Fish as Bioindicators to Assess the Effects of Pollution inTwo Southern Brazilian Rivers Using the Comet Assay

and Micronucleus Test

Vanessa Moraes de Andrade,1* Juliana da Silva,1,2 Fernanda R. da Silva,1

Vanina D. Heuser,1 Johnny F. Dias,3 Maria Lucia Yoneama,4

and Thales R.O. de Freitas1

1Departamento de Genetica, Instituto de Biociencias, Universidade Federal do RioGrande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

2Laboratorio de Genetica Toxicologica, PPG em Ensino de Ciencias e Matematica,Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil

3Laboratorio de Implantacao Ionica, Instituto de Fısica da Universidade Federal doRio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

4Programa de Pos-Graduacao em Geologia, Universidade do Vale do Rio dosSinos, Sao Leopoldo, Rio Grande do Sul, Brazil

Industrial effluents, agricultural runoff, and municipalwastewaters contain unknown substances and com-plex mixtures that are released into the environmentand can lead to contamination of surface and subsur-face waters. In the present report, we have used thealkaline Comet assay and the micronucleus (MN) testto detect the genotoxicity due to multiple sources ofpollution in the peripheral blood of two native estua-rine fish (mullet and sea catfish) and evaluated possi-ble interactive genotoxic effects from multiple contam-inants and the seasonal variation of the genotoxicity.Mullet and sea catfish were captured in the Traman-daı and Mampituba Rivers in the southern Brazilianstate of Rio Grande do Sul. Reference animals wereobtained from the Armazem lagoon. Fish captured inthe two estuaries during the four seasons over a pe-riod of 2 years had increased levels of DNA damageand MN frequencies relative to the reference fish. Ingeneral, the alkaline Comet assay was more sensitive

to the genotoxicity of the river contaminants than theMN test. The Comet assay demonstrated significantdifferences in fish captured at different seasons and atthe two river sites, while the MN test showed signifi-cant differences only for the annual average for mulletfrom both sites and fish from the control site. Theincreases in DNA damage appear to be related to theincrease in the number of people in the towns close tothe study areas during the warm spring and summerseasons. Although no specific cause-effect relation-ships were established, comparison of the chemicalcontaminants and physical variations in the rivers withthe genotoxicity data indicate that there may be someassociation between hydrocarbons, metals, pH, andwater temperature and the level of damaged cellsobserved in mullet and sea catfish from the Tramandaıand Mampituba estuaries. Environ. Mol. Mutagen.44:459–468, 2004. © 2004 Wiley-Liss, Inc.

Key words: fish; Comet assay; micronucleus test; mullet (Mugil sp.); sea catfish (Netuma sp.);biomonitoring

INTRODUCTION

Industrial effluents, agricultural runoff, and municipalwastewaters contain unknown substances and complex mix-tures that are released into the environment and can lead tocontamination of surface and subsurface waters [Houk,1992; Kale et al., 1995; White et al., 1996]. Compoundspresent in polluted water are capable of causing biologicalalterations that can affect particular populations and entireecosystems [Rajaguru et al., 2003]. Organisms inhabitingareas influenced by effluent discharges can suffer geneticdamage, and humans using polluted water are at risk ofsimilar genotoxic effects and cancer development [Stahl,1991; De Flora et al., 1993]. The continued production andrelease of pollutants into the aquatic environment have

made investigating the genotoxic potential of inland andcoastal waters a major task of environmental pollution-control monitoring [Rajaguru et al., 2002]. Although it has

Grant sponsor: Coordenacao de Aperfeicoamento de Pessoal de NıvelSuperior (CAPES).

*Correspondence to: Vanessa Moraes de Andrade, Departamento de Ge-netica, Instituto de Biociencias, Universidade Federal do Rio Grande doSul, Av. Bento Goncalves 9500, CP 15053, Porto Alegre, RS, 91501-970,Brazil. E-mail: vmoraesdeandrade@yahoo.com.br

Received 20 April 2004; provisionally accepted 5 June 2004; and in finalform 13 August 2004

DOI 10.1002/em.20070

Published online 29 October 2004 in Wiley InterScience (www.interscience.wiley.com).

Environmental and Molecular Mutagenesis 44:459–468 (2004)

© 2004 Wiley-Liss, Inc.

proven difficult to establish a direct linkage between theecological effects of pollution and human health, the use ofwild species as bioindicators or sentinels has provided theconceptual basis for this connection [Minissi et al., 1996;Bickham et al., 2000]. Fish are often used as sentinelsbecause they play a number of roles in the trophic web,bioaccumulate toxic substances, and respond to low con-centrations of mutagens [Al-Sabti and Metcalfe, 1995;Stegeman, 2000].

