Toxicological & Environmental Chemistry, Oct.–Dec.

2006; 88(4): 729–737

Induction of micronuclei in tadpoles of Odontophrynus

americanus (Amphibia: Leptodactylidae) by the
pyrethroid insecticide cypermethrin

MARIANA C. CABAGNA1, RAFAEL C. LAJMANOVICH2,

PAOLA M. PELTZER2, ANDRÉS M. ATTADEMO2, & EZEQUIEL ALE3
1
Faculty of Biochemistry and Biological Sciences – FBCB-UNL, Cathedra of Normal
Morphology, Pje. El Pozo s/n (3000), Santa Fe, Argentina, 2Faculty of Biochemistry and
Biological Sciences – ESS-FBCB-UNL, National Council for Scientific and Technical Research
(CONICET), Pje. El Pozo s/n (3000), Santa Fe, Argentina, and 3Laboratory of General
Citogenetic, (UNaM), Félix de Azara 1552 (3300), Posadas (Misiones), Argentina

Abstract
Cypermethrin (CY) is an active cyano pyrethroid effective against a wide range of pests encountered
in agriculture and forestry. Although CY is not mutagenic in in vitro assays for gene mutation, in vivo
assays showed conflicting results. In vivo genotoxicity of the synthetic pyrethroid CY in erythrocytes of
Odontophrynus americanus tadpoles was examined. The frequency of micronuclei (MN) was recorded
in blood smears obtained from tadpoles exposed in vivo to four different nominal concentrations
5, 10, 20 or 40 mg L
1 of the compound and fixed at two sampling times 48 and 96 h. As a positive
control larvae were exposed to 40 mg L
1 of cyclophosphamide (CP). Tadpoles exposed to all CY
treatments showed a significant increase in single small MN compared to the negative control group
after 48 h and at 5 and 10 mg L
1 of CY at 96 h. Results obtained here demonstrated the genotoxic
effects of the commercial formulation CY in the anuran larvae analyzed. Thus, data suggest that
measurements of MN and other erythrocytes morphological aberrations performed in circulating
blood samples of O. americanus tadpoles is a method for detecting cytogenetic damage in other native
species.

Keywords: Odontophrynus americanus, cypermethrin, tadpoles

Correspondence: Rafael C. Lajmanovich, Faculty of Biochemistry and Biological Sciences – FBCB-UNL, Laboratory of
Ecotoxicology, Pje. El Pozo s/n (3000), Santa Fe, Argentina. Tel.: þ54 342 4740152. E-mail: rafalajmanovich@yahoo.com.ar
ISSN 0277-2248 print/ISSN 1029-0486 online ß 2006 Taylor & Francis
DOI: 10.1080/02772240600903805
730 M. C. Cabagna et al.

Introduction
Pesticide contamination is considered an important factor in the decline of amphibian
populations in agricultural regions [1]. Pyrethroid insecticides are synthetic molecules
structurally related to natural pyrethrins, sharing many characteristics of their actions with
DDT [2]. Pyrethroids have become increasingly popular for insect control in land and
aquatic agricultural systems. Although pyrethroids are considered relatively non-toxic to
birds and mammals, they are extremely toxic to aquatic organisms, including invertebrates,
fish, and amphibians [3,4].
Cypermethrin (CY) [(RS)-alpha-cyano-3-phenoxybenzyl (1RS)-cis-,trans-3-(2,2,-
dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] is a highly active synthetic pyrethroid
insecticide. Because of its effectiveness against a wide range of arthropods, CY is routinely
used not only in agriculture, but also in the control of human head lice and animal external
parasite infestations [5]. CY is one of the most light-stable pyrethroids [6] and its
degradation rates was similar in both laboratory and field surveys [7]. The average rate of
CY application to crops is 10–200 g of active ingredient (CY ha
1) [8]. Aside from direct
deposition or drift, pyrethroid insecticides reach aquatic habitats via runoff, which depend
on soil conditions, rainfall, and slope of the catchments area [9]. Four hours after
overspraying ponds with CY (100 g ha
1), a concentration of 100 mg L
1 was measured in
the surface water (depth of 2.5–10 cm; [10]). The range of half-lives in the model ecosystem
of CY was 4.7–30.8 days [7]. It was also noted that the degradation rates of the pyrethroids
followed first-order kinetics, and that only fenvalerate and CY residues remained at
detectable levels 56 days post-application [11].
Although CY is not mutagenic in in vitro assays for gene mutation [12], in contrast in vivo
assays showed conflicting results. Positive results were obtained in a mouse bone marrow
micronuclei test (MNT) following oral and dermal administration of CY and increased
frequency of sister chromatid exchanges in vivo [13,14] and in Drosophila melanogaster using
the alkaline Comet assay [15]. In addition, [16] reported a dose-dependent micronuclei
(MN) induction by pyrethroid lambda-cyhalothrin in Rana catesbeiana. Current
genotoxicity tests in amphibians are based on the observation of MN in vivo [17,18].
The objective of this study was to evaluate the effects of commercial formulation CY in
aquatic organisms using the MN test in erythrocytes of Odontophrynus americanus tadpoles.
This study integrated a project to investigate lack of effects from exposure to pesticides on
wild populations of amphibians in Paraná River floodplain, Argentina [19–21].

