Chemosphere 182 (2017) 730e737

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Chemosphere
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Genotoxic and mutagenic effects of Atrazine Atanor 50 SC on

Dendropsophus minutus Peters, 1872 (Anura: Hylidae) developmental
larval stages
Macks Wendhell Gonçalves a, d, Calebe Bertolino Marins de Campos d,
Vinícius Guerra Batista e, Aparecido Divino da Cruz d, Paulo de Marco Junior b, c,
Roge
rio Pereira Bastos b, c, e, Daniela de Melo e Silva a, c, d, )
a
Programa de Po
s-Graduaça ~o em Gen
etica e Biologia Molecular, Laborato
rio de Mutag^enese, Instituto de Ci^encias Biolo
gicas, Universidade Federal de
Goia
s, Goia
^nia, Goia

s, Brazil
b
Programa de Po
s-Graduaça ~o em Ecologia e Evoluça~o, Instituto de Ci^
gicas, Universidade Federal de Goia
encias Biolo
s, Goia^nia, Goia
s, Brazil
c
Programa de Po
s-Graduaça ~o em Biodiversidade Animal, Universidade Federal de Goia
s, Goia
^nia, Goia
s, Brazil
d
Núcleo de Pesquisas Replicon, Departamento de Biologia, Pontifícia Universidade Cato
lica de Goia
s, Goia^nia, Goia

s, Brazil
e
Programa de Po
s-Graduaça ~o em Ecologia de Ambientes Aqua
ticos Continentais, Universidade Estadual de Maringa
, Parana
, Brazil

h i g h l i g h t s

Environmental contamination contributes to amphibian population declines.

Mutagenic and genotoxic effects of atrazine was investigated in the current study.

Tadpoles of Dendropsophus minutus were divided in groups.

Premetamorphic and metamorphic stages were more sensitive to atrazine.

Atrazine was genotoxic and mutagenic for D. minutus dependent on larval stages.

a r t i c l e i n f o a b s t r a c t

Article history: The potential mutagenic and genotoxic effects of the herbicide atrazine were investigated in different
Received 13 March 2017 developmental stages of Dendropsophus minutus tadpoles. These animals were exposed to 4 nominal
Received in revised form concentrations of atrazine (2.25, 4.5, 9, and 18 mg/L) and 40 mg/L of Cyclophosphamide as a positive
11 May 2017
control, for 96 h. Negative controls were also added to the experiment. The tadpoles were divided into
Accepted 12 May 2017
three groups according to Gosner's developmental stages, namely GS 25-33 as premetamorphic, GS 36-
Available online 16 May 2017
39 as prometamorphic, and GS 42-43 as metamorphic climax. Our results showed that the pre-
Handling Editor: David Volz metamorphic and metamorphic stages were more sensitive than the prometamorphic stage to the
herbicide. A comet assay and micronucleus test for the sensitive stages demonstrated DNA damage in a
Keywords: concentration-dependent curve. Although a dose-response effect was not observed for the prom-
Herbicide etamorphic stage, a statistically significant difference was found between the treatment of 18 mg/L and
Susceptibility the negative control. Moreover, the highest concentration of atrazine showed both the largest amount of
Genotoxicity DNA damage and the highest micronucleus frequency regardless of the developmental stage of
Mutagenicity
D. minutus. In conclusion, atrazine was genotoxic and mutagenic for D. minutus in a dose-sensitive
Tadpole
manner, dependent on larval developmental stages. Considering the prometamorphic stages showed
no dose-response effect to atrazine, we suggest caution when using this stage in biomonitoring studies in
order to avoid false negative results. Amphibians have been proven to be useful bioindicators, and we
suggest replicating biomonitoring studies using different species to represent ecosystems' environmental
impacts.
© 2017 Elsevier Ltd. All rights reserved.