Monitoring genotoxicity in the environment using indig-enous organisms as sentinels requires sensitive assays [Al-Sabti and Metcalfe, 1995; Pandrangi et al., 1995]. DNAdamage (e.g., strand breaks) has been proposed as a usefulbiomarker for assessing the genotoxic properties of envi-ronmental contaminants in biomonitoring studies [Minissiet al., 1996; Mersch and Beauvais, 1997; Bombail et al.,2001], mainly because such lesions are considered poten-tially premutagenic [Claxton et al., 1998; Mitchelmore andChipman, 1998; Kammann et al., 2000]. There are a numberof assays for detecting DNA damage but the single-cell gelelectrophoresis assay or Comet assay is the most commonlyused technique, because it has advantages that make it idealfor use in sentinel organisms [Mitchelmore and Chipman,1998; Silva et al., 2000a; Bombail et al., 2001; Heuser et al.,2002]. Although it has been successfully applied to nucle-ated red blood cells in several fish species exposed togenotoxic agents in vivo and in vitro [Nacci et al., 1996;Belpaeme et al., 1998; Mitchelmore and Chipman, 1998;Andrade et al., 2004], there are few aquatic biomonitoringstudies using the Comet assay on field-sampled fish [Pan-drangi et al., 1995; Devaux et al., 1998; Russo et al., 2004].

The sensitivity of the Comet assay is comparable withthat of the well-established micronucleus (MN) test, whichmeasures structural and numerical chromosome damage[Fenech, 1997; Kirsch-Volders et al., 1997]. The MN testhas been used since the beginning of the 1980s to estimatethe genotoxic effects of exposure to environmental contam-inants [Al-Sabti and Metcalfe, 1995]. In environmental mu-tagenesis investigations, the MN test using fish has provedto be a simple, reliable, sensitive, and inexpensive proce-dure to assess the biological impact of freshwater pollution[Al-Sabti and Metcalfe, 1995; Minissi et al., 1996; Ayllon etal., 2000; Russo et al., 2004].

In the present study, we have characterized the genotox-icity associated with two polluted sites situated in estuarinezones in the southern Brazilian state of Rio Grande do Sul(RS). We have used the Comet assay and MN test toevaluate genotoxicity in two native fish, the gray mullet(Mugil sp.) and the sea catfish (Netuma sp.). In a laboratorystudy, we recently established that the Comet assay detectedincreased DNA damage in erythrocytes from mullet and seacatfish exposed to methyl methanesulfonate [Andrade et al.,2004]. In addition, the influence of seasonal variation andsome of the agents involved with the contaminating siteswere studied.

MATERIALS AND METHODS

Site Selection and Description

We studied the estuaries of the Tramandaı and Mampituba Rivers, bothof which are important southern Brazilian nursery areas for many com-mercial and recreational fish species. These estuaries are located in thesouthern Brazilian state of Rio Grande do Sul near the small coastal townsof Tramandaı and Torres, respectively. These towns experience increasesin the number of inhabitants and the discharge of pollutants during thesummer (December to March). The control region with no recent history ofexposure to pollutants was the Armazem lagoon, located about 5 km fromthe Tramandaı River (Fig. 1).

Fish Species Analyzed and Sampling Methods

Gray mullet (Mugil sp.) and sea catfish (Netuma sp.) were chosen for thegenotoxicity studies because of three reasons. One, both genera are estu-ary-dependent [Lenanton and Potter, 1987], being reliant on the estuarinehabitat both as a nursery area and for feeding. If the estuary becomescontaminated, these species may be exposed to pollutants for a significantproportion of their lifetimes and hence may be useful as indicators ofpollution.

Two, both genera are generally available all year round and this mini-mizes the difficulties involved in locating and sampling fish in the field.Also, the temporal and spatial distribution and abundance of the principalspecies of these genera have been described for the Tramandaı estuary bySilva [1982]. Although there is no convenient way to speciate fish in thesegenera, Silva [1982] indicates that the sampling sites used in this studyhave equal proportions of the major species, canceling out any species-related effects on the measured responses.

Three, both genera are of commercial and recreational significance forTramandaı and Torres, which means that the information provided by ourstudy may be important both to people using the estuarine resources and tothose responsible for fishery management. We recently established that theComet assay detects increases in DNA damage in erythrocytes from these

Fig. 1. Location of sampling sites.

460 Andrade et al.

genera exposed in the laboratory to known genotoxicants (methyl meth-anesulfonate) [Andrade et al., 2004].

We began monitoring the fish for genetic damage in July 2001 (winter)and continued each season until April 2003 (autumn), one sampling pro-gram being undertaken per season per site (note that in the southernhemisphere, spring is September to December; summer, December toMarch; autumn, March to June; and winter, June to September). Thereference animals were collected only during the course of a single year,one sampling per season, and data from these samples were combined. Bythe end of two years, about 370 fish had been captured per season, with theproportion of each genera sampled dependent on the availability of theparticular genera at the time. Peripheral blood samples were drawn fromthe cardiac vein of the mullet and the caudal vein of the sea catfish usingheparinized syringes and the blood cells used for the Comet assay and theMN test.

