Ecological Indicators 49 (2014) 83–87

Genotoxic evaluation in two amphibian species from Braziliansubtropical wetlands

Marcelo Estrella Josende a, Alexandro Marques Tozetti b, Marcelo Tavares Alalan a,Volnei Mathies Filho a, Simone da Silva Ximenez a, Samantha Eslava Martins a,*aUniversidade Federal do Rio Grande, Instituto de Ciências Biológicas, Av. Itália km 8, Rio Grande, RS 96203-900, BrazilbUniversidade do Vale do Rio dos Sinos, Avenida Unisinos, 950, São Leopoldo, RS 93022-000, Brazil

A R T I C L E I N F O

Article history:Received 22 November 2013Received in revised form 21 September 2014Accepted 9 October 2014

Keywords:Nuclear abnormalitiesPseudis minutaLeptodactylus gr latransConservation unitWetlands

A B S T R A C T

Biomarkers analysis serves as an early warning system for the presence of pollutants because theirresponses appear before irreversible damage to the ecosystem takes place. The genotoxic effects ofpollutants may occur at cellular pollutant concentrations that are well below levels that would causegross cytotoxicity, making this a useful tool to detect early effects of toxic environmental agents.Combining the importance of Brazilian wetlands to the conservation of amphibian biodiversity with thepotential negative impacts of irrigated rice fields in the surrounding areas, the aim of the present studywas to evaluate genotoxic damage in two amphibian species, Pseudis minuta, and Leptodactylus gr latrans,from the southern Brazilian wetlands. Adult specimens from both Anuran species were capturedfrom preserved (Taim Ecological Station = TAIM) and non-preserved (Senandes) wetlands. Nuclearabnormalities were quantified in erythrocytes, and the results were compared using the Mann–WhitneyU test. There was a higher incidence of micronucleated erythrocytes in P. minuta, and of notched nuclei inL. gr latrans that were collected in TAIM when compared to those that were collected in Senandes, despitethe fact that TAIM is a conservation unit. These findings indicate that Anurans are coping with genotoxicsubstances in their habitats, and underscore the need to implement monitoring programs in TAIM todetermine which compounds or mixtures might be causing cell damage and to investigate the effects ofsuch compounds on other anuran species and animal groups.

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1. Introduction

The world decline in amphibian communities was firstdetected during the 1990s (Mackey and Boone, 2009), but thereasons for this decline remain controversial (Stuart et al., 2008).Generally, the decline has been associated with habitat loss andpollution. Both factors are related to the growing development ofagricultural activities that replace the natural vegetation coverand generate habitat pollution with fertilizers and pesticides(Relyea and Mills, 2001). These toxic substances cause theanimals to be borne and develop with malformations and/ordevelop behavior alterations, which may limit their reproductivesuccess (Bridges, 1999; Stuart et al., 2008).

As a function of their biology and life cycle, amphibians dwellin water and on land and feed on plants and animals that mayserve as important routes of pollutants uptake. Furthermore,

* Corresponding author. Tel.: +55 5332935162.E-mail address: samanthamartins@furg.br (S.E. Martins).

http://dx.doi.org/10.1016/j.ecolind.2014.10.0071470-160X/ã 2014 Elsevier Ltd. All rights reserved.

they possess a semi-permeable skin, which could advance theentry of harmful organic substances such as pesticides andhydrocarbons (Wells, 1977). These features make amphibians tobe appropriate models for ecotoxicological studies (Burlibaşa andGavril�a, 2011).

Despite the fact that Brazil shelters the highest anuranbiodiversity in the world (SBH, 2013), there have been few studiesconcerning amphibian population trends in wetland habitats(Oliveira et al., 2013). Most of the southern Brazilian wetlands havebeen diminished in size due to the expansion of agricultural areas,primarily irrigated rice fields (Maltchik et al., 2003). This situationis problematic because these wetlands store water, and they areessential hotspots of biodiversity (Guadagnin, 1999).