) Corresponding author. Laborato
rio de Gene
tica e Mutage^nese, Estrada do

Campus, s/n, Universidade Federal de Goia
s, Depto. de Gene
tica, Instituto de
^ncias Biolo
Cie
gicas, ICB I e Room: 218/228, Campus Universita

rio, CEP: 74690-900,
Goia^nia, Goi

as, Brazil.

http://dx.doi.org/10.1016/j.chemosphere.2017.05.078
0045-6535/© 2017 Elsevier Ltd. All rights reserved.
 M.W. Gonçalves et al. / Chemosphere 182 (2017) 730e737
1. Introduction
The increased use of pesticides, associated with habitat loss,
global warming, and diseases, has been considered one of the main
factors driving the decline of amphibian populations over recent
decades (Relyea, 2003; Collins and Storfer, 2003; Hayes et al., 2010).
The extensive use of pesticides in agriculture adversely affects
nontarget organisms such as amphibians (Sparling et al., 2010).
During their aquatic phase, amphibian species inhabiting agricul-
tural surroundings may be exposed to pesticides (Cerejeira et al.,
2003; Johansson et al., 2006; Relyea, 2006).
Several studies have suggested that amphibians are one of the
groups most sensitive to a number of pesticides (Wojtaszek et al.,
2004; Hayes et al., 2010; Gunderson et al., 2011; Nikoloff et al.,
2014; Ruiz De Arcaute et al., 2014; Thompson, 2004; Siddiqua
et al., 2013; Yadav et al., 2013) including atrazine (Brodeur et al.,
2013; Buck et al., 2015; Coady et al., 2005; Ji et al., 2016).
Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine)
is one of the most widely used herbicides in the world for corn,
sorghum, and sugarcane crops (Solomon et al., 1996; Collins and
Storfer, 2003; Miller et al., 2000). However, the use of atrazine
has been banned or restricted in several European nations due to its
persistent contamination of groundwater. The herbicide reaches
aquatic systems via atmospheric deposition and runoff from agri-
cultural fields, and it has a half-life ranging from 2 to 800 days,
depending on the pH and other environmental factors (Coady et al.,
2005). There is no evidence that atrazine is acutely genotoxic or
mutagenic at environmentally relevant concentrations, but chronic
exposure to the herbicide has been suggested to cause endocrine
disruption in amphibians (Hayes et al., 2002a, 2006, 2002b;
Langlois et al., 2009). Furthermore, other studies have reported
incidences of gonadal anomalies in atrazine exposed frogs (Tavera-
Mendoza et al., 2002a, 2002b; Carr et al., 2003).
However, there are significant knowledge gaps on comparative
sensitivities to atrazine at different larval developmental stages in
anuran species. It has been shown that pesticide exposure produces
significantly different survival rates at distinct developmental
stages (Harris et al., 2000). In regard to larval susceptibility to
atrazine, there are some studies that evaluate sensitivities between
the early and later larval stage (Howe et al., 1998; Storrs and
Kiesecker, 2004; Brodeur et al., 2009; Svartz et al., 2012; Siddiqua
et al., 2013). No studies have been carried out yet to systemati-
cally evaluate the genotoxicity and mutagenicity effects of atrazine
exposure on anuran amphibians at different larval stages. Accord-
ing to McDiarmid and Altig (2000), the Gosner stages (GS) can be
divided into three distinct groups: premetamorphosis (GS 25-33),
prometamorphosis (GS 36-39), and metamorphic climax (GS 42-
43).
The model organism used in this study, Dendropsophus minutus
(Peters, 1872), is one of the most common amphibians in South
America, inhabiting the Andean slopes, Amazon Basin, Guiana
Shield, and even the Atlantic Forest of Southeast Brazil. Gehara et al.
(2014) discovered high levels of diversity in the neotropical tree
frog D. minutus through use of a mitochondrial dataset. Given
numerous divergent lineages and the high degree of differentiation
revealed, the authors infer that more than one species could be
hidden behind the name Dendropsophus minutus. In view of its
wide distribution and tolerance of a broad range of habitats, this
species has been listed as a low concern and unlikely to decline
(Silvano et al., 2010).
The present study was undertaken to further evaluate the short-
term effects (mutagenic and genotoxic) of Atrazine Atanor 50 SC
exposure in different larval stages of D. minutus tadpoles.
731
2. Materials and methods
2.1. Collection of test animals
For each series of tests, several jelly egg masses and either
amplexed adults or early larval stages of Dendropsophus minutus
were collected in two different lentic permanent ponds (pond 1: 6
eggs' masses - 14 080 31.2700 S/47 380 55.4800 O; pond 2: 3 amplexed
adults - 14 070 50.0800 S/4741053.2900 O) in Caldas Novas, Goia
s mu-
nicipality, state of Goia
s, Brazil. The voucher numbers are ZUFG
10451, ZUFG 10457, ZUFG 10458, ZUFG 10461, ZUFG 10462 and
ZUFG 10467 and all the experiments were approved by the Bra-
zilian Ethics Committee for the Use of Animals (Protocol number
032/15).
The locations were far from agricultural or urban development
and had no history of pesticide exposure. The ponds were very
similar (physical structure) and presented high vegetation nearby.
The egg masses were transported in bags with native water to the
laboratory in Pontifícia Universidade Cato
lica de Goia

