Parasitol Res (2007) 100:1267–1270

DOI 10.1007/s00436-006-0418-2

ORIGINAL PAPER

Effects of solvents and surfactant agents on the female

and larvae of cattle tick Boophilus microplus
Karla Gonçalves & Eduardo Toigo & Bruna Ascoli &
Gilsane von Poser & Vera Lucia Sardá Ribeiro

Received: 16 August 2006 / Accepted: 21 November 2006 / Published online: 20 January 2007
# Springer-Verlag 2007

Abstract Many natural compounds have low water solu- Introduction

bility and need to be dissolved in organic solvents, or
surfactant agents must be used before addition into Boophilus microplus (one-host tick) constitutes a major
experimental systems. Therefore, it is necessary to deter- problem to veterinary health in various countries of the
mine their toxicity. Experiments were performed aiming to world as a debilitating parasite or as a vector of a great
select solvents to be used in the bioassays, searching new variety of diseases or both. Blood loss and reduction in
acaricide agents from plants. Laboratory tests were carried weight gain resulting from tick feeding represent one of the
out on larvae and adults of the cattle tick Boophilus most important pathological constraints to livestock pro-
microplus to determine the sensibility of B. microplus duction. The parasites also directly affect the quality of skin
female and larvae to different solvents (acetone, methanol, (Ducornez et al. 2005).
ethanol and 1% dimethyl sulfoxide) and surfactant agents Highly potent chemical substances such as cypermethrin
(1% Tween 80 and 5% Triton X-100) using the larval (pyrethroid) and amitraz (formamidine) have been used to
immersion test (LIT) and adult immersion test (AIT). In the eliminate the ectoparasite. However, the development of
AIT, the effect of the treatments on engorged females was acaricide resistance and the environmental pollution limit
assessed by measuring egg production and hatching rate. their use. Alternative anti-tick products and or strategies are
Acetone was toxic to the adults promoting mortality of therefore warranted.
100%. Methanol and ethanol caused 45.3 and 14.2% of An alternative of lower environmental impact to the
mortality, respectively. The other tested substances were not control of cattle ticks would be to use active principles of
toxic to the engorged females of B. microplus. In the LIT, it plants with acaricidal properties, and some interesting
was observed that the larvae were more resistant; after 48 h, results have been obtained (Chungsamarnyart and Jiwajinda
about 100% of the larvae were alive in all the treatments 1992; Prates et al. 1993; Chungsamarnyart and Jansawan
except with acetone that caused a mortality of 10%. 1996; Chagas et al. 2002; Borges et al. 2003; Castrejón et
al. 2003; Pereira and Famadas 2004; Fernandes et al. 2005).
In laboratory bioassays, the use of organic solvents is
unavoidable, as many natural compounds have low water
solubility and need to be dissolved in organic solvents
before addition into experimental systems. Nevertheless,
K. Gonçalves : V. L. S. Ribeiro with conventional solvent extracts, the tests are difficult to
Laboratório de Entomologia, Faculdade de Veterinária, UFRGS,
perform because the solvents employed are toxic for
Avenida Bento Gonçalves, 9090,
91540-000 Porto Alegre, RS, Brazil biological systems. So, the selection of a suitable solvent
for the bioassays is governed by the solubility of the
E. Toigo : B. Ascoli : G. von Poser (*) samples (extracts, fractions or isolated compounds) and by
PPG-Ciências Farmacêuticas, UFRGS,
the stress imposed on test organisms by organic solvents.
Avenida Ipiranga, 2752,
90610-000 Porto Alegre, RS, Brazil Most reports on the comparative toxicity of solvents
e-mail: [email protected] towards test organisms deal with the effects of solvents on
1268 Parasitol Res (2007) 100:1267–1270

fish and aquatic invertebrates with some data available for room temperature). Water was used as the control. Ticks
algae (Stratton 1989; Tadros et al. 1994; Jay 1996; Ma and were recovered from the solutions, dried and randomly
Chen 2005), insects (Gorb et al. 2000) and for the tropical placed in each Petri dish (5.5-cm diameter, 1.5 cm high).
horse tick Anocentor nitens (Beadles et al. 1973). Only two The Petri dishes were incubated at 27–28°C, 70–80%
papers dealing with the solvent toxicity on B. microplus were relative humidity. After 14 days, the number of females
found (Chagas et al. 2003; Freitas and Fernandes 2005). laying eggs was recorded, and the eggs were collected and
The present study was conducted to establish the effect weighed. The eggs were incubated, and the percentage of
of acetone, methanol, ethanol, DMSO, Tween 80 and Triton hatched eggs was estimated.
X-100 on B. microplus using the methodology proposed by Mortality (percent inhibition of egg laying) was calcu-
Sabatini et al. (2001) with some modification. lated as follows:
Index of egg laying ðIEÞ
Materials and methods ¼ weight of eggs laid ðgÞ=weight of females ðgÞ

