this period in
Bahia Blanca city was 22 °C (range 9–33 °C), and in
Charrúas, the average was 31 °C (range 14–42 °C).
Autumn field trials were carried out during May and
June 2013, in the experimental apiary of Arthropods
Laboratory (National University of Mar del Plata), near
Mar del Plata city. The average temperature recorded
during this period was 14 °C (range −2–20 °C).
Field trials. Forty-eight beehives were used to assess
product efficacy. Three experiments were conducted to
test the efficacy of a new formulation with oxalic acid
(Aluen CAP) made by Cooperativa de Trabajo Apícola
Pampero Ltda. In the first two trials, the treatment
efficacy was assessed during the summer season: one
trial was performed in Bahía Blanca (15 colonies divid-
ed in three groups of five colonies each one: a control
group BA^without treatment, a second control group
BB^with celulose strips embebed with glycerin and a
treatment group BC^with Aluen CAP) and the other in
Charrúas (19 colonies divided in two groups: one of
nine (control group) and the other of ten colonies (treat-
ment group,Aluen CAP)). In a third trial, the honey and
wax was tested for any OA residues (apiary located at
Mar del Plata, 14 colonies divided in two groups of 7
colonies: control and treatment groups). All colonies
used during trials were previously equalized for bee
population, brood area, and honey and pollen stores.
Hive bottoms specially adapted for the collection of
dead mites were placed in each colony. In each trial,
control groups without oxalic treatment were
established. Apiaries were selected based on geographic
locations at least 5 km away to avoid reinfestation
phenomena.
At the beginning of the experiment, the new formulation
were applied to the treatment group. Aluen Cap treatment
consists of four strips U-shaped. The matrix of these
strips is composed of cellulose (45 cm×3 cm×1.5 mm);
each one contains 10 g of OA mixed with 20 mL of
glycerin. Each strip was placed astride on frames 2, 4, 6,
and 8 of the brood chamber (Figure 1). Falling mites
were counted after 7, 14, 21, 28, 35, and 42 days
using the hive bottoms specially adapted for the col-
lection of dead mites. After the last count, the strips
were removed and at the same day, colonies received
four flumethrin strips according to the instructions of
the manufacturer (registered trade name: Flumevar®,
M. Maggi et al.

supplied by APILAB SA, Argentina, http://
www.apilab.com/flumevarcostarica.pdf). No resistance
phenomena to flumethrin had been reported previously
in these apiaries or in the region where assays were
performed. This treatment was left in the colonies for
45 days. Falling mites also were counted throughout
this period.
The efficacy of oxalic acid treatment was calculated as a
percentage: ((number of dead mites during oxalic acid
treatment)/(number of dead mites collected during the
treatment with OA and flumethrin))×100. The cumula-
tive mite fall after oxalic acid and flumethrin treatment
was assumed to be 100 %. Data on percent efficacy
were analyzed by analysis of variance (ANOVA) after
arcsine transformation in the case of percentages, to
reduce the heterogeneity of the variance.
Colony population development. The progress of the
hives treated with the oxalic acid was monitored prior to
each treatment application and also 1 week after the
acaricide application. As an example, at T
0
, the colonies
were inspected and the first treatment application was
applied. Any change was compared with the untreated
control hives. All other conditions (weather, nourish-
ment, and supervision) were identical. The parameters
to quantify the general state of the colonies during the
evaluation were as follows: number of combs fully
covered with bees and open and sealed brood areas.
These procedures were satisfactory to assess colony
development in previous research (Maggi et al. 2013;
Negri et al. 2015). All colonies were checked by visual
observations for dead brood bees and queens after treat-
ment. Data were analyzed by ANOVA to evaluate if OA
treatment produces changes in the variables measured.
Oxalic acid extraction from bees, honey, and bees-
wax. During the autumn assay, samples of honey, bees,
and wax were collected from colonies to detect possible
residues generated by oxalic acid treatment. The bees
were analyzed as a whole. Samples of each colony were
taken prior the oxalic treatment application (control,
timepoint T
0
) and 42 days after it (timepoint T
2
).
Samples were collected according to the protocol of
the European Working Group (2001) and were stored
at −80 °C until analysis. Ten grams of sample was then
diluted with 80 % ethanol. Beeswax sampled was heat-
ed in a water bath 62±2 °C for 15 min); the samples had
been previously acidified with 100 μL of pure HCl
(Merck®). Tubes were shaken vigorously for 1 min.
