Research Article Genotoxic Potential and Physicochemical ...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 209737 6 pageshttpdxdoiorg1011552013209737

Research ArticleGenotoxic Potential and Physicochemical Parameters of SinosRiver Southern Brazil

Madalena C S Scalon1 Ciliana Rechenmacher2 Anna Maria Siebel2

Michele L Kayser3 Manoela T Rodrigues3 Sharbel W Maluf4 Marco Antonio SRodrigues5 and Luciano Basso da Silva6

1 Programa de Pos-graduacao em Qualidade Ambiental Universidade Feevale 93352-000 Novo Hamburgo RS Brazil2 Curso de Ciencias Biologicas Universidade Feevale 93352-000 Novo Hamburgo RS Brazil3 Curso de Biomedicina Universidade Feevale 93352-000 Novo Hamburgo RS Brazil4 Laboratorio de Citogenetica Servico de Genetica Medica Hospital de Clınicas de Porto Alegre90035-903 Porto Alegre RS Brazil

5 Programa de Pos-graduacao em Qualidade Ambiental Grupo de Pesquisa em Tecnologias Ambientais93352-000 Novo Hamburgo RS Brazil

6 Programa de Pos-graduacao em Qualidade Ambiental Grupo de Pesquisa em Indicadores de Qualidade AmbientalUniversidade Feevale RS 239 No 2755 93352-000 Novo Hamburgo RS Brazil

Correspondence should be addressed to Luciano Basso da Silva lucianosilvafeevalebr

Received 27 August 2013 Accepted 16 September 2013

Academic Editors T Coccini and T Gupta

Copyright copy 2013 Madalena C S Scalon et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study aimed to evaluate the physicochemical parameters and the genotoxic potential of water samples collected inthe upper middle and lower courses of the Sinos River southern Brazil The comet assay was performed in the peripheral bloodof fish Hyphessobrycon luetkenii exposed under laboratory conditions to water samples collected in summer and winter in threesampling sites of Sinos River Water quality analysis demonstrated values above those described in Brazilian legislation in Parobeand Sapucaia do Sul sites located in the middle and in the lower courses of the Sinos River respectively The Caraa site located inthe upper river reach presented all the physicochemical parameters in accordance with the allowed limits in both sampling periodsComet assay in fish revealed genotoxicity in water samples collected in the middle course site in summer and in the three sites inwinter when compared to control groupThus the physicochemical parameters indicated that the water quality of the upper coursecomplies with the limits set by the national guidelines and the ecotoxicological assessment however indicated the presence ofgenotoxic agents The present study highlights the importance of combining water physicochemical analysis and bioassays to rivermonitoring

1 Introduction

Rivers receive a huge amount of wastes from urban areas andindustrial and agricultural activities [1] Hence water bodieswill be contaminated with complex ill-defined mixtures ofchemicals and most freshwater organisms will be exposed tovarying degrees to this contamination [2] In this contextchemical analysis of water is not sufficient to assess theirtoxic potential for wildlife and humans This is because

the bioavailability the biological activities and interactionsbetween different environmental chemicals are not com-pletely understood and considered when hazard assessmentsand predictions of possible ecotoxicological effects are madebased on concentrations alone [3] Therefore a simultaneousapplication of bioassays is a good complement to chemicalanalyses and a useful tool to establish the ecological effectsto environment as it provides the complete response of testorganisms to all the compounds in the water [4]

2 The Scientific World Journal

Among the methods available to assess the many possibletoxic effects caused by the chemicals present in the environ-ment the analysis of DNA alterations in aquatic organismshas been shown to be a highly suitable method for evaluatingthe genotoxic contamination of environments being ableto detect toxicity at low concentrations of contaminants ina wide range of species [5] The pollution-induced geneticdamage might cause adverse effects to species and affect thestability of ecosystems [6ndash8]The comet assay or single cell gelelectrophoresis is a rapid sensitive and relatively inexpensivemethod to study DNA damage in different cell types andhas found wide application in genotoxicity evaluation in fishexposed to various xenobiotics in the aquatic environment[1 9] The comet assay is based on the ability of negativelycharged fragments of DNA to be drawn through an agarosegel in response to an electric field The extent of DNAmigration depends directly on the DNA damage present inthe cells [1]