Materials and methods

Chemical
Cypermethrin (CAS No. 52315-07-8) commercial grade, trade name ‘Cipermetrina’,
was obtained from Argengric, Argentina. The commercial product consisted of 2.5% w/w
CY formulated in aqueous xylene. As a positive control, tadpoles were exposed to
cyclophosphamide (CP) (CAS No. 50-18-0, Filaxis) at a concentration of 40 ppm
(mg L
1). All test solutions were freshly prepared before each experiment.

Anuran tadpoles
Tadpoles of O. americanus were selected as the test organisms. This anuran has an extensive
Neotropical distribution [22], and inhabits natural areas, agricultural land and urban
territories [23]. Indeed, this species is easy to handle and acclimates to laboratory conditions.
 Induction of micronuclei in tadpoles by cypermethrin 731

The tadpoles used in the bioassay were collected from a temporary pond of the Paraná
River floodplain (31 430 S; 60 340 W-Argentina). Prometamorphic larvae (from stages 26 up
to 36) [24] were used for the bioassay. The average total size (snout-tail) was 15  0.5 mm
and weight was 0.07  0.02 g. The tadpoles were acclimatized to a 12 : 12 h light : dark cycles
in glass tanks (12.5 cm diameter and 13.5 cm high) with artificial pond water (APW) [25]
of pH 6.8, conductivity 175 mmhos cm
1, dissolved oxygen concentration 4.5  1 mg L
1,
hardness 50.5 mg L
1 of CO3Ca at 24  2 C for 7 days.

Exposure design
The 96 h sub-lethal tests were conducted according to USEPA Standard Methods [26],
with 10 prometamorphic larvae (from stages 26 up to 36) [24] per treatment group. In the
sub-lethal test, the nominal concentrations used were: 5, 10, 20 or 40 mg of CY L
1.
Negative controls were conducted in APW during the same period. CP was used as a
positive control, at a concentration of 40 ppm (mg L
1). All test solutions were prepared in
triplicate immediately before each experiment. The water, containing the compound and
the food (boiled lettuce) changed for every 24 h. The MN frequency in each group was
measured after 48 and 96 h.

Micronuclei test
Red blood cells (RBCs) in amphibians are nucleated and undergo cell division in the
circulation, particularly during the developmental stages [27]. The blood was obtained
directly from the heart of anesthetized tadpoles (30% ethyl alcohol). Peripheral blood smears,
two for each tadpole, were prepared on clean slides, fixed and stained by the May–Grunwald–
Giemsa method [28]. It is important to note that, [29] found that no significant difference
between data from preparations stained by Giemsa and acridine orange. The MN frequency
was determined in 1000 erythrocytes from each tadpole using 1000 magnification [16,30].
Coded and randomized slides were scored blind by a single observer. The criteria for MN
determinations are (1) the intensity of stained MN and the diameter of the MN should
be less than one-third of the main nuclei, (2) the intensity of stained MN is similar to the main
nuclei, (3) is not connected to the main nuclei, (4) is round with a nuclear membrane,
(5) there is no overlap with the main nuclei, and (6) is within the cytoplasm [31,32].
In addition, other alterations of the erythrocytes were recorded.

Data analysis
The non-parametric Kruskal–Wallis test [33] was used to analyze data from controls and
experimental groups. The criterion for significance was p 5 0.05.

Results
The MN frequencies and time-responses at different concentrations of test compounds are
shown in Figure 1. Tadpoles exposed to all CY treatments showed a significant increase in
single small MN compared to the negative control group after 48 h and at 5 and 10 mg L
1
of CY at 96 h. Tadpoles exposed to CP showed a significant increase in micronucleated
erythrocytes at 48 and 96 h exposure.
Control tadpole’s mature erythrocytes are oval cells with centrally placed and similarly
shaped nuclei (Figure 2a). The nuclei are clearly structured and possess a well-defined
boundary, which facilitates the identification of fragments in cytoplasm. For all treatments
732 M. C. Cabagna et al.

Figure 1. Induction of micronuclei in red blood cells (per 1000 cells) of O. americanus larvae
treated with different concentrations of test compounds (Cypermethrin) (*p 5 0.05 in relation to the
negative control).

Figure 2. May–Grunwald–Giemsa stained blood smear of O. americanus tadpoles (1000).