An aliquot of 50 μL was placed in a 2-mL vial and
Figure 1. The application of the new formulation Aluen CAP, an organic product containing oxalic acid in strips of
cellulose and glicerine to a hive.
Varroa destructor control using oxalic acid

evaporated until dried, using a nitrogen stream. The
extract was resuspended in 1 mL of ethanol: acid solu-
tion. Vials were placed in incubator at 50±2 °C for 5 h.
GF-FID analysis. A gas chromatograph HP 6890
with a FID detector and an autosampler for 100 samples
was used. For GF-FID analysis, an Austosampler
Agilent 7683 was used. The injector conditions were:
temperature, 100 °C; pressure, 12.45 p.s.i.; split ratio,
10:1; and gas saver, 20 mL/min. An Agilent Column
(30 m×0.25 m×0.25 μm HP-5MS Ultra Inerte (p/n
19091S-433UI)) was employed using Helium 5.0
(Praxair) in constant pressure mode (flow, 1 mL/min;
oven temperature, 50 ° C; injection volume, 1 μL;
detector: FID: temperature, 250 °C) as gas carrier. The
average recovery of the oxalic acid ranged from 87 to
110 %, and detection limits were honey: detection limit,
0.5 mg/kg–quantification limit, 1 mg/kg; wax: detection
limit, 1.5 mg/kg–quantification limit, 12 mg/kg; bees:
detection limit, 0.8 mg/kg–quantification limit, 2 mg/kg.
Data were analyzed by ANOVA to evaluate if OA
treatment produces changes in the variables measured.
3. RESULTS
Efficacy. The efficacy of the treatment in Var r o a
control was determined by comparing the number
of falling mites recorded during the oxalic acid
treatment period with the total number of falling
mites recorded during the whole trial (including
the flumethrin treatment). Results are presented in
Table I. The average final efficacy in colonies
treated with the new oxalic acid formulation was
94 % (in the experimental apiary located near to
Charrúas dowtown, summer trial), 92.7 % (in
experimental apiary placed near to Bahia Blanca
city, summer trial) and 92.8 % (in experimental
apiary placed near to Mar del Plata city, autumn
trial). Final efficacy of the OA treatment had
significant differences to the control (P<0.05).
All trials had a low variability in the final efficacy
(range between 85.9±98.8 %). The highest mor-
tality for the three assays was recorded during the
first 22 days, with an average partial efficacy of
74.4 %.
Effects on colony population Colony population
parameters before and after treatment are shown
in Table II. For summer trials, treated colonies
(oxalic acid treatment) finished with an average
of 4.5 brood combs and 8.2 frames of bees. In the
autumn trial, colonies finished with 3.2 combs
covered with brood (open+sealed brood) and 8
frames covered with adult bees. Adverse effects
on the colonies (dead bee brood or queens) were
not detected during and after treatment. Adverse
effects on the bee populations were not detected in
the treated colonies in all three trials (P>0.05).
Oxalic acid detection from bees, honey and
beeswax. Table III summarizes the measure-
ments of oxalic acid in all samples taken
before and after treatment. The natural oxalic
acid content varied between 2.5 and
33.8 mg/kg. There was no increase in oxalic
acid content of honey, wax, and bees after
treatments, in all three trials (P>0.05). All
samples of bees and beeswax were negative
before and after the OA treatment.
4. DISCUSSION
The new formulation with oxalic acid as the
active ingredient, presents some advantages over
the current methods of organic acid treatments.
Polymer-cellulose matrices not only have all the
benefits of the oxalic acid liquid treatments but
also delay the release of OA so that it can remain
longer in the colony (Eguaras et al. 2003). This
reduces the number of visits to apiaries to reapply
OA treatments. In this study, we report a new
formulation based on oxalic acid with high acar-
icide activity against V. destructor. However, tak-
ing into account that mite fall was registered with
hive bottom specially adapted, the estimated effi-
cacy results not exclusively from the application
of the test medication but also from a combinatory
effect (natural mite mortality). Nonetheless,
strong differences were detected with the control
group, demonstrating an excellent acaricide activ-
ity of the new treatment.
Other research has demonstrated that good ef-
ficacy has been obtained using at least three ap-
plications of liquid oxalic acid per colony. In other
cases, three or more applications were not enough
for effective Varroa control (Rademacher and
M. Maggi et al.

Table I. Number of falling mites with oxalic acid treatment and flumethrin treatment for each of the three field assays.