The subtropical Sinos River is one of the main riverslocated in Brazilrsquos southernmost state Rio Grande do Sul andis an example of an impacted watercourse The Sinos Riverbasin presents an area of approximately 4000 km2 whichcovers 32 municipalities and provides drinking water for12 million inhabitants and for one of the most importantindustrial centers in Brazil [10] In the downriver directionthe river is divided into upper middle and lower coursesfollowing a gradient of pollution The Sinos River in theupper course has low demographic density and suffers mod-erate impacts from small amounts of agricultural waste andrelatively little influence from domestic sewage The middlecourse has a major impact with large areas used for ricecultivation and cattle ranching and has a larger number ofinhabitants In the lower course the demographic density andconcentration of industries are high [10] The most denselypopulated urban areas include three cities with more than200000 inhabitants (Novo Hamburgo Sao Leopoldo andCanoas) which are located in the lower course of the river

Studies by the State Environmental Protection Agency(Fundacao Estadual de Protecao Ambiental FEPAM)revealed high loads of domestic and industrial sewage inthe lower course of the Sinos River [10] Moreover somechemical parameters in the middle course revealed that itswater quality index was similar to that of the lower region ofthe basin [11] Studies investigating the genotoxicity of watersamples collected from the Sinos River have yielded bothpositive [12ndash14] and negative [14ndash17] results

In our previous investigation of genotoxicity of SinosRiver the results suggested that the sampling site nearthe river spring is not appropriate as reference site to fishgenotoxicity bioassays [13] Therefore the aim of the presentstudy was to evaluate the water quality of Sinos River throughphysicochemical parameters and genotoxic assay using thefish Hyphessobrycon luetkenii as organism test

2 Materials and Methods

21 Sampling Sites Water samples were collected in Marchand July 2008 (summer and winter resp) at three sampling

sites in the upper Sinos River course near the river spring(town of Caraa S 29∘4310158402610158401015840 and W 50∘16101584046) in the middlecourse (town of Parobe S 29∘4110158400510158401015840 andW50∘5010158405210158401015840) and inthe lower river course (town of Sapucaia do Sul S 29∘4710158405310158401015840and W 51∘1110158402410158401015840)

22 Fish Bioassay Healthy juvenile specimens of indige-nous fish species Hyphessobrycon luetkenii (Boulenger 1887)(Characidae) were obtained from a local fish farm Ani-mals were randomly divided into four groups of about12 specimens and placed in a 10 L aquarium with well-oxygenated dechlorinated tap water at 21∘C plusmn 2∘C for a96 h acclimatization period they were then released into10 L aquariums with undiluted water samples from each sitewithin 4 h after sampling Tap water was used as negativecontrol group The exposure period was 48 h with no foodsupply

23 Comet Assay After the exposure period the comet assaywas performed on peripheral erythrocytes according to Ticeet al [19] Slides were precoated with normal melting pointagarose A mixture of 5 120583L of blood sample collected fromcaudal veins of fish with 95120583L low melting point agarose(07) was added to the slide and immediately covered with acoverslip and then kept for 10min in a refrigerator to solidifyAfter solidification of the gel coverslips were gently removedand the slides were immersed in cold freshly made lysingsolution (25MNaCl 100mM EDTA and 10mM Tris pH102 to which 1 Triton X-100 and 10 DMSO had beenadded) and refrigerated at 4∘C for 6ndash24 h After the lysisthe slides were placed in a horizontal electrophoresis boxside by side The tank was filled with fresh electrophoresisbuffer (300mMNaOH and 1mMEDTA pH gt 13) at 4∘CTheliquid covered the slides which were then left in the solutionfor 20min before the power was turned on Electrophoresiswas performed at 25V and 300mA (sim095Vcm) for 20minThe steps above were carried out under red light to avoidinduction of DNA damage After electrophoresis the slideswere gently removed from the tank and neutralizing buffer(04M Tris pH 75) was added to the slides dropwise threetimes letting it sit for 5min each timeThe slides were rinsedthree times with distilled water air-dried for at least 24 h andthen fixed and stained with silver stain according to Nadin etal [20]

For evaluation of DNA damage 100 cells per individualwere analyzed under an optical microscope at 400x magni-fication The analysis of the slides was conducted using thequality assurance criteria suggested by Tice et al [19] Allslides were coded and examined blind by the same observerThe cells were visually scored according to tail length intofive classes from undamaged (class 0) to complete damage(class 4) [21] Value was assigned to the different categories(from class 0 = 0 to class 4 = 4) and a damage index wascalculated [(No of class 0 cells times 0) + (No of class 1 cells times 1)+ (No of class 2 cells times 2) + (No of class 3 cells times 3) + (No ofclass 4 cells times 4)] for each fish [22] Therefore the total scoreper individual ranged from 0 (all undamaged) to 400 (allmaximally damaged) The damage frequency was calculated

The Scientific World Journal 3

as the percentage of damaged cells (eg the sum of cells withdamage classes 1 to 4)