One of the most important continuous area of wetland insouthern Brazil is the Taim Ecological Station (TAIM), which isprotected as a conservation unit. This area shelters high animal andplant biodiversity and many migratory birds use this area to restand feed. Extensive areas with irrigated rice fields, in which largeamount of pesticides are applied, cattle farms, and the exploitationof non-native plants such as Pinus spp. and Eucalyptus spp., have

84 M.E. Josende et al. / Ecological Indicators 49 (2014) 83–87

occurred in the surroundings of TAIM. These activities coulddisrupt ecological processes and increase the risk of vulnerabilityand extinction of many species, so it is crucial to assess the healthof such ecosystems and their resident organisms. With regard towetlands, even the maintenance of large areas under theprotection of the law does not assure a high-quality ecosystembecause of the inputs of pollutants from allochthonous sources ofwater (Ramsar Convention Secretariat., 2007). This scenarioreinforces the urgency of studies on species associated withwetlands, particularly assessments of the health of wild popula-tions (Oliveira et al., 2013).

Biomarker analysis has been successfully used in biomonitoringprograms. It serves as an early warning system for the presenceof pollutant compounds because biomarker responses appearbefore irreversible damage to the ecosystem takes place. Thegenotoxic effects of pollutants may occur at cellular pollutantconcentrations well below those that cause severe cytotoxicity(Al-Sabti and Metcalfe, 1995), which makes this tool particularlyuseful to detect the early effects of environmental toxic agents. Themicronucleus (MN) test is a rapid, effective, low-cost assay todetermine mutagenicity in several cell types (Ferrier et al., 1998;Gauthier, 1996). Micronuclei are derived from chromosomalfragments or whole chromosomes that do not migrate to themain nucleus during cell division (Schmid, 1975). Other nuclearabnormalities result from analogous damage (Serrano-Garcia andMontero-Montoya, 2001) and may be taken into account to assessthe exposure of organisms to genotoxic contaminants (Marqueset al., 2009).

In the present study, we evaluated wild populations of twoanuran species, Leptodactylus sp. (gr latrans) and Pseudis minuta.These species occupy either preserved or disturbed habitats,suggesting that they present plasticity in coping with changes intheir habitats. L. gr latrans distributes along the Pampa biome inSouth America, being a relative large semiaquatic anuran thatfeeds on a wide variety of prey (Heyer et al., 1990). P. minutainhabits the Pampa biome in South America, being a mostly aquaticanuran, small in size, which feeds on aquatic invertebrates(Kwet, 2000). Considering these ecological features and therelative high abundance of these species in Brazilian wetlands,we believe that they depict good models to evaluate genotoxicityin wetland anuran populations.

Combining the importance of subtropical Brazilian wetlandsto the conservation of amphibian biodiversity, and the potentialnegative impacts of irrigated rice fields in the surroundingareas, the main goal of the present study was to verify thesuitability of using genotoxic damages as biomarkers of pollutanteffects in amphibian as bioindicators of such effects. Also, weverified if the anthropogenic activities in the surroundings ofTAIM affect the anuran population in regards of nuclearabnormalities.

2. Material and methods

2.1. Study area

The study was conducted in a region of the Pampa biome, whichis characterized by an extensive area of wetlands, temporarylagoons, lakes, and associated marshes (Waechter, 1985). Todetermine the possible relationship between the intensificationof agricultural activities and genotoxicity in Anurans, amphibianswere collected in two sampling sites: (1) the Taim EcologicalStation (TAIM) (32�500S; 52�260W), which is an extension of thelowlands that are protected as a conservation unit. Most of theprotected area (approximately 33,000 ha) is surrounded by farms,so this habitat is indirectly affected by agricultural activities.It should be noted that TAIM is the most important conservation

unit in southern Brazil and encompasses the last remains ofrelatively well-preserved subtropical Brazilian wetlands; and(2) the wetlands in Senandes neighborhood (32�080S; 52�110W),which is composed of permanent and temporary floodedgrasslands (Carvalho and Ozorio, 2007) in a rural and urban areasthat is composed of a mosaic of natural wetlands, rice fields, andcattle fields.