s, Brazil. After,
tadpoles larval stages were collected and they were placed in an
80 L glass aquarium that was then filled with dechlorinated water
for acclimation purposes until the tadpoles hatched. After hatching,
the larvae were transferred to three separate glass aquaria and
reared until they had reached the specific developmental stage
required. A commercial dry food (AlconBASIC®) was fed to the
tadpoles, and all holding water was 100% replaced once a week.
We used Gosner (1960) also preferred by McDiarmid and Altig
(2000), to grade larval development in the test animal. In order
to compare the different developmental stages according to the
metamorphic process, for each series of tests, we selected the
Gosner stages of GS 25-33 (premetamorphic), GS 36-39 (prom-
etamorphic), and GS 42-43 as metamorphic climax stages.
2.2. Chemicals
The atrazine herbicide Atanor 50 SC® contained 50% of the
active ingredient. This commercial formulation (soluble in water)
was diluted directly in reconstituted water for the tests. All expo-
sure concentrations were prepared from a stock solution of 2.5 mL
of atrazine (500 g/L) diluted in deionized water in order to prepare
250 mL of atrazine stock solution. The same water was used as
control group.
2.3. Experimental design and determination of LC50
At the laboratory, we monitored water quality parameters,
including dissolved oxygen, pH, water temperature, and conduc-
tivity, which were recorded both before adding the test organisms
to the treatments and at the end of experiments (Table 1). The air
temperature was 28 ± 2  C with a photoperiod of 12 h light/12 h
dark.
Several sets of pilot experiments were performed in order
choose the start point concentration. For LC50 determination, 16
tadpoles with representatives of some Gosner development stages
in quadruplicates were maintained in a 2 L glass aquarium for each
experimental point. This strategy allowed us to find the LC50, which
represents all groups, therefore performing a factorial analysis
model rather than analyzing the larval stages separately. We pre-
pared nine nominal atrazine concentrations (1, 2, 3, 6, 12, 24, 48, 96,
and 192 mg/L) during 96 h. The negative control consisted of 16
tadpoles kept in dechlorinated tap water (pH ¼ 7.6) conducted
simultaneously. All test solutions were prepared immediately
before use. The tadpoles were not fed throughout the experiment.
No renewal of test solution was performed during the 96-h expo-
sure period. The tadpoles were visually examined, and narcosis was
732 M.W. Gonçalves et al. / Chemosphere 182 (2017) 730e737

Table 1
Comparison of tadpole's weight/length and physical-chemical parameters among treatments.