24 Physicochemical Analysis Immediately after collectionwater samples were analyzed according to Standard Methodsfor the Examination of Water and Wastewater [23] Thefollowing parameters were analyzed pH chemical oxygendemand (COD) five-day biological oxygen demand (BOD

5)

conductivity chlorides hardness total nitrogen ammoniacalnitrogen phosphorus aluminum lead chromium coppernickel iron zinc sodium total solids dissolved solids totalvolatile solids nitrite nitrate turbidity dissolved oxygen(DO) total coliforms and fecal coliforms (Escherichia coli)

25 Statistical Analysis Statistical analysis was performedusing ANOVA followed by Tukey multiple comparison testwhen appropriate All analyses were carried out using theStatistical Package for the Social Sciences (SPSS) 150 forWindows considering a significance level of 119875 le 005

3 Results

The values of physicochemical variables measured in thethree sampling sites during the summer and winter seasonsare shown in Table 1The values were compared to CONAMAResolution 3572005 by the Environment National Council[18] for classifying the national water bodies In this legis-lation the freshwater systems were allocated to five classes(from the class 1 to the class 4 water quality decreases andrestrictions to water use as drinking supply increase) andupper limits were fixed for different water variables for eachcategory (some of them for class 2 category are shown inTable 1) Freshwater systems within class 2 category may beused for human supply (after treatment) aquatic biologicalcommunity protection recreation irrigation aquacultureand fishing activities

Concerning the nineteenwater parameters with referencevalues defined by CONAMA Resolution n 357 higher valuesthan standards were observed in sites Parobe and Sapucaia doSul Higher values were observed in Parobe for aluminumiron and fecal coliforms in the two sampling periods fortotal phosphorus in summer and for BOD in winterThe twowater samples of Sapucaia do Sul showed BOD conductivitytotal phosphorus iron dissolved oxygen and fecal coliformsvalues at odds with the CONAMA resolution in addition toaluminum and copper in the winter sample The Caraa sitepresented all the physicochemical parameters in accordancewith the allowed limits in both sampling periods

The results of damage frequency () and damage indexestimated by the comet assay in the erythrocytes of Hluetkenii exposed to water samples from three sampling sitesin the Sinos River and to tap water (control) are summarizedinTable 2 In the summer period damage frequency at Parobesite was significantly higher than that found at Caraa and inthe control group however no differences in damage indexwere observed The fish exposed to water samples collectedat the three sampling sites in winter have shown significant

increase in both damage index and damage frequency whencompared to control group

4 Discussion

Resolution n 357 [18] is a guiding tool used to evaluate waterquality in Brazil and in the present study values above thosedescribed in resolution were observed at sites Parobe andSapucaia do Sul located in the middle and lower courses ofthe Sinos River respectively Thus water quality tended todecrease downstream as the river passes through the denselyurbanized and industrial areas and receives discharge oftreated and untreated domestic and industrial effluents [24]According to the Sinos River BasinManagement Committeeat present domestic sewage is considered to be the majorproblem affecting water quality of the Sinos River becauseonly 10 of domestic sewage is treated The results obtainedfor dissolved oxygen indicated anoxic conditions of waterin Sapucaia do Sul the lower course site Moreover Parobeand Sapucaia do Sul showed fecal coliforms and BOD

5

values higher than the limits possibly as a consequence ofhigh organic matter input from untreated domestic sewagedischarge [25]

Apart from the domestic sewage the Sinos River alsoreceives pollutants from industrial and rural waste whichhave been reported as a source of toxic and mutagenic effectson the environment [5 26] Studies on the genotoxicity ofSinos River basin have yielded conflicting results Absenceof genotoxic activity was found in the Salmonellamicrosometest [15] themicronuclei analysis in theV79 cell line (Chinesehamster lung fibroblasts) [16] the wing somatic mutationand recombination test (SMART) inDrosophila melanogaster[17] and the Allium cepa test (micronucleus test) [14] On theother side Vargas et al [15] have found genotoxic effects inthe microscreen phage-induction assay and Lemos et al [12]using Salmonellamicrosome assay found indicative muta-genic response including in the headwaters of a tributary ofthe Sinos River Moreover Nunes et al [14] found positiveresults in V79 cell line (comet assay and micronucleus test)

Although the in vitro tests used in most studies assess-ing Sinos River genotoxicity are recommended methods toevaluate the mutagenic activity of environmental samplessuch tests do not provide an assessment of genotoxicity inecologically relevant organisms [27] such as fish species Inthe first study in which Sinos River water genotoxicity wasassessed using fish biomarkers the comet assay has shownsignificant differences between sampling periods with highervalues in water samples collected at sites in the upper andmiddle course during the spring season [13] The resultssuggested that sites near the river spring are not appropriateas reference sites to fish genotoxicity bioassays In additionbased on the monitoring of physicochemical parameters ofSinos River water Blume et al [11] have concluded that thesampling sites in the upper course should no longer be usedas a reference point in future studies