Trial Treatment Colony No. of falling mites with treatment at day Total No. of falling
mites with
shock treatment
Efficacy of
formulation
(%)
Mean efficacy of
formulation (%)
7 1421283542
Summer trial
(Bahia Blanca
2010)
Oxalic acid
formulation
1 32 0 56 8 11 18 125 15 89.3 92.7
2 395 852 872 212 77 52 2460 40 98.4
3 55 97 123 89 94 80 538 44 92.4
4 211 228 221 44 68 54 826 65 92.7
5 107 30 97 139 86 10 469 61 88.5
Control I (strips
with glicerine)
0 04530012 42 22.2 20.6
2 2681113444 49 47.3
1 1310005 164 2.9
12 1253201638 106 26.4
1 1210037 169 3.9
Control II (strips
without glicerine)
11 00477523 77 23 28.9
12 00204612 84 12.5
13 201001013 60 17.8
14 2586121851 142 26.4
15 14 8 11 9 17 74 133 72 64.9
Summer trial
(Charrúas, Bahia
Blanca 2011)
Oxalic acid
formulation
16 54 185 144 4 15 1 403 21 95 94
17 0 282 46 14 0 1 343 14 96.1
18 8 695 595 15 3 3 1,319 16 98.8
19 19 186 112 18 3 4 342 1 99.7
20 3670105337615 96.1
21 0 915 348 41 1 2 1,307 0 100
22 88 620 340 815 253 221 2,337 70 97
23 42 42 93 2 3 1 183 20 90.1
24 26 10 33 1 3 0 73 23 76
25 46 67 49 22 5 0 189 18 91.3
Untreated 26 8 14 8 1 10 0 41 2032 1.9 5.2
27 2069113251 1295 3.8
Varroa destructor control using oxalic acid

Tab le I (continued)
Trial Treatment Colony No. of falling mites with treatment at day Total No. of falling
mites with
shock treatment
Efficacy of
formulation
(%)
Mean efficacy of
formulation (%)
7 1421283542
28 163100222 592 3.6
29 625620176 1920 3.8
31 19211317 121 12.3
32 20 11 4 5 16 1 57 897 5.97
33 5492008192 1120 7.5
34 81692026 796 3.1
35 28 6 8 11 13 0 66 1234 5.1
Autumn trial
(Mar del Plata
2013)
Oxalic acid
formulation
36 3448271001109 92.4 92.8
37 120 106 24 1 0 0 251 21 92.2
38 56 214 56 0 2 0 328 13 96.1
39 12472220083 1 98.8
40 87 119 17 1 1 2 227 42 84.3
41 2042214271 3 95.9
42 4112900062 7 89.8
Untreated 43 1301207 246 2.7 13.7
44 0100416 326 1.8
45 00190010 128 7.2
40 00720211 147 6.9
446 283122027 56 32.5
47 3140008 16 33.3
48 13401312 91 11.6
M. Maggi et al.

Harz 2006). Also, during the summer season, the
efficacy obtained by liquid treatments is reduced
as oxalic acid does not kill the mites in sealed
brood cells. In addition, Hatjina and Haristos
(2005) have shown a detrimental effect on brood
development plus low effectiveness using the
trickling method of OAwhen open brood is pres-
ent. They suggest that this method is not as safe as
has been reported in the past.
Marinelli et al. (2006) evaluated the efficacy of
cellulose strips with oxalic acid in comparison
with other oxalic acid treatments (trickling and
vaporization methods). In spring trials, the effec-
tiveness ofthe cellulose strips was not statistically
different from the natural fall of Va r ro a . Similarly,
the autumn trials had the same results. However,
when spraying, trickling, and vaporizing oxalic
acid, good control of Varr o a was seen in central
Italy. They conclude that the unsatisfactory effi-
cacy of cellulose strips versus the good control
results of trickling and vaporization could be ex-
plained by the high acidity of the oxalic acid water
solutions. A pH around 1 may be responsible for
the best oxalic acid activity against the mites.