Treatments (Mean ± Standard deviation) p

NC 2.25 mg/L 4.5 mg/L 9.0 mg/L 18.0 mg/L

Gosner (25-33) stages

Weight (g) 0.23 ± 0.01 0.22 ± 0.03 0.22 ± 0.06 0.22 ± 0.08 0.28 ± 0.07 0.46
Length (mm) 21.74 ± 0.71 20.66 ± 1.31 19.69 ± 1.36 20.57 ± 1.51 20.29 ± 1.45 0.09
Water temperature ( C) 24.22 ± 0.38 25.04 ± 0.58 23.94 ± 1.14 24.88 ± 0.56 23.59 ± 0.86 0.03
pH 7.10 ± 0.03 7.16 ± 0.06 7.17 ± 0.03 7.16 ± 0.06 7.17 ± 0.07 0.11
Conductivity (mS cm1) 0.06 ± 0.01 0.09 ± 0.01 0.07 ± 0.005 0.08 ± 0.01 0.08 ± 0.001 <0.001
Dissolved oxygen (mgL1) 8.16 ± 1.24 7.18 ± 0.90 7.15 ± 0.88 7.75 ± 0.55 6.61 ± 0.61 0.15
Gosner (36-39)
Weight (g) 0.38 ± 0.03 0.34 ± 0.03 0.36 ± 0.06 0.37 ± 0.08 0.42 ± 0.06 0.29
Length (mm) 26.86 ± 0.31 27.14 ± 1.27 25.86 ± 1.29 26.57 ± 1.39 25.73 ± 1.53 0.34
Water temperature ( C) 24.69 ± 0.06 24.05 ± 0.58 24.53 ± 0.42 23.89 ± 0.56 23.8 ± 0.54 0.5
pH 7.15 ± 0.07 7.19 ± 0.04 7.21 ± 0.08 7.23 ± 0.04 7.16 ± 0.03 0.18
Conductivity (mS cm1) 0.08 ± 0.001 0.07 ± 0.006 0.07 ± 0.001 0.07 ± 0.01 0.07 ± 0.01 0.03
Dissolved oxygen (mgL1) 8.37 ± 1.24 7.20 ± 0.93 7.04 ± 0.88 7.62 ± 0.55 6.67 ± 0.59 0.15
Gosner (42-43)
Weight (g) 0.48 ± 0.03 0.47 ± 0.04 0.46 ± 0.08 0.51 ± 0.10 0.52 ± 0.07 0.75
Length (mm) 26.88 ± 1.16 31.52 ± 2.77 31.72 ± 1.73 32.17 ± 0.85 31.73 ± 0.77 0.03
Water temperature ( C) 24.94 ± 0.06 24.07 ± 0.58 24.53 ± 0.42 23.87 ± 0.56 23.77 ± 0.54 0.02
pH 7.20 ± 0.07 7.33 ± 0.09 7.42 ± 0.09 7.27 ± 0.07 7.43 ± 0.05 <0.01
Conductivity (mS cm1) 0.07 ± 0.001 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.02 0.07 ± 0.01 0.46
Dissolved oxygen (mgL1) 8.88 ± 1.32 7.59 ± 0.94 7.29 ± 0.97 8.00 ± 0.58 7.07 ± 0.62 0.14

*Kruskal-Wallis.