Comet Assay

The blood samples were diluted 1:120 (v/v) with RPMI-1640 mediumand used immediately. The alkaline Comet assay was performed as de-scribed by Tice et al. [2000], with adaptations for fish cells described byAndrade et al. [2004]. Briefly, 5 �l of each diluted blood sample wereadded to 95 �l of 0.75% (w/v) molten low-melting-point agarose, and aportion of the mixture was spread on a microscope slide precoated with1.5% (w/v) normal-melting-point agarose and covered with a coverslip.After a brief period on ice to solidify the agarose, the coverslip wascarefully removed, and the slide placed in lysis buffer [2.5 M NaCl,100 mM EDTA, and 10 mM Tris, pH 10.0–10.5, with freshly added 1%Triton X-100 and 10% dimethyl sulfoxide (DMSO)] for a minimum of 1 hrat 4°C. The slide then was incubated in freshly made alkaline buffer(300 mM NaOH and 1 mM EDTA, pH � 13) for 10 min. The slides werethen subject to electrophoresis in the same buffer: 20 min at 300 mA and20 V (0.7 V/cm) for mullet blood cells and 15 min at 270 mA and 25 V(0.9 V/cm) for sea catfish blood cells. All these steps were carried outunder dim indirect yellow light. Following electrophoresis, the slides wereneutralized in 400 mM Tris (pH 7.5) and the DNA stained with a solutioncontaining 2 �g/ml ethidium bromide. The slides were coded for blindanalysis and analyzed immediately.

To demonstrate the sensitivity of the assay, negative and positive con-trols from human blood were included in each electrophoresis. For thepositive control, 50 �l of whole human blood were mixed with 13 �l of asolution containing methyl methanesulfonate (MMS, M4016; Sigma,St. Louis, MO) in Hank’s balanced salt solution (HBSS; Sigma) to give afinal MMS concentration of 8 � 10�5 M, and the mixture incubated for2 hr at 37°C. The results from each electrophoresis were considered validonly if the negative and positive controls yielded negative and positiveresults, respectively.

Leukocytes were evaluated for human blood and erythrocytes for fishblood. To calculate image length (IL), 100 randomly selected cells (50from each of two replicate slides) from each sample were assessed using afluorescence microscope fitted with a BP546/12 nm excitation filter and a590 nm barrier filter and an eyepiece micrometer (1 unit � 5 mm at 400�magnification) to estimate the length of the comet image (nuclear region �tail) in micrometers.

To calculate a damage index (DI), cells were visually allocated into fiveclasses according to tail size (0 � no tails; 4 � maximum-length tails),which resulted in a single DNA damage score for each sample (in the caseof the piscines, one fish) and for each control group (i.e., MMS concen-tration at a specified time and temperature). Visual scoring is considered tobe an acceptable alternative to image analysis for evaluating DNA damagein the Comet assay [Hartmann et al., 2003]. Thus, the DI of the group couldrange from 0 (completely undamaged � 100 cells � 0) to 400 (maximumdamage � 100 cells � 4) [Collins et al., 1995, 1997; Silva et al., 2000a].The percentage damage frequency (DF) was calculated for each samplebased on the number of cells with a tail versus those with no tail.

Micronucleus Test

The MN test was performed on peripheral blood as a second monitoringsystem for comparison with the alkaline Comet assay. The MN test wasperformed according to published protocols [Al-Sabti and Metcalfe, 1995;Minissi et al., 1996]. Peripheral blood samples were drawn and immedi-ately smeared onto microscope slides, air-dried, and stained for 10 minwith a mixture of 10 ml Giemsa and 90 ml phosphate buffer (pH 6.8). Theslides then were rinsed with distilled water, air-dried, and examined usingoptical microscopy. Two blood smears were prepared from each fish andcoded for blind analysis. A total of 4,000 erythrocytes was examined foreach fish.

Chemical Analysis

For each sampling site and for each season, the pH and temperature (°C)of the water were determined in situ, and water and sediment samples werecollected in precleaned amber bottles and stored in the dark at 4°C untilanalysis. The concentration of total hydrocarbons (HCs) in the water wasmeasured by gas chromatography [American Public Health Association,1995] at the Latox Toxicological Laboratory (Porto Alegre, RS, Brazil).

The metal content of the sediment samples was analyzed through theparticle-induced X-ray emission (PIXE) technique [Johansson et al., 1995]due to its high sensitivity, simplicity, and the ability to detect multiplemetals simultaneously. Briefly, sediment samples were dried at 60°C for24 hr, ground into a fine powder, and mixed 1:1 with 20% normal-melting-point agarose. Then the samples were homogenized and pressed intoapproximately 3 mm thick pellets, which were placed in the target holderinside the reaction chamber. During the experiments, the pressure insidethe reaction chamber was about 10�5 mbar. The experiments were carriedout at the Ion Implantation Laboratory of the Physics Institute of theFederal University of Rio Grande do Sul. A 3 MV Tandetron acceleratorprovided a 2.0 MeV proton beam with an average current of 5 nA at thetarget. The X-rays produced in the samples were detected by a Si(Li)detector with an energy resolution of about 155 eV at 5.9 keV. The spectrawere analyzed with the GUPIX software package [Maxwell et al., 1989,1995; Campbell et al., 2000]. Data are expressed in arbitrary units ratherthan ppm to facilitate comparisons [Heuser et al., 2002].

Statistical Analysis

The normality of variables was evaluated by the Kolmogorov-Smirnovtest. Statistical analysis was performed using the Kruskal-Wallis test forcomparison of DNA IL, DI, DF, and the MN frequency for each of theseasons and sites. Pearson correlation and linear regression were carriedout to compare DI and water pH as well as temperature.