Here, we show that one application of the Aluen
Cap formulations is enough to have good mite
control even with the presence of bee brood. In
this study, the oxalic acid formulation had a high
efficacy and it was possible to keep low Va r ro a
prevalence indices in the colonies. The matrix of
the OA strip may help to keep an adequate acid
concentration inside the colonies for a long time
(42 days). In addition, the combination of glycerin
with AO could help to maintain the drug for a
longer time inside the beehive and consequently
with mites (including those that are emerging
from brood cells). As it was reported by Segur
and Oberstar (1951), glycerin possesses a high
viscosity. This chemical property would help to
disseminate the acid among bees during a longer
time in comparison with other strip formulations
without glycerin, increasing its efficacy (these
hypotheses should be examined in later studies).
During the season when this work was done, the
temperature range was −2–42 °C, and in the three
trials, there were no problems with the higher
temperatures in the colonies.
This new product offers a good alternative for
Varr o a control as it does not have the two most
Table II. Colony population parameters before and after treatment for the three trials.
Colony population
parameter
Treatment Apiary I (Bahía Blanca) Apiary II (Charrúas, Entre Ríos) Apiary III (Mar del Plata)
Before treatment After treatment Before treatment After treatment Before treatment After treatment
Number of combs covered
with adult bees
OA 9.8a±0.4 (5) 8.6a±0.4 (5) 8.4a±2.2 (10) 7.9a±2.1 (10) 8.7a±1.2 (7) 8a±0.5 (7)
Control 9.8a±0.4 (10) 8.8a±0.4 (10) 7.4b±1.6 (9) 7.4b±1.7 (9) 9a±0.4 (7) 8.6a±0.2 (7)
Number of combs covered
with brood
OA 6.4c±2 (5) 2.9d±.2 (5) 4.6d±1.4 (10) 4.7d±1.8 (10) 4.6d±0.8 (7) 3.2d±0.4 (7)
Control 5.8c±2.4 (10) 4.1d±1.7 (10) 4.8d±1.3 (9) 5d±1 (9) 4.7d±0.3 (7) 3.1d±0.3 (7)
Mean±standard deviations. The number of colony sampled are enclosed in parentheses. Uppercase (a–d) letters indicate statistical differences among treatments (ttest, P<0.05)
Varroa destructor control using oxalic acid

frequent disadvantages of organic products used
for mite control. First, it is easy to use, safe for
beekeepers, and presents low variability between
colonies in its efficacy. Second, it does not present
a risk to colony development and so it can be used
to safely reduce the damage done by Va rr o a mites
even during the summer season.
In Argentina (as in many other parts of the
world), the use of synthetic acaricides is being
restricted due to the emergence of resistant mite
populations (Maggi et al. 2009,2010a,2011;
Sammataro et al. 2005), as well as honey and
wax contamination (Bogdanov et al. 2003;
Wallner 1999;Medicietal.2015). Oxalic acid is
a natural constituent of honey; values between 8
and 17,000 mg/kg have been found in different
honeys (Mutinelli et al. 1997;Bernardiniand
Gardi 2001;Bogdanovetal.2002).
Most vegetables contain much higher amounts
of oxalic acid than honey so the total daily intake
is negligible. Thus, from a nutritional point of
view, oxalic acid should, like formic acid, also
have a generally recognized as safe (GRAS) sta-
tus. Moreover, no significant residues are expect-
ed after oxalic acid treatments as demonstrated in
our research. Indeed, there is no risk of honey
residues after all types of oxalic acid treatments
(Radetzki 1994; Mutinelli et al. 1997; Del Nozal
et al. 2000; Bernardini and Gardi 2001;Radetzki
and Barmann 2001; Bogdanov et al. 2002).
Spraying and trickling of oxalic acid are accept-
ed for use against V. d e s t r u c t o r in most European
countries and are widely used by beekeepers
throughout Europe (Charriere and Imdorf 2002).
This new formulation is not dangerous for the user
or for the bee colony. Thus, Aluen CAP could be a
valid alternative for Varr oa mite control.
ACKNOWLEDGMENTS
The authors would like to thank the Universidad
Nacional de Mar del Plata (UNMDP) and Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET). This research was supported by a grant
of ANPCyT, PICT 2011 to M.M.