2.2. Sample collections

From March 2011 to August 2012, adult specimens from theanuran species P. minuta (n = 30; average body length = 29 � 3 mm;average body weight = 2.9 � 1.1 g) and L. gr latrans (n = 30; averagebody length = 65.4 �11.8 mm; average body weight = 32.4 �14.1 g)were captured from wetlands after a visual search (Tozetti andToledo, 2005). Sample collections were in accordance to Brazilianregulations (SISBIO license number 27,755-1).

2.3. Nuclear abnormalities assays

After length, weighting, and sex determination, Anurans wereanesthetized and blood was obtained by heart puncture. Twoperipheral blood smears for each sampled specimen wereprepared on clean slides, fixed with methanol for 10 min anddyed with Giemsa stain (10% v/v). Slides were viewed under anoptic microscope (1000� magnification). A total of 1000 eryth-rocytes per slide were scored. For MN quantification, two slides peranimal were scored. The criteria for MN determinations followedthose proposed by Lajmanovich et al. (2005). Kidney shaped nuclei(KS), lobed nuclei (LB), and notched nuclei (NT) were identifiedfollowing Carrasco et al. (1990) and blebbed nuclei (BL) wasquantified according to the criteria adopted by Strunjak-Perovicet al. (2009). All experimental procedures involving animalhandling were approved by the Ethics Committee on AnimalUse of FURG (permit number 23,116.005645/2012-89).

2.4. Statistical analysis

Individuals of each species from TAIM were compared to thosethat were captured in Senandes. Moreover, the incidence ofmicronuclei in P. minuta was compared to that of L. gr latrans for thesame sampling area. Data were fitted to a non-parametricstatistical analysis, so the Mann–Whitney U test was used. Avalue of p < 0.05 was adopted to indicate significance.

3. Results and discussion

Similarly to other Anurans (Lajmanovich et al., 2005), bothP. minuta and L. gr latrans have oblong-oval shaped matureerythrocytes with a centric nucleus (Figs. 1 and 2A). The MNincidence ranged from 0 to 22 in 2000 analyzed cells for P. minutaand from 0 to 15 in 2000 analyzed cells for L. gr latrans. Theobserved MN were spherical fragments that were smallerand separated from the principal nucleus (Figs. 1 and 2B).Although, individual MN were common in the analyzed cells,multiple MN were rarely observed. In regard of the other nuclearabnormalities investigated, incidence in 1000 analyzed cellsfrom P. minuta were as follows: NT ranged from 0 to 16.4;LB ranged from 0 to 7.8; KS ranged from 0 to 2.9; and BL rangedfrom 0 to 3.9. In L. latrans erythrocytes (1000 per individual),the anomalies were found as following: NT ranged from 0 to 7;LB ranged from 0 to 3.9; KS ranged from 0 to 2; and BL rangedfrom 0 to 2. Figs. 1 and 2 show the nuclear morphology in any ofthe abnormality found.

Previous studies have shown a very low incidence of MN inAnurans that were not exposed to contaminants, with a frequency

Fig. 1. Nuclear abnormalities in erythrocytes from P. minuta. (A) normal appearance cell, (B) micronucleus, (C) notched nucleus, (D) lobed nucleus, (E) kidney shaped nucleus,(F) blebbed nucleus. Slides were analyzed under an optic microscope at 1000� magnification (easy path, model vision plus).

M.E. Josende et al. / Ecological Indicators 49 (2014) 83–87 85

often lower than 1 in 1000 cells, but most of these studies wereperformed with tadpoles (Da Rocha, 2011; Lajmanovich et al., 2005;Marques et al., 2009; Rudek and Rozek, 1992). In the present study,all specimens were adults, and the incidence of micronucleatedcells were high in specimens taken from TAIM, as shown in Table 1.The incidence of the other nuclear abnormalities was also higherwhen compared to studies performed with different anuranspecies, except for blebbed nuclei (Pérez-Iglesias et al., 2014;Marques et al., 2009, Table 1).