defined as lack of sudden swimming response when gently
touched, in regard to control organisms. Mortalities were recorded
daily. To determine the susceptibility of D. minutus tadpoles, a
regression probit analysis was used to calculate LC50 96 h, as this
concentration has an effect on 50% of organisms tested (Farah et al.,
2004).
Based on the LC50 value, four atrazine concentrations were
tested: 2.25, 4.5, 9, and 18 mg/L. For the negative control, dech-
lorinated tap water was used during the same period of exposure.
Cyclophosphamide (CP) was used as a positive control at a con-
centration of 40 mg/L. Each treatment had five replicates. The same
pattern was used in order to perform three series of experiments
for each Gosner developmental stage. For the GS 25-33 stages, there
were ten tadpoles per aquarium, totaling 300 individuals.
Regarding the GS 36-39 stages, a total of 150 tadpoles were
exposed, with five in each aquarium. For the series of experiments
with the GS 42-43 stages, there were three tadpoles for each
aquarium (total of 90).
The tadpoles of D. minutus were anesthetized for approximately
2 min in a solution of benzocaine (300 mg/L), and blood samples
were obtained by a transversal cut in the tail. Animal care was
performed under the approval of the Ethical Committee on Animal
Use (CEUA-UFG), in accordance with the National Council for Ani-
mal Experiments Control (CONCEA). Voucher specimens were
deposited in the zoological collection of the Universidade Federal
de Goia
s (ZUFG).
2.4. Cell viability test
A cell viability test for each treatment and the Gosner stages was
performed before and immediately after exposure to the atrazine
herbicide. The viability was determined by a membrane integrity
analysis using propidium iodide as a marker for dead cells. The cell
suspension (500 ml) was stained with the propidium iodide solution
(1 mg/mL) for 20 min in the dark, at room temperature. After dye
incubation, the stained cell suspension was immediately analyzed
in a C6 Accuri flow cytometry (Accuri Cytometers, Ann Arbor, MI,
USA). A minimum of 5000 cells per sample was acquired with the
FL3-H channel.
2.5. Micronuclei assay
Two blood smears for each animal were prepared with clean,
new blades and were fixed in methanol and air-dried. The slides
were stained 12 h after alcohol fixation with Giemsa 10%, for
10 min. Slides were coded, randomized, and blind-scored by a
single researcher. The criteria employed for micronuclei (MN)
identification were as follows: a diameter smaller than 1/3 of the
main nuclei diameter, nonrefractability, staining intensity similar to
or lighter than that of the main nuclei, no connection or link with
the main nuclei, and no overlapping with the main nuclei (Fenech,
2000; Ferreira et al., 2004; Cabagna et al., 2006). The frequency was
determined by analyzing 1000 cells from each tadpole, using
1000  magnification as suggested elsewhere (Cabagna et al.,
2006).
2.6. Comet assay
The comet assay was performed using the alkaline method, as
described by Singh et al. (1988), with a few modifications. Fifteen ml
erythrocytes mixed with 0.5% LMP agarose were placed on normal
1.5% agarose microscope slides. The essential steps of comet assay
involved at least 3 h of cell lysis by detergent at a high salt con-
centration (1% Triton X-100, 10% DMSO, stock lysis solution
pH ¼ 10; at 4  C). Electrophoresis was performed under alkaline
conditions (300 mMNaOH, 1 mM EDTA, pH > 13, 25 min unwind-
ing, 25 min electrophoresis at 300 mA and 25 V, at 4  C). Nucleoids
were stained with 20 mg/mL of ethidium bromide (EB). We analyzed
100 nucleoids per slide, totaling 200 nucleoids per sample. The
analysis was performed by a fluorescence microscopy system called
Axioplan-Imaging®, using the Isis software with an excitation filter
of 510e560 nm and a barrier filter of 590 nm, with a 20 objective.
For the evaluation of genomic damages, we used the TriTek
Comet Score™ program, version 1.5. This software evaluated pixel
intensity to provide corresponding values to estimate genomic
damage as arbitrary units (AU). We quantified genomic damages
with tail length (TL), the percentage of DNA in the tail (% DNA), and
the Olive tail moment (OTM) [Collins, 2004].
 M.W. Gonçalves et al. / Chemosphere 182 (2017) 730e737
2.7. Statistical analyses
After obtaining the results of genotoxicity and mutagenesis, we
performed P-P plots (Shapiro-Wilk) and Levene's test in order to