RESULTS

The Comet assay data for mullet and sea catfish collectedbetween winter 2001 and autumn 2003 from the Tramandaıand Mampituba River estuaries are summarized in Tables Iand II. The concurrent control data (Armazem lagoon) fromthe different seasons were similar (no significant differ-ences), thus they were combined into a single group. Dataon the mean DNA IL, DI, and DF for each season and siteand the annual averages for mullet are presented in Table I.When subjected to the Kruskal-Wallis test, the mean mulleterythrocyte IL, DI, and DF values for the Tramandaı andMampituba groups for the spring and summer were signif-icantly higher than the values for mullet from the control

Fish as Biomarkers of Pollution 461

Armazem lagoon. For autumn only, the Tramandaı mullethad significant increases in DNA IL, DI, and DF in relationto the control, with the Mampituba mullet having a signif-icant increase with respect to the control fish only for the DFvalues. The results for fish sampled during the winter sam-pling periods were similar for both the estuarine groups andthe control group. When the comparisons were made be-tween the seasons, all the DNA damage parameters for fishsampled in spring and summer were significantly higherthan those for fish sampled in winter for both estuaries,while DNA damage in autumn fish was significantly higherthan for winter fish only for the Tramandaı estuary. Inrelation to spring values, there was a significance increase inthe DF values of Tramandaı estuary fish only during au-tumn, while the spring IL and DI and summer IL, DI, andDF values for the Mampituba estuary fish were statisticallygreater than the autumn values. Based on these results, aseasonal pattern for mullets could be distinguished in whichthe level of DNA damage followed the sequence summer �autumn � spring � winter for the Tramandaı fish, andsummer � spring � autumn � winter for the Mampitubafish.

When erythrocytes from Tramandaı and Mampituba es-tuary mullets were compared by season for DNA damageparameters, we found significant differences for spring(Mampituba � Tramandaı for IL and DI values), summer

(Mampituba � Tramandaı for IL values), and autumn (Tra-mandaı � Mampituba for DF values), as shown in Table I.The analysis of the overall mean values obtained from theaverage number of damaged cells per season indicated asignificant increase in IL (P � 0.05), DI (P � 0.01), and DF(P � 0.001) values for the Tramandaı and Mampituba Rivermullet as compared to the Armazem lagoon controls. Nodifferences in annual averages were observed between theTramandaı and Mampituba River groups, although in gen-eral the Mampituba fish had slightly higher levels of DNAdamage than the Tramandaı fish.

Table II shows data on the mean DNA damage in seacatfish for each season and the annual averages. Compari-sons of the mean IL, DI, and DF values for each season andgroup (Mampituba, Tramandaı, and Armazem lagoon)showed that, with the exception of the summer MampitubaDF values, sea catfish from the Tramandaı and MampitubaRiver estuaries had significantly increased levels of DNAdamage during the spring and summer. In fish sampled inthe autumn, only the ID and DF values of the Tramandaıcatfish were significantly greater than the control. Withrespect to comparisons between the seasons, the IL and DIvalues of both the Tramandaı and Mampituba sea catfishgroups were significantly higher in spring and summer thanin winter, while the DF parameter was higher in spring andsummer than in winter only for the Tramandaı group. Only

TABLE I. Detection of DNA Damage in the Erythrocytes of Mullet Captured Between Winter 2001 and Autumn 2003 FromArmazem Lagoon, the Control Site, and the Tramandaı and Mampituba Rivers

Season Site nmDNA imagelength (�m)n

Damage index(0–400)n

Damagefrequency

(%)nMean

water pHMean water

temperature (°C)

Armazem lagoon 15 24.9 � 4.4 135.6 � 50.5 47.0 � 16.9Wintero Tramandaı 20 24.4 � 5.5 141.8 � 77.4 58.6 � 18.6 9.0 21.0

Mampituba 11 24.8 � 4.2 157.2 � 63.7 60.9 � 15.9 8.7 16.5Springo Tramandaı 31 29.0 � 5.7a,e 191.4 � 82.3a,d 72.6 � 18.5c,d 7.5 21.0

Mampituba 21 35.0 � 8.9c,f,h,j 237.1 � 51.0c,e,h,j 79.5 � 9.6c,e 7.0 24.8Summero Tramandaı 26 29.3 � 6.5a,e 212.1 � 79.0b,e 76.4 � 16.6c,e 8.0 25.0

Mampituba 20 34.2 � 6.2c,f,h,k 278.2 � 104.0c,f,k 85.4 � 16.1c,f,i 6.4 27.0Autumno Tramandaı 23 28.3 � 4.6a,e 205.7 � 57.3b,e 85.7 � 9.4c,f,g,l 7.8 21.5

Mampituba 21 25.7 � 4.0 158.9 � 54.1 69.8 � 16.6b 7.3 19.0Annual average Tramandaı 100 27.9 � 5.8a 190.2 � 78.5b 74.3 � 18.5c 8.1 � 0.6 22.1 � 1.9

(2001–2003) Mampituba 73 29.9 � 7.5b 210.1 � 90.5b 74.0 � 17.7c 7.3 � 0.9 21.8 � 4.9

aData significant in relation to Armazem lagoon at P � 0.05. In all cases, significance was tested using the Kruskal-Wallis test.bP � 0.01.cP � 0.001.dData significant in relation to winter from the same site group at P � 0.05.eP � 0.01.fP � 0.001.gData significant in relation to Tramandaı spring at P � 0.001.hData significant in relation to Tramandaı from the same season group at P � 0.05.iData significant in relation to Mampituba autumn at P � 0.05.jP � 0.01.kP � 0.001.lData significant in relation to Mampituba from the same seasonal group at P � 0.01.mNumber of animals per group.nMean values � SD obtained from average of 100 erythrocytes per fish.oMean values obtained from average of two fish captures per season per site.