To our knowledge, this is the first report on the incidence ofgenotoxic damage in P. minuta or L. gr latrans. Therefore, there areno previous data about the threshold level of abnormal nucleifor these species. Taking into account that the mean incidence

Fig. 2. Nuclear abnormalities in erythrocytes from L. gr latrans. (A) normal appearancenucleus, (F) blebbed nucleus. Slides were analyzed under an optic microscope at 1000

of micronuclei in individuals collected from Senandes was near1 MN/1000 cells, we suggest this micronuclei frequency as abackground level for both species, but further studies arenecessary to confirm this hypothesis.

The MN test is a fast and powerful tool to assess the ecologicalrisks caused by pollutants (Ferrier et al., 1998; Gauthier, 1996),which has been used as a biomarker of mutagenic damage indifferent groups of organisms. Although the presence of MNindicates irreversible cell damage, an increased number ofmicronucleated erythrocytes may display a prompt response topollutants. In this sense, ecosystems with a low level of impact canbe identified, and management may be initiated to avoid anincrease in degradation, restoring good environmental health.

cell, (B) micronucleus, (C) notched nucleus, (D) lobed nucleus, (E) kidney shapedx magnification (easy path, model vision plus).

Table 1Results of the nuclear abnormalities assays in erythrocytes from P. minuta and L. gr latrans.

Damage Data P. minuta L. gr latrans

SENANDES TAIM SENANDES TAIM

MN Upper extremea 2 11 7.5 5Lower extremeb 0 0 0 0Mean (�SE) 0.979 � 0.18* 2.493 � 0.46c,* 0.982 � 0.18 1.484 � 0.27d

Number of animals 9 21 15 15

NT Upper extremea 2 0 5.3 7Lower extremeb 1 16.4 0 0Mean (�SE) 1.65 � 0.19 3.83 � 0.8 1.33 � 0.51* 2.32 � 0.45*

Number of animals 6 21 11 14

LB Upper extremea 2.9 7.8 3.9 3Lower extremeb 0 0 0 0Mean (�SE) 1.15 � 0.36 2.71 � 0.43c 0.8 � 0.33 1.41 � 0.26d

Number of animals 6 21 11 14

KS Upper extremea 1 2.9 1.9 2Lower extremeb 0 0 0 0Mean (�SE) 0.49 � 0.2 0.89 � 0.2 0.44 � 0.19 0.63 � 0.19Number of animals 6 21 11 14

BL Upper extremea 3 3.9 2 2Lower extremeb 0 0 0 0Mean (�SE) 0.83 � 0.43 3.46 � 0.59 0.45 � 0.19 0.42 � 0.16Number of animals 6 21 11 14

a Maximum incidence in a single animal (per 1000 cells).b Minimum incidence in a single animal (per 1000 cells); SE denotes standard error. MN = micronuclei; NT = notched nuclei; LB = lobed nuclei; KS = kidney shaped nuclei;

BL = blebbed nuclei. Different letters (c,d) indicate significant difference (p < 0.05) between P. minuta and L. gr latrans from the same sample site.* Denotes significant difference (p < 0.05) between TAIM and Senandes, for the same species.

86 M.E. Josende et al. / Ecological Indicators 49 (2014) 83–87

The P. minuta individuals from TAIM showed a higherincidence of micronucleated blood cells than those fromSenandes. Also, L. gr latrans specimens from TAIM showed ahigher incidence of notched nuclei than those from Senandes(Table 1). Although MN and NT were the only nuclear abnormali-ties in which significant differences were found to be higher inTAIM than in Senandes, the Pearson product moment correlationpointed out strong positive correlations between the incidenceof MN and the incidence of other nuclear abnormalities inTAIM, as follows: MN � NT = 0.841 (p < 0.001); MN � LB = 0.77(p < 0.001); MN � KS = 0.552 (p = 0.009); and MN � BL = 0.540(p = 0.01).