verify the normality and homogeneity of variance, respectively. The
Kruskal-Wallis test was used to compare physical chemical pa-
rameters among treatments for each stage group. A two-way
analysis of covariance (ANCOVA) was used to compare the geno-
toxic and mutagenic effects among treatments and the Gosner
developmental stages. After getting significant differences in the
ANCOVA analysis, Tukey's post hoc test was performed to determine
differences within treatments at the 0.05 significance level.
Following, to test the estimates of genomic damage (TL, % DNA
and OTM) and nuclei lesions (MN, SN, KSN and ENA) among
treatments, we also performed analysis of covariance (ANCOVA)
followed by Tukey's post-hoc tests. The covariates included were
water temperature and conductivity. We also performed the t-test
between the negative and positive control. For all experiments, the
level of significance adopted was 5% (p < 0.05).
3. Results
3.1. LC50 determination
After 96 h of exposure to Atrazine Atanor 50 SC®, the probit
analysis of the mortality data yielded an LC50 value of 36.41 mg/L
(IC-95%, 27.76e48.92) as shown in Fig. 1. The LC50 values for 96 h
were calculated considering the total mortalities from atrazine
exposure. No mortalities or any kind of effects were observed in
controls for all tested larval developmental stages.
3.2. Short-term genotoxicity test
For the short-term genotoxicity (96 h) tests, we measured the
average weight/length of the tadpoles and the physical character-
istics of water (temperature, pH, conductivity, and dissolved oxy-
gen) for each Gosner stage. There were significant differences
among treatments in the covariates' water temperature (p ¼ 0.03)
and conductivity (p < 0.001) for GS 25-33 and conductivity
(p ¼ 0.03) for GS 36-39. On the other hand, the average pH
measured in the test solution remained within the range, from 7.05
up to 7.38, with the temperature varying between 22.82 and
Fig. 1. Concentrationeresponse curves describing LC50 after 96 h of Atrazine exposure
obtained by probit analysis.
733
Table 2
CL50 in distinct Gosner stages and Atrazine treatments in tadpoles of D. minutus.
Gosner stages Treatments (mg/L)
NC 2.251 4.5 9.0 18.0
GS 25-33 99.68 99.3 99.46 99.62 98.68
GS 36-39 99.82 99.78 99.86 99.74 99.66
GS 42-43 96.96 94.8 94.6 95.76 93.02
25.95  C and dissolved oxygen varying from 5.72 to 10.22 (Table 1).
For all samples, we observed cell viability equal to or higher than
94%. No significant differences in cell viability were found among
treatments (Table 2). The GS 25-33 and GS 36-39 had very similar
percentages of cell viability, higher than 98%. However, we
observed that GS 42-43 had a lower percentage of cell viability,
although higher than 94%, which was acceptable.
3.2.1. Comet assay
A descriptive analysis of % DNA among treatments for each
Gosner stage is shown in Fig. 2. Tadpoles exposed to atrazine
showed a higher amount of DNA damage when compared to tad-
poles kept under control conditions. There was a significant in-
crease of DNA damage among treatments (ANCOVA: F ¼ 10.8;
p < 0.001). The concentrations of 9 and 18 mg/L were significantly
higher than NC (Tukey: p ¼ 0.001 and p < 0.001, respectively). We
found significant differences of DNA damage among Gosner stages
(ANCOVA: F ¼ 29.0; p < 0.001). The GS 25-33 and GS 42-43 showed
a greater sensitivity when compared to GS 36-39 (Tukey: p < 0.001
and p < 0.001 respectively). Although there was significant differ-
ences in DNA damage overall, no significant differences (Tukey:
p ¼ 0.52) were found between earlier (GS 25-33) and metamorphic
climax stages (GS 42-43). These two Gosner stage groups had a
better concentration-dependent increase in the occurrence of DNA
damage. On the other hand, we did not find statistically significant
differences of DNA damage for GS 36-39. We found that the effect of
atrazine exposure was not equal among the three developmental
stages (ANCOVA: F ¼ 2.5; p ¼ 0.02) [Fig. 3].
There was no variation in physic-chemical parameters between
NC and the positive control group (CP). Significant increases of DNA
damage were observed in CP, with respect to NC observed
considering GS 25-33, GS 36-39, and GS 42-43 (t-test: p ¼ 0.001 and
p ¼ 0.01 and p < 0.001, respectively).
Fig. 2. Descriptive statistics of % DNA among treatments for each Gosner stages groups.
734 M.W. Gonçalves et al. / Chemosphere 182 (2017) 730e737