462 Andrade et al.

the autumn Tramandaı group had significant increases in DIand DF values compared to winter values. The spring IL,DI, and DF values for the Tramandaı group were greaterthan the summer and autumn values, while the spring DNAdamage for the Mampituba group was only greater than theautumn values. The summer IL and DI values for theMampituba fish also were greater than the autumn values. Asimilar seasonal pattern in DNA damage was observed forfish from both the Tramandaı and Mampituba estuaries,where seasons with higher temperatures were associatedwith higher levels of DNA damage than seasons with lowertemperatures.

Comparison of DNA damage in sea catfish from theTramandaı and Mampituba estuaries by season indicatedsignificant differences in DNA damage for samples taken inthe spring and autumn. In comparison to control sea catfishfrom the Armazem lagoon, the overall mean IL, DI, and DFvalues were increased for sea catfish from the TramandaıRiver (P � 0.001), while the DI (P � 0.01) and DF (P �0.05) were increased for Mampituba sea catfish. In addition,there were differences in the amount of DNA damage in fishfrom these two estuaries, with sea catfish from the Traman-daı River having significantly higher (P � 0.01) levels ofDNA damage than those from the Mampituba River(Table II).

Tables I and II also show the mean water pH and tem-perature (°C) measurements for each site. For mullets, thecorrelation coefficients for the Tramandaı (r � 0.57) andMampituba (r � 0.97) estuaries indicated a significant as-sociation with water temperature only for Mampituba (P �0.05). Linear regression analysis conducted with the datafrom each site gave Y � �22.6 � 9.5� for Tramandaı andY � �52.6 � 11.9� for Mampituba, indicating a trend forboth estuaries for more damaged mullet cells occurring athigher temperatures. However, when the regression coeffi-cients were evaluated using Students’ t-test, only the Mam-pituba data were significant (P � 0.05). For sea catfish, thet-test indicated that the correlations between DNA damageand water temperature for the Tramandaı (r � �0.09) andMampituba (r � 0.89) estuaries were not significant. Therelationship for linear regression of the Tramandaı data wasY � 118.9 � 1.8�, while for Mampituba it was Y ��112.8 � 7.9�. Although higher temperatures at the Mam-pituba site correlated with increased DNA damage in seacatfish cells, Students’ t-test indicated that there was nosignificant correlation for either river. When correlationanalysis was conducted for DNA damage in mullets andwater pH, linear regression produced lines of Y � 514.0 �40.5� for the Tramandaı River (r � �0.8) and Y �576.6 � 50.1� for the Mampituba River (r � �0.81);

TABLE II. Detection of DNA Damage in the Erythrocytes of Sea Catfish Captured Between Winter 2001 and Autumn 2003From Armazem Lagoon, the Control Site, and the Tramandaı and Mampituba Rivers

Season Site noDNA imagelength (�m)p

Damage index(0–400)p

Damagefrequency (%)p

Meanwater pH

Mean watertemperature (°C)

Armazem lagoon 14 21.1 � 1.4 14.8 � 14.1 8.6 � 10.3Winterq Tramandaı 18 22.7 � 4.1 40.7 � 57.6 16.2 � 19.6 9.0 21.0

Mampituba 11 22.1 � 3.4 31.5 � 49.6 21.1 � 31.3 8.7 16.5Springq Tramandaı 24 32.7 � 9.2c,f,h,j,l 128.5 � 59.9c,f,h,i,l 43.1 � 16.2c,f,g,i,m 7.5 21.0

Mampituba 21 24.9 � 5.7a,d,j 63.9 � 60.1b,e,i 21.4 � 17.9a,i 7.0 24.8Summerq Tramandaı 22 25.3 � 6.3b,d 74.9 � 74.7c,d 29.3 � 22.1c,d 8.0 25.0

Mampituba 20 28.6 � 8.1c,e,j 121.0 � 107.8c,f,j 42.1 � 30.7 6.4 27.0Autumnq Tramandaı 19 23.1 � 4.8k 72.2 � 42.7c,d,l 28.7 � 15.3c,d,m 7.8 21.5

Mampituba 21 19.9 � 4.1 26.9 � 35.6 10.0 � 10.4 7.3 19.0Annual average Tramandaı 83 26.4 � 7.7c,n 82.4 � 67.6c,n 30.3 � 20.6c,n 8.1 � 0.6 22.1 � 1.9

(2001–2003) Mampituba 73 24.1 � 6.6 63.9 � 78.9b 23.7 � 25.7a 7.3 � 0.9 21.8 � 4.9

aData significant in relation to Armazem lagoon at P � 0.05. In all cases, significance was tested using the Kruskal-Wallis test.bP � 0.01.cP � 0.001.dData significant in relation to winter from the same site group at P � 0.05.eP � 0.01.fP � 0.001.gData significant in relation to Tramandaı summer at P � 0.05.hP � 0.01.iData significant in relation to autumn from the same site group at P � 0.05.jP � 0.001.kData significant in relation to Mampituba from the same season group at P � 0.05.lP � 0.01.mP � 0.001.nData significant in relation to Mampituba River from annual at P � 0.01.oNumber of animals per group.pMean values � SD obtained from average of 100 erythrocytes per fish.qMean values obtained from average of two fish captures per season per site.