These results were unexpected because TAIM is a conservationarea, with a low level of expected environmental stress. However,agricultural and livestock activities take place in the surroundingareas, and the irrigated rice fields in the watershed near TAIM maybe especially harmful to the ecosystem. To maintain this level offarming, several associated water bodies are used for irrigation,with a water withdrawal of approximately 2 l/ha per day over thecourse of five months (Fragoso Jr. et al., 2011). Beyond the highdemand for water and associated nutrient inputs, the pesticidesused during cultivation, such as glyphosate, may leach to land orwater bodies inside TAIM, leading to toxic effects in the residentorganisms. This hypothesis is only speculative since there is noinformation to date on chemical analysis in this area, but it isknown that pesticides induce genotoxicity in anuran erythrocytes(Lajmanovich et al., 2005). According to IRGA (2013), an area ofapproximately 70,000 ha is used for rice production in areassurrounding TAIM. Nevertheless, high levels of nuclear abnormali-ties were found in amphibians collected from TAIM, indicating theneed of further studies to associate cause-effect relationships andmanage the risk.

Chemical pollution causes genetic malformations that maylead to adverse reproductive effects in wild populations (Burlibaşaand Gavril�a, 2011). The use of pesticides is a likely contributor to

the worldwide decline in amphibian populations (Hayes et al.,2010; Mann et al., 2009).

Both species studied are semi aquatic amphibians, and despitetheir ability to move, they seem to spend most of their lives incontact with water. The higher level of water body connectivity inTAIM might concentrate the chemical pollutant inputs into thesystem. As a consequence, Anurans in these habitats might bemore vulnerable to pollution than those who inhabit Senandes.There is a lack of studies about contamination levels in TAIM, andour results reinforce the importance of determining the waterquality of this and other wetland systems.

As shown in Table 1, the MN and LB incidence in P. minutawas higher than in L. gr latrans in specimens collected from TAIM.Other studies also revealed that different anuran species may showdifferent sensitivities to pollutants, according to their detoxifyingsystems (Attademo et al., 2014).

Although data concerning the sensitivity of amphibians topollutants are contradictory (Bridges et al., 2002), Anurans aresuitable for the use as biological models to assess environmentalquality. Most species present two life stages (aquatic andterrestrial), no shelled eggs, and semipermeable skin (Duellmanand Trueb, 1986). Their presence in the habitat usually indicates anarea with a low level of impact. However, early responses todegradation, such as the appearance of micronuclei and othernuclear abnormalities in erythrocytes, indicate that these animalsare affected by environmental pollution, and although they do notdie in the presence of these chemicals, they may experiencereproductive and behavioral alterations, which could lead tolong-term population and community declines. Furthermore,amphibians may serve as a transfer component of pollutants tohigher levels of the food web. In this sense, cellular aberrations inamphibians represent a significant fragility in these wetlandecosystems. Wetlands and other habitats formed by lakes andtransient swamps offer high trophic complexity. Some animalgroups, including Anurans, feed on the water–land interface and

M.E. Josende et al. / Ecological Indicators 49 (2014) 83–87 87

gather food webs from both compartments, carrying pollutantsalong with energy and nutrients.

The present study showed that the frequency of micronucleatedcells and lobed nuclei cells were higher in P. minuta than inL. gr latrans from TAIM, suggesting the use of P. minuta as asensitive organism to detect mutagenic and genotoxic effects ofpollutants, primarily through the micronucleus test. P. minuta ispresent only in Uruguay, Argentina, and southern Brazil (Langone,1994), reinforcing the importance of conservation of the ecosys-tems containing this species. Moreover, our results indicate thefragile ecosystem of TAIM in face of environmental stressors. Wesuggest that these nuclear abnormalities raised specially from theuse of pesticides in rice crops, but research on contamination levelsare lacking in the study area, highlighting the need for monitoringprograms to better assess the health of the environment.

4. Conclusions

Although it is a conservation unit, there was a high incidence ofmicronucleated and lobed nuclei erythrocytes in the P. minutaindividuals that were collected in TAIM. This finding indicates thatAnurans are coping with genotoxic substances in their habitats.These results highlight the need for the implementation ofmonitoring programs in this ecological station to determine whichcompounds or mixtures might be causing cell damage and toinvestigate other effects of such compounds. In this sense,P. minuta showed to be suitable for using in monitoring programsto evaluate pollution in wetland ecosystems in South America.

Acknowledgements

The authors wish to thank Mauro C. L. M. de Oliveira for hiscontributions. This study was supported by the Brazilian ResearchCouncil (CNPq), process 476575/2011-7.

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