3.2.2. Micronuclei assay

The frequencies of MN are summarized in Fig. 4. MN frequencies
in the blood samples of tadpoles exposed to atrazine were signifi-
cantly higher than those to NC. The higher atrazine concentration,
18 mg/L, showed greater MN frequencies for all Gosner stages.
Significant differences were found in MN frequencies among
treatments (ANCOVA: F ¼ 5.99; p < 0.001) involving the compari-
son of 9 and 18 mg/L to NC (Tukey: p ¼ 0.02 and p ¼ 0.002,
respectively). When comparing MN frequencies among Gosner
stages, we also found significant differences (ANCOVA: F ¼ 4.31;
p ¼ 0.02). The GS 25-33 showed greater sensitivity when compared
to GS 36-39 and GS 42-43 (Tukey: p ¼ 0.04 and p ¼ 0.02). However,
no significant differences were observed between GS 36-39 and GS
42-43 (Tukey: p ¼ 0.86). The effect of atrazine exposure was equal
among the three Gosner stage groups (ANCOVA: F ¼ 2.23; p ¼ 0.99)
[Fig. 5].
Significant increases of MN frequencies in the CP were observed
for GS 25-33, GS 36-39, and GS 42-43 (t-test: p ¼ 0.02; p ¼ 0.02 and
p ¼ 0.007, respectively) (see Figs. 6 and 7). Fig. 5. Contrasting MN frequencies among treatments and Gosner stages groups.