Fish as Biomarkers of Pollution 463

neither of these correlations was significant. For sea catfish,linear regression of DNA damage and pH measurementsproduced lines described by Y � 472.2 � 48.7� for theTramandaı (r � �0.86) and Y � 308.7 � 33.7� for theMampituba estuary (r � �0.75). Although there was a trendtoward more damaged catfish cells at lower pH values,again, neither of these relationships was significant.

Smears for the MN assay were made with the same bloodsamples used for the Comet assay. The MN data for mulletare summarized in Table III, while the MN data for seacatfish are given in Table IV. No significant differencesbetween seasons were observed for either of the genera. Inrelation to the Armazem lagoon controls, the annual averageof micronucleated cells was significantly increased (P �0.05) only for mullets from both sites (Table III); no sig-nificant difference was seen for sea catfish at either site(Table IV).

Water samples from the Tramandaı estuary had a higherannual mean concentration of total HCs (1.5 �g/L) thanMampituba estuary water. In fact, HCs from MampitubaRiver were below the level of detection (1.0 �g/L).

The metal content of sediments from both sites is shownin Table V. Most of the metals related to human activitieswere present in the samples, and, in general, the concentra-tions of K, Ti, Mn, and Fe were higher in the Tramandaıestuary samples than the samples from the Mampitubaestuary. The saline composition of the estuarine zones dif-

fered from that of the open oceans, with the concentration ofsodium and magnesium showing variations between thesites, which were possibly related to fluctuations in waterlevels. Data on water levels, however, were not collected.

DISCUSSION

Assessing environmental risk requires systems that quan-titatively and qualitatively reflect the effects of exposure.Organisms that are in direct contact with contaminatedenvironmental compartments (sediment, water, air) are wellsuited for inclusion in such systems [Rajaguru et al., 2003].Fish are especially useful for assessing the biological risk ofhazardous and toxic wastes in water and sediment since theyare in direct contact with the contaminants in their environ-ment and are sensitive to anthropogenic compounds. Thissensitivity may lead to induction of metabolizing enzymes,inhibition of reproduction and growth, accumulation of haz-ardous substances, genetic damage, and possibly death[White and Rasmussen, 1998; Bickham et al., 2000].

In the present study, we used mullet (Mugil sp.) and seacatfish (Netuma sp.) as sentinel organisms because these fishhave been shown to be sufficiently sensitive to anthropo-genic compounds in laboratory tests [Andrade et al., 2004]and therefore suitable for biomonitoring studies. Both gen-era are known to feed at least in part on organisms anddetritus in the sediment and are thus exposed to toxic agents

TABLE III. Mean Frequencies of Micronucleated Cells (MN) Observed in the Erythrocytesof Mullet Captured Between Winter 2001 and Autumn 2003 From Armazem Lagoon, theControl Site, and the Tramandaı and Mampituba Rivers

Season

Armazem lagoon Tramandaı Mampituba

na MN/4,000 cells na MN/4,000 cells na MN/4,000 cells

Winter 20 1.2 � 1.3 21 1.2 � 1.7Spring 31 1.6 � 2.2 16 1.2 � 1.4Summer 26 2.0 � 2.8 23 1.9 � 1.5Autumn 23 1.1 � 1.7 16 0.9 � 1.2Annual average

(2001–2003) 17 0.6 � 0.7 100 1.5 � 2.2b 76 1.4 � 1.4b

aNumber of animals per group.bSignificant in relation to control region (Armazem lagoon) at P � 0.05, Kruskal-Wallis test.

TABLE IV. Mean Frequencies of Micronucleated Cells (MN) Observed in Erythrocytes ofSea Catfish Captured Between Winter 2001 and Autumn 2003 From Armazem Lagoon, theControl Site, and the Tramandaı and Mampituba Rivers

Season

Armazem lagoon Tramandaı Mampituba

na MN/4,000 cells na MN/4,000 cells na MN/4,000 cells

Winter 18 1.3 � 1.4 12 1.2 � 1.3Spring 23 1.8 � 1.8 23 1.2 � 1.3Summer 22 1.4 � 2.5 23 1.8 � 5.1Autumn 18 1.0 � 0.9 22 0.7 � 1.2Annual average

(2001–2003) 20 0.5 � 0.9 81 1.4 � 1.8 80 1.2 � 2.9

aNumber of animals per group.

464 Andrade et al.

in both water and sediment. In addition, these genera arewidespread and abundant in the study area and known to beestuarine-dependent fish [Silva, 1982].