Une nouvelle formulation de l'acide oxalique appliquée
dans les colonies d' Apis mellifera pour lutter contre
Varroa destructor,enprésenceducouvain
Acari / lutte antiparasitaire / couvain d'abeille /
Argentine
Eine neue Oxalsäure-Formulierung zur Bekämpfung
von Varroa destructor in Völkern von Apis mellifera
mit Brut
Varroa destructor / Bekämpfung / Oxalsäure /
Bienenbrut / Argentinien
REFERENCES
Bacandritsos, N., Papanastasiou, I., Saitanis, C., Nanetti,
A., Roinioti, E. (2007) Efficacy of repeated trickle
applications of oxalic acid in syrup for varroosis con-
trol in Apis mellifera : Influence of meteorological
conditions and presence of brood. Vet. Parasitol. 148 ,
174–178
Table III. Oxalic acid residues in wax, bees and honey after treatment.
Treatment Matrix Before treatment After treatment
Control Wax 0 (7) 0 (7)
Bees 0 (7) 0 (7)
Honey 14.2
b
±11.6 (7) 25.6
c
±12.5 (7)
OA Wax 0 (7) 0 (7)
Bees 0 (7) 0 (7)
Honey 7.5
a
±5.2 (7) 17.3
b
±15 (7)
Mean±standard deviations (SD) and number of samples (n). Honey: detection limit, 0.5 mg/kg–quantification limit, 1 mg/kg; wax
detectionlimit, 1.5 mg/kg–quantification limit, 12 mg/kg; bees: detection limit, 0.8 mg/kg–quantification limit, 2 mg/kg.Uppercase
letters (a–c) indicate statistical differences among treatments (ttest, P<0.05)
M. Maggi et al.

Barbero, R., Panella, F., Bonizzoni, L. (1997) Ácido
oxálico y el tratamiento de limpieza radical de otoño-
invierno. Vida Apícola 85 ,8–13
Bernardini, M., Gardi, T. (2001) Influence of acaricide
treatments for Varroa control on the quality of honey
and beeswax. Apitalia 28(7–8), 21–24
Bogdanov, S., Charriere, J., Imdorf, A., Kilcheman,
V., Fluri, P. (2002) Determination of residues in
honey after treatments under field conditions with
formic and oxalic acid. Apidologie 33 (4), 399–
409
Bogdanov, S., Kilchenmann, V., Butikofer, U. (2003) De-
termination of acaricide residues in beeswax: Collabo-
rative study. Apiacta 38 (3), 235–245
Charrière, J.D. (1997) Potentiel et limites de l’emploi des
acides organiques. Sanitaire 161,219–227
Charrière, J.D., Imdorf, A. (2002) Oxalic acid treat-
ment by trickling against Varroa de structor :rec-
ommendations for use in central Europe and un-
der temperate climate conditions. Bee World
83 (2), 51–60
Del Nozal, M., Bernal, J., Diego, J., Gomez, L.,
Ruiz, J., Higes, M. (2000) Determination of ox-
alate, sulfate and nitrate in honey and honeydew
by ion-chromatography. J. Chromat. A 881 ,629–
638
Eguaras, M., Ruffinengo, S. (2006) Estrategias para el
control de Var r o a . Ed. Martin, Mar del Plata.
Eguaras, M., del Hoyo, M., Palacio, A., Ruffinengo, S.,
Bedascarrasbure, E. (2001) A new product with formic
acid for Varroa jacobsoni control. I Efficacy. J. Vet.
Med., (Series b), 48 ,11–14
Eguaras,M.,Palacio,A.,Faverin,C.,Basualdo,M.,
Del Hoyo, M., Velis, G., Bedascarrasbure, E.
(2003) Efficacy of formic acid in gel for Var ro a
control in Apis mellifera : importance of the dis-
penser position inside the hive. Vet. Parasitol.
111 ,241–245
Gregorc, A., Planinc, I. (2001) Acaricidal effect of oxalic
acid in honeybee (Apis mellifera) colonies. Apidologie
32 ,333–340
Gregorc, A., Planinc, I. (2002) The control of Varroa
destructor using oxalic acid. Vet. J 163 ,306–310
Hatjina, F., Haristos, L. (2005) Indirect effects of oxalic
acid administered by trickling method on honey bee
brood. J. Apic. Res. 44 (4), 172–174
Higes, M., Meana, A., Suárez, M., Llorente, J. (1999)
Negative long-term effects on bee colonies treated with
oxalic acid against Varroa jacobsoni Oud. Apidologie
30 ,289–292
Imdorf, A., Charrière, J.D., Kilchenmann, V., Bogdanov,
S., Fluri, P. (2003) Alternative strategy in central Eu-
rope for the control of Varroa destructor in honey bee
colonies. Apiacta 38 ,258–278
Maggi, M., Ruffinengo, S., Damiani, N., Sardella, N.,
Eguaras, M. (2009) A first detection of Va rr oa
destructor resistance to coumaphos in Argentina.