Fig. 6. Results of tow-way ANCOVA comparing MN frequencies among treatments and

Fig. 3. Contrasting % DNA among treatments and Gosner stages groups. Gosner stages groups.

Fig. 4. Mean and Confidence Interval (95%) of MN frequencies among treatments for
each Gosner stages groups. Fig. 7. Comparative analysis of MN frequencies between negative and positive control.
 M.W. Gonçalves et al. / Chemosphere 182 (2017) 730e737
4. Discussion
Until now, previous short-term amphibian toxicity studies have
focused only on the lethal effects of the herbicide atrazine. Here, we
move forward and evaluate the genotoxicity and mutagenicity of
this herbicide in tadpoles of Dendropsohus minutus. After 96 h of
exposure to four nominal concentrations of atrazine, results
revealed that the premetamorphic (GS 25-33) and metamorphic
(GS 42-43) stages of D. minutus were the most sensitive phases
tested. Analyses of comet assay and micronucleus tests for these
two Gosner stage groups showed a concentration-dependent in-
crease in the occurrence of DNA damage. On the other hand, we did
not find a dose-response effect for GS 36-39, and a significant dif-
ference was evident only when comparing 18 mg/L with the
negative control. Moreover, it was also evident that the highest
concentration of the herbicide atrazine had the largest amount of
DNA damage and the highest micronucleus frequency regardless of
the developmental stage of the model organism. In a toxicity study,
Howe et al. (1998) found that later larval stages of the American
frogs Anaxyrus americanus and Lithobates pipiens showed greater
sensitivity to atrazine. However, the authors found that the early
stages were less sensitive than the late stages (Howe et al., 1998). In
another study by Brodeur et al. (2009), the early stages (GS 25) of
Rhinella arenarum were found to be more sensitive than the late
stages (GS 38-39) after 14 and 21 days of being exposed to atrazine.
The 96 h LC50 value of 36.41 mg/L, obtained in the present study
for all Gosner stages of D. minutus, is lower than the values found in
the early stage of A. americanus (66.4 mg/L) and higher than the late
stage of A. americanus (15.8 mg/L). Regarding L. pipiens, both early
and late stages showed a higher LC50 when compared to D. minutus
(69.7 and 45.3 mg/L, respectively) [Howe et al., 1998]. On the other
hand, the LC50 value of D. minutus was higher than Xenopus laevis
(maximum of 25.6 mg/L) and X. tropicalis (maximum of 31.8 mg/L)
[Fort et al., 2004], R. arenarum (maximum of 27.1 mg/L) [Brodeur
et al., 2009] and R. marina (maximum of 24.4 mg/L), and Limno-
dymastes peronii (maximum of 16 mg/L) [Siddiqua et al., 2013].
The metamorphosis process in amphibians is controlled by the
thyroid hormone and based on the production of the thyroid hor-
mone, which is classified into three distinct periods: pre-
metamorphosis, prometamorphosis, and metamorphic climax
(Etkin, 1968; Shi, 2000). Premetamorphosis is characterized by the
absence of the thyroid hormone and includes embryos and early
larval stages. In the prometamorphosis occurrence, the differenti-
ation of the toes in the hind limbs is influenced by an increased
concentration of thyroid hormones (Shi, 2000). The metamorphic
climax is characterized by rapid morphological changes headed by
a peak of the endogenous thyroid hormones (Shi, 2000). Therefore,
tolerance to the herbicide atrazine may vary according to the
anuran species and their different larval developmental stages. Our
data demonstrates that premetamorphic and metamorphic climax
stages of D. minutus showed greater sensitivity to atrazine than
prometamorphic stages evaluated by a micronuclei test and comet
assay. In the present study, the highest extension of DNA damage
was found in GS 42-43 at a concentration level of 18 mg/L of
atrazine. The greater sensitivity of the metamorphic climax stage to
atrazine may be due to added physiological stress during the
metamorphosis process (Howe et al., 1998). As shown in our re-
sults, the premetamorphic stage also had a significantly higher
amount of DNA damage and micronucleus frequency, probably
resulting from the short length of their larval development and
their physiological response to atrazine exposure (Siddiqua et al.,
2013). All data presented here highlight the importance of evalu-
ating the effects of the herbicide atrazine and other pesticides at
different developmental stages, aiming to avoid an underestimated
conclusion. From the results obtained in the present study, we can
735
also suggest that GS 25-33 may be the best choice to use as a bio-
indicator of environmental health in biomonitoring studies. Ac-
cording to the OECD (2009), chronic toxicity tests should be carried
out by exposing them at Gosner stage 29 (McDiarmid and Altig,
2000).
In Brazil, in order to protect human health and aquatic life, both
the National Council for the Environment (CONAMA, 2005) and the
Ministry of Health e Brazil (2004) recommend a maximum atra-
zine level of 2 mg/L in surface waters. Although the 96 h LC50 found
in the present study is above the maximum level permitted by law,
the effect of lower concentrations of atrazine on aquatic organisms
remains the concern, with emphasis on how lower concentrations
of atrazine cause DNA damage, as shown in our results. Another
concern is the indirect effects of atrazine on zooplankton, which
may cause changes in food sources once atrazine primary mode of
action is inhibition of photosynthesis (Solomon et al., 1996; Vryzas
et al., 2009).
The potential genotoxicity and mutagenicity of the herbicide
atrazine for all nominal concentrations of each Gosner stage were
verified by the results as significantly different from the negative
control. The genotoxicity effects of atrazine, which were evaluated
by evidence of DNA damage, were also demonstrated by Ribas et al.
(1995), Garaj-Vrhovac and Zeljezic, 2002. Our data corroborated
what was relayed by Clements et al. (1997) on using single cell
electrophoresis. Those authors demonstrated that atrazine (AAtrex
Nine-O) induced significant DNA damage in the erythrocytes of
Lithobates catesbeianus (Clements et al., 1997). Ribas et al. (1995)
showed that the herbicide atrazine induced DNA damage in hu-
man lymphocytes at concentrations of 50e200 mg/L. The herbicide
atrazine also induced DNA damage in mouse leucocytes as
demonstrated by Tennant et al. (2001). Furthermore, our results are
in agreement with other authors (Garaj-Vrhovac and Zeljezic,
2002), demonstrating that employees who were occupationally
exposed to atrazine presented significant increases in DNA migra-
tion of blood cells measured by comet assay. Considering the gen-
otoxic and mutagenic results of the herbicide atrazine for
D. minutus, which were obtained by a micronuclei test and comet
assay, we can infer that the mechanism of the micronuclei induc-
tion resulted from the atrazine clastogenic effect. However, we
cannot affirm that this effect was the exclusive mechanism for such
induction.
5. Conclusions
In conclusion, we can suggest that the herbicide atrazine is
genotoxic and mutagenic for D. minutus, and its sensitivity may
vary at different larval developmental stages. Moreover, the pre-
metamorphic and metamorphic stages appear to be more sensitive
to high concentrations of atrazine than prometamorphic stages.
Considering that the prometamorphic stage showed no dose-
response effect to atrazine, the authors suggest caution when us-
ing this stage of D. minutus in biomonitoring studies, in order to
avoid false negative results. Amphibians have proved to be useful
biomonitors, and we strongly suggest replicating biomonitoring
studies using different species to represent environmental insults
of distinct ecosystems.
Acknowledgments
The authors acknowledged Mr. S. Quail for proofreading this
manuscript. We also thank Projeto Girinos do Brasil (Edital SIS-
BIOTA: Processos CNPq 563075/2010-4 and FAPESP 2010/52321-7)
and to FAPEG (Processos: 201210267001094 e Universal/2012 and
201210767000812 - Pronex). ADC, DMS, and RPB thank the CNPq
fellowship, and MWG thank the individuals and organizations that
736 M.W. Gonçalves et al. / Chemosphere 182 (2017) 730e737
have granted their scholarships.
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