(a) Control, normal erythrocytes and heterophils (HT). (b) Erythrocytes exposed to the sub-lethal
doses of CY (5 mg CY L
1 – 48 h), arrow indicating one small MN. (c) Binucleated cells (BN) in short
exposed (5 mg CY L
1 – 48 h) erythrocytes. (d) Long exposed (10 mg CY L
1 – 96 h) erythrocytes
showing multiple MN.
 Induction of micronuclei in tadpoles by cypermethrin 733

Figure 2. Continued.

single MN were predominant in the erythrocytes analyzed (Figure 2b). Indeed, binucleated
cells and erythrocytes showing multiple MN in the way of apoptotic-like cells [34] were
found in tadpoles exposed to all CY concentrations in low frequencies (520%) at different
times (Figure 2c,d), except at 20 and 40 mg L
1 of CY at 96 h treatment because the
tadpoles did not survive.

Discussion
Pesticides have become indispensable in modern agriculture and are also harmful
pollutants, especially in aquatic environments [9,35,36]. Results obtained here showed
genotoxic effects of the CY on erythrocytes of O. americanus tadpoles.
Most toxicological studies on pyrethroids have been performed in the laboratory with
rodents and limited data are available for other vertebrates [37]. Pyrethroids are more
hydrophobic than other classes of insecticides and this feature indicates that the site of
action is biological membranes [38]. It is possible that pyrethroids are transported
through blood to the liver for metabolism and may produce cellular damage to
erythrocytes [39].
734 M. C. Cabagna et al.

In fact, it is well known that CY is toxic at lower concentrations, particularly for tadpoles
of different amphibian species [34]. In the present study CY, at 96 h exposure produced
larvae mortality. This elevated mortality at longer periods and in higher concentrations
simulated biological animal responses to CY and confirmed mutagenic effects induced by
MN test [32]. Although the occurrence of MN was relatively lower (1–3 MN/1000 cells),
this result is to find in tadpoles exposed to pyrethroids, copper, acetylaminofluorene, and
organochlorines [16,32,40,41].
CP produced a significant increase in the frequency of micronucleated erythrocytes at
almost all times observed. This substance, a bifunctional alkylating agent, is extensively
used for genetic effects in a wide variety of animal species [42]. This drug produced a
marked increase in the number of MN and is recommended for use as a positive
control [43,44] in amphibian tadpoles genotoxicological test at concentrations of
5–40 mg L
1 [16,41].
Different studies on the genotoxicity of technical grade CY are available in the
literature [13,45] but few reports used aquatic animals for evaluation. To our
knowledge the results presented here appear to be the first to demonstrate the
genotoxic effect of CY on erythrocytes of anurans tadpoles. On the other hand,
Vanderkerken et al. [46] classified MN into two major types of morphology, classifying
small and large MN relative to cell size. They postulated that aneugens induced MN
of the large type while clastogens induced small MN. Further, Ramadan et al. [47]
postulated that, generally, the clastogens induced many more MN than aneugens,
mostly of the small type, and more frequently induced multiple MN. Data suggest a
clastogenic effect of CY [48] on erythrocytes of O. americanus.
Moreover, the finding of apoptotic-like cells at 96 h treatment was similar to the results
of [49]. The authors demonstrated the CY induction (12.9–100 mg L
1) of apoptotic cell
in prometamorphic larvae developing brain of another sympatric leptodactylid,
Physalaemus biligonigerus. During the process of apoptosis and at the stage of chromatin
condensation the original nuclei splits into a number of dense MN, scattered throughout
the cytoplasm [50]. These MN generally appear surrounded by a double membrane
system, externally outlined by ribosomes. The functional role of these MN is still
unknown, but it is generally accepted that they contain sequestered inactive genetic
material [51]. Consequently, in the MNT a possible explanation is that the very early
steps of chromatin condensation due to apoptosis are not easily distinguishable from MN
induced by chemicals using Giemsa staining [31]. Moreover, Simko et al. [52] showed
that the increase in apoptotic cells is positively correlated with the appearance of MN.
It is important to note that the occurrence of nuclear morphological aberrations in
O. americanus, as well as the general degenerative changes in the erythrocytes during the
tadpole stages studied, corresponded to a period of intense hematopoiesis with active cell
division in the circulating blood [18,53].
Finally, measurements of MN and other erythrocytes morphological aberrations
performed in circulating blood samples of O. americanus tadpoles is a method for
detecting cytogenetic damage. The general measurement of tadpole’s blood character-
istics, including erythrocyte morphology, may provide a sensitive means of early warning
for some water quality changes adverse to aquatic life [40,54,55]. Extrapolation from the
present study is easy, by the use of a representative species of native anuran commonly
found in agroecosystems [56]. Before definitive conclusions are made about the genotoxic
effect of CY on anuran larvae, more assessments are urgently need to be conducted under
laboratory conditions and on agricultural systems with other native species. Finally,
further work needs to be undertaken on the factors affecting rates of increased MN in
 Induction of micronuclei in tadpoles by cypermethrin 735

tadpoles subject to the range of chemical and physical conditions found in the
environment.

Acknowledgment
We thank Dr Sergio Guerrero, Chief of CIEN-FBCB, for providing laboratory
facilities and Lic. Hugo G. Pitoco for helping with the translation. We also thank Sam
Kacew PhD.

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