Exp. App. Acarol. 47 (4), 317–320
Maggi, M., Ruffinengo, S., Negri, P., Eguaras, M. (2010a)
Resistance phenomena to amitraz from populations of
the ectoparasitic mite Varroa destructor of Argentina.
Parasitol. Res. 107 (5), 1189–1192
Maggi, M., Ruffinengo, S., Gende, L., Sarlo, G., Bailac, P.,
Ponzi, M., Eguaras, M. (2010b) Laboratory evalua-
tions of Syzygium aromaticum (L.) Merr. et Perry
essential oil against Varroa destructor . J. Ess. Oil
Res 22 ,119–122
Maggi, M., Ruffinengo, S., Mendoza, Y., Ojeda, P.,
Ramallo, G., Floris, I., Eguaras, M. (2011) Suscepti-
bility of Varroa destructor (Acari: Varroidae) to syn-
thetic acaricides in Uruguay: Va rr o a mites’potential to
develop acaricide resistance. Parasitol. Res. 108 ,815–
821
Maggi, M., Negri, P., Plischuk, S., Szawarski, N., De
Piano, F., De Feudis, L., Eguaras, M., Audisio,
C. (2013) Effects of the organic acids produced
by a lactic acid bacterium in Apis mellifera
colony development, Nosema ceranae control
and fumagillin efficiency. Vet. Microbiol. .
doi:10.1016/j.vetmic.2013.07.030
Marinelli, E., Formato, G., Vari, G., De Pace, F.M. (2006)
Va r ro a control using cellulose strips soaked in oxalic
acid water solution. Apiacta 41 ,54–59
Medici, S., Maggi, M., Sarlo, E., Ruffinengo, S., Marioli, J.,
Eguaras, M. (2015) Presence of syntethic acaricides in
beeswax and its relationship with the development of
resistance in Varroa destructor. J. Apic. Res, in press
Mutinelli, F., Baggio, A., Capolongo, F., Piro, R., Prandin,
L., Biaison, L. (1997) A scientific note on oxalic acid
by topical application for the control of varroosis.
Apidologie 28 (6), 461–462
Nanetti, A., Büchler, R., Charriere, J.-D., Fries, I., Helland,
S., Imdorf, A., Korpela, S., Kristiansen, P. (2003)
Oxalic acid treatments for Var ro a control (review).
Apiacta 38,8
1–87
Negri, P., Maggi, M., Ramirez, L., De Feudis, L., Szwarski,
N., Eguaras, M., Lamattina, L. (2015) Abscisic acid
enhances the immune response in Apis mellifera and
contributes to the colony fitness. Apidologie 46(4),
542–557
Rademacher, E., Harz, M. (2006) Oxalic acid for the con-
trol of varroosis in honey bee colonies –areview.
Apidologie 37 ,98–120
Radetzki, T. (1994) Oxalsäure, eine weitere organische
Säure zur Varroabehandlung. Allgemeine Deutsche
Imkerzeitung 28 (12), 11–15
Radetzki, T., Bärmann, M. (2001) Verdampfungsverfahren
mit Oxalsäure. Feldversuch mit 1509 Völkern im Jahr
2000. Allgemeine Deutsche Imkerzeitung 35 (9), 20–
23
Ruffinengo, S., Maggi, M., Fuselli, S., De Piano, F., Negri,
P., Brasesco, C., Satta, A., Floris, I., Eguaras, E. (2014)
Bioactivity of microencapsulated essentials oils and
perspectives of their use in the control of Var roa
destructor. Bull. Insectol. 67(1), 81–86
Varroa destructor control using oxalic acid

Sammataro,D.,Untalan,P.,Guerro,F.,Finley,J.
(2005) The resistance of Va rro a mites (Acari:
Varroidae) to acaricides and the presence of es-
terase.Int.J.Acarol.31 (1), 67–74
Segur, J., Obrstar, H. (1951) Viscosity of glycerol and its
aqueous solutions. Ind. Eng. Chem. 43 (9), 2117–2120
Wallner, K. (1999) Varroacids and their residues in bee
products. Apidologie 30 ,235–248
M. Maggi et al.