The study area consisted of the Tramandaı and Mampi-tuba estuaries, which are located in close proximity to twocoastal towns. The areas around both these estuaries receivea large influx of people during the summer (December toMarch), increasing their normal populations of about 30,000to 35,000 by about 10-fold. The effects of this large increasein inhabitants during the summer was a question addressedby our study, i.e., is this influx related to more environmen-tal contamination of water, resulting in greater DNA dam-age in fish?

Our results for mullet and sea catfish captured in thesetwo estuaries during the four seasons over a period of2 years demonstrated increased DNA damage (Tables Iand II) and an increase in the frequency of micronucleatedcells (Table III and IV) relative to control fish from areference location. In general, the alkaline Comet assaydisplayed greater sensitivity than the MN test, with theComet assay detecting significant differences between theseasons and also between sites, while the MN test showedsignificant differences only between the annual average formullets from both test sites and fish from the control site.Despite these findings, if we compare Table I with III(mullet) and Table II with IV (sea catfish), it is possible toobserve a general correspondence between the detection ofDNA damage in the Comet assay and the MN test. Formullet erythrocytes, the greatest responses for both assaysand both sites occurred during the summer, while for seacatfish erythrocytes, the highest values were observed inboth assays during spring for fish from the Tramandaı siteand during summer for fish from the Mampituba site. Thedifference between the responses in the Comet assay andMN test indicates that micronucleated cells were not alwayspresent in the blood of the test fish.

Although differences in DNA damage levels could berelated to ecophysiological effects, the formation of DNA

strand breaks in fish erythrocytes may also result from theabsorption and biotransformation of genotoxic environmen-tal pollutants [Akcha et al., 2003]. Various mechanismshave been proposed for the formation of DNA strand breaksin higher vertebrates, and this wide range in potential mech-anisms for strand break induction could make the Cometassay more sensitive to potentially genotoxic damage thanthe MN test [Bombail et al., 2001].

Our Comet assay results for mullet and sea catfish raiseseveral interesting points. The most important observationis the seasonal variations in DNA damage detected by theComet assay, which is consistent with its being a moresensitive method for detecting genotoxicity than the MNtest. The seasonal variation in the Comet assay responsesappears to be strongly related to the increase in the numberof people in the towns close to the test areas during thewarm spring and summer seasons. Both fish genera showedsignificant increases in DNA damage during spring andsummer. These results agree with our previous work [An-drade et al., 2004], where we found an increase in baselineDNA damage associated with high temperature. In addition,the DNA damage at the Tramandaı site was almost alwaysgreater than the DNA damage for the Mampituba site,which is consistent with Tramandaı being a larger town thanTorres. These are related with an increase in the number ofinhabitants, which was presumably associated with in-creased discharge of effluents and municipal wastewater.

Comparisons between the seasons in terms of water pHindicated that during the warm seasons the mean pH waslower than during the cool seasons. When correlations weremade, the results showed a trend linking lower pH values tomore damaged cells. One of the major effects of lower pHis an increase in the dissolution of cheated metal ions and aconcomitant increase in the concentration of metal ions inthe water bed [Rajaguru et al., 2002].

The concentration of metals at the Tramandaı and Mam-pituba sites reinforces the association between warm sea-sons and high levels of DNA damage: the total metal

TABLE V. Concentrations in Arbitrary Units of Metals Found in Sediment Samples From Tramandaı andMampituba Rivers

Season Site

Metalsa

Sodium(Na)

Magnesium(Mg)

Aluminium(Al)

Silicium(Si)

Potassium(K)

Titanium(Ti)

Manganese(Mn) Iron (Fe)

Winter Tramandaı 0.0 � 0.0 0.0 � 0.0 12.1 � 0.6 422.3 � 5.5 4.8 � 0.3 6.9 � 0.2 0.4 � 0.1 18.3 � 0.3Mampituba 0.0 � 0.0 2.9 � 0.4 10.3 � 0.5 394.5 � 5.1 4.6 � 0.2 1.5 � 0.1 0.0 � 0.0 16.4 � 0.2

Spring Tramandaı 0.0 � 0.0 3.0 � 0.3 10.0 � 0.5 411.1 � 5.2 11.0 � 0.4 20.0 � 0.4 1.0 � 0.1 28.3 � 0.3Mampituba 0.0 � 0.0 6.0 � 0.4 12.6 � 0.6 416.5 � 5.4 9.3 � 0.4 1.7 � 0.1 0.3 � .01 22.7 � 0.3

Summer Tramandaı 92.2 � 8.2 5.0 � 0.4 16.7 � 0.6 348.9 � 4.5 22.1 � 0.6 1.1 � 0.1 0.8 � 0.1 7.9 � 0.1Mampituba 0.0 � 0.0 3.2 � 0.4 9.3 � 0.5 402.3 � 5.2 5.0 � 0.3 1.4 � 0.1 0.0 � 0.0 13.6 � 0.2

Autumn Tramandaı 0.0 � 0.0 3.9 � 0.4 12.8 � 0.6 379.8 � 5.0 12.8 � 0.4 1.3 � 0.1 0.9 � 0.1 23.4 � 0.3Mampituba 0.0 � 0.0 3.5 � 0.4 16.3 � 0.6 394.7 � 5.1 10.9 � 0.4 2.8 � 1.3 0.4 � 0.1 30.8 � 0.3

aThe values represent net values, i.e., discounting the background due to the agarose used as a binder. Limits of detection: Na � 0.7; Mg � 0.7; Al �0.7; Si � 0.2; K � 0.1; Ti � 0.06; Mn � 0.06; Fe � 0.07.