Test samples
%Mortality
The solvents employed included acetone, ethanol, methanol
¼ IE control group  IE treated=IE control group 100
and dimethyl sulfoxide (DMSO). All the test samples were
analytical reagent. Their concentrations are given as percent
volume. Water was used as control.
Statistical analyses
Preparation of ticks
The significance of the data was evaluated by the ANOVA
one-way test.
Engorged female ticks were collected from infested
animals, washed with water and dried by paper towelling.
The average weight of engorging ticks was 0.30 g. These
Results
females were used in the adult immersion test (AIT) or
incubated at 27–28°C and 70–80% relative humidity for
Data on the effects of organic solvents on B. microplus
about 2 weeks until the egg laying. These eggs afforded the
engorged females are shown in Table 1. Acetone and
larvae used in the larval immersion test (LIT).
methanol were the most toxic solvents tested; absolute
ethanol was also found to be of moderate toxicity towards
Larval immersion test
B. microplus.
In the AIT, the effect of the treatments on engorged
The modified LIT was conducted as follows: approximately
females was assessed by measuring egg production and
200 embryonated eggs (0.01 g) were placed into pockets
hatching rate. Acetone was toxic to the adults promoting
made with pieces (6×6 cm) of TNT fabric. The pockets
mortality of 100%. Methanol and ethanol caused 45.3 and
were incubated at 27–28°C and 70–80% relative humidity
14.2% of mortality, respectively. The other tested sub-
for about 14 days, until the eggs hatched. After another
14 days, the pockets containing the larvae ready for testing
were immersed for 5 min in 10 ml of the solutions test
Table 1 Index of egg laying and percent mortality of engorged
(absolute acetone, methanol and ethanol, 1% DMSO in
females of Boophilus microplus treated with solvents and surfactant
water, 1% Tween 80 in water, Triton 5% X-100 in water agents
and water used as control). After approximately 1 h to
allow the solvents to evaporate, the pockets were incubated Index of egg laying % Mortality
at 27–28°C and 70–80% relative humidity for 48 h, and Control (H2O) 0.521±0.0293 0
then larvae (alive and dead) were counted to assess percent Acetone 100% 0* 100
mortality. Each treatment contained four replicates. Methanol 100% 0.285±0.0458* 45.3
Ethanol 100% 0.447±0.0196 14.2
Adult immersion test 1% DMSO 0.528±0.0279 0
1% Tween 80 0.496±0.0527 4.9
5% Triton X-100 0.509±0.0204 2.3
Each group of 16 engorged females (5.0 g) was weighed
and immersed for 5 min in the solutions (10 ml) in a 50-ml Significant difference in relation to the control group (ANOVA one
Becker flask and gently agitated (four replicates each at way, p=0.001)
Parasitol Res (2007) 100:1267–1270 1269

stances were not toxic to the engorged females of B. As cholesterol is freely soluble in acetone, the wax lipid
microplus. The experiments were continued, observing was destroyed, and the 100% mortality of the females
daily the egg masses for evidence of hatching. The observed after the contact with this solvent occurred by
treatment with methanol resulted in some infertile eggs, dehydratation. The mortality observed in the treatment of
but this solvent as well as the other samples tested did not methanol and ethanol could also be attributed to a similar
alter significatively the eclodibility. effect of these solvents in the female wax layer, as
The data indicate that DMSO, Triton X-100 and Tween cholesterol is somehow soluble in these solvents.
80 in the concentration tested would be suitable solvents/ In general, the females are more sensible to the solvents
surfactant agents to be used in bioassays involving B. than the larvae, and according to Chagas et al. (2003), it can
microplus because of their low toxicity, especially DMSO. be attributed to the difference in the cuticle composition:
The other solvents probably would be not toxic in lower The wax layer does not occur in the larvae, and the solvents
concentrations, but it should be tested before the use. do not cause dehydratation as it was seen in the females.
In the larvae immersion test, larval survival percent was
estimated, and in general, no significative effect of the
solvents was observed. Methanol and ethanol did not affect Conclusion
the larvae. The 1% solution of DMSO killed 5% of the
larvae. Among the surfactant agents, 1% Tween 80 did not The results obtained in this work indicate that acetone and
affect the larvae, whereas 5% Triton X-100 killed 3% of the methanol would not be suitable solvents in the AIT using B.
larvae. Acetone was the most toxic agent killing 10% of the microplus engorged females. The larvae were more resis-
larvae after 48 h. In general, similar results were obtained in tant, and all the tested agents except acetone could be used
the experiments performed by Chagas et al. (2003) and in the LIT searching new acaricide agents.
Freitas and Fernandes (2005). Some differences can be due
to the different concentrations of the solvents/surfactant Acknowledgements The authors are grateful to CNPq, Fapergs and
Propesq/UFRGS.
agents tested in the bioassays.

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