Fish as Biomarkers of Pollution 465

concentrations were higher in the spring and summer thanthe fall and winter. These results suggest that the two rivers,which both receive domestic sewage and other kinds ofcontaminants such as agricultural drainage and municipalwastewaters, are more contaminated during the seasons inwhich there are more people in the cities. It is known thattrace elements, many of which are persistent, toxic, andbioaccumulate, are being increasingly discharged intocoastal seas by anthropogenic activities; the concentrationof heavy metals in several estuarine environments is knownto be increasing due to industrialization and increased pop-ulation density around rivers [Gatti et al., 1999]. Severalheavy metals are considered essential elements for life,while others do not have a defined biological function.These latter metals are generally toxic to a variety of or-ganisms, and different ecosystem compartments have dif-ferent, and limited, capacities to receive such metals withouttoxic effects [Martin et al., 1998]. The concentrations ofsome elements in the two rivers are shown in Table V.These data indicate that the concentrations of metal ions arevery variable in the different seasons and at the differentsites, which makes it difficult to correlate metals with DNAdamage. One possibly significant observation, however, isthat higher amounts of iron, manganese, potassium, andtitanium occurred at the Tramandaı site than the Mampitubasite, and such metals are known to be associated with highlevels of human activity [Valkovic and Bogdanovic, 1996].

As there are so many different chemicals in crude oil andin other petroleum products, it is not practical to measureeach one separately. Therefore, the concentration of totalHCs at a site is often measured as an index of contaminationwith petroleum products (Agency for Toxic Substances andDisease Registry, toxicological profile for total petroleumhydrocarbons, http://www.atsdr.cdc.gov/toxfaq.html). Thehypothesis that the Tramandaı site is more exposed tocontamination than the Mampituba site is supported by theobservation that water from the Tramandaı site had a highertotal HC concentration than water from Mampituba site,where HC concentrations were below the limit of detection.The HCs could be associated with pumping gasoline, spill-age of oil, or the use of HCs at home or in industry. Thereis also some evidence that HCs can be synthesized bymicrobes and plants [Yu, 2001], but the most common HCsproduced in this way are not strongly genotoxic [Freedman,1995]. Manganese is associated with petroleum productsand this metal was also detected at the Tramandaı site(Table V).

Our study did not attempt to define which specific con-taminants in the polluted river water were responsible forgenetic damage. Our more modest objective was to detectoverall genotoxic effects from multiple contaminants, be-cause the two estuaries are subjected to multiple sources ofmunicipal pollution. The Comet assay, which detects theDNA damage produced by a large number of genotoxicagents in a dose-dependent manner, is considered to be an

appropriate and reliable tool for detecting genotoxicity inaquatic environments [Pandrangi et al., 1995; Kamer andRinkevich, 2002; Rajaguru et al., 2003]. Several studieshave been conducted over the past few years that employthe Comet assay or the MN test to detect genotoxicity inbioindicator fish species [Pandrangi et al., 1995; Minissi etal., 1996; Ayllon et al., 2000; Bombail et al., 2001; Grisoliaand Starling, 2001; Russo et al., 2004]. However, there havebeen few studies employing these methods as field biomar-kers in fish sampled from aquatic environments duringdifferent seasons. Our results confirm the potential value ofboth assays for environmental biomonitoring. Based on ourexperience, we propose using the alkaline Comet assay as ascreening tool for biomonitoring contamination from com-plex mixtures at regular periods and, if the results arepositive, utilizing the MN test to confirm the results andverify the individual responses.

A challenge associated with biomonitoring is the identi-fication of the individual compounds that may be responsi-ble for possible adverse effects associated with exposure toenvironmental agents. Samples obtained from environmen-tal sources are complex mixtures of organic and inorganiccompounds, consisting of thousands of individual compo-nents that may interact to produce additive, synergistic, orantagonistic effects [Fent, 2003]. Our study generatedchemical and biological data from areas exposed to multiplesources of pollutants. Although no specific cause-effectrelationships were established, the data indicate that theremay be associations between HCs, metals, pH, and watertemperature and the level of damaged cells in mullet and seacatfish from the Tramandaı and Mampituba estuaries. Ourresults agree with previous data on sites contaminated bydomestic sewage, which show increases in DNA damage,with our data showing seasonal and site differences in theamount of damage [Stahl, 1991; De Flora et al., 1993].Finally, the results of our study indicate that the alkalineComet assay is more sensitive than the MN assay fordetecting genotoxicity in an aquatic environment, and thatmullet and sea catfish are useful sentinel organisms withsufficient sensitivity to be effective monitors of biologicalhazards.

ACKNOWLEDGMENTS

The authors thank Mr. Fabio Iranco, Mr. Juliano Silveira,Ms. Julianna Marko da Silva, and Ms. Roseli de Oliveiraand all fishermen of Tramandaı and Torres city who helpedthem during field work and Dr. Robert Heflich for helpfulcomments on this manuscript.

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