In the present study the water samples collected in thesummer in the middle course site and in the winter in thethree sites have shown genotoxicity in the peripheral blood of


Table 1 Physicochemical parameters of water samples from different sites of Sinos River in the summer and winter of 2008

Parameter CONAMACaraa

Upper courseParobe

Middle courseSapucaia do SulLower course

Summer Winter Summer Winter Summer WinterBOD5 mgO2 L

minus1 50 lt10 1 1 7lowast 7lowast 12lowast

COD mgO2 Lminus1 mdash 8 2 10 7 15 34

Chlorides mg Lminus1 250 31 29 29 35 122 107Conductivity 120583S cmminus1 100 3275 29 6905 6845 14285lowast 1197lowast

Hardness mgCaCO3 Lminus1 mdash 7 13 22 29 31 42

Total nitrogen mg Lminus1 mdash 13 lt05 228 lt05 391 lt05Ammoniacal nitrogen mg Lminus1 37 nd lt05 nd lt05 146 lt05Total phosphorus mg Lminus1 01 nd lt0012 0105lowast 006 0201lowast 1075lowast

Aluminum mg Lminus1 01 005 nd 059lowast 018lowast 005 20lowast

Lead mg Lminus1 001 nd nd nd 00015 nd 0004Chromium mg Lminus1 005 nd nd nd nd nd 004Copper mg Lminus1 0009 nd nd 0002 nd 0004 004lowast

Nickel mg Lminus1 0025 nd nd nd nd nd 001Iron mg Lminus1 03 008 023 193lowast 066lowast 113lowast 688lowast

Zinc mg Lminus1 018 001 nd 001 nd 001 008Sodium mg Lminus1 mdash 5 54 6 69 134 141Dissolved solids mg Lminus1 500 105 77 355 91 17 110Total solids mg Lminus1 mdash 515 197 1175 215 169 870Total volatile solids mg Lminus1 mdash 345 144 755 133 105 227pH 60 to 90 746 706 721 728 689 689Nitrates mg Lminus1 100 109 012 283 142 218 128Nitrite mg Lminus1 10 nd 0003 004 004 016 012Turbidity NTU 100 042 002 174 09 124 505Dissolved oxygen mg Lminus1 50 813 853 731 711 447lowast 203lowast

Total coliforms NMP100mL mdash 5012 697 101120 68930 56300 23100Fecal coliforms NMP100mL 1000 85 84 2690lowast 8620lowast 3000lowast 5200lowast

Values were obtained from one water sample nd not detected lowastValues at odds with the CONAMA resolution n 35705 upper limits to class 2 water category[18]

Table 2 Damage frequency (the sum of cells with damage classes 1 to 4 in ) and damage index estimated by the comet assay (mean plusmnstandard deviation) in the erythrocytes of H luetkenii exposed to water samples from the Sinos River and to tap water (control)

Sampling site Summer WinterDamage frequency (n) Damage index Damage frequency (n) Damage index

Caraa(Upper course) 506 plusmn 93 (12)a 581 plusmn 145 663 plusmn 133 (11)a 695 plusmn 142

a

Parobe(Middle course) 613 plusmn 63 (12)b 646 plusmn 74 629 plusmn 160 (8)a 653 plusmn 179

a

Sapucaia do Sul(Lower course) 533 plusmn 104 (12)ab 608 plusmn 160 596 plusmn 115 (11)a 625 plusmn 150

a

Control 471 plusmn 115 (12)a 511 plusmn 142 404 plusmn 160 (8)b 404 plusmn 160b

P 0005 0107 0002 0002The number of fish analyzed in each sample is presented between brackets P values referring to ANOVA between sampling sites and respective control group(abvalues with different letters have significant difference in the same column)


H luetkenii when compared to negative control confirmingthe results of other studies that have detected contaminationof genotoxic compounds in the Sinos River basin includingthe upstream course [12 13] Thus the results of this studyindicate that native fish species might be at risk for genotoxicdamage in the Sinos River However the genotoxic poten-tial of contaminants is an organism-specific and exposure-dependent phenomenon and interspecies variability for toxicresponses as a result of differences in uptake accumulationmetabolism excretion and DNA repair efficiency should beconsidered [8]

Many studies investigated the genotoxic effects of ruralindustrial and urban effluents however their relative contri-bution and comprehensive chemical characterization remaina major challenge [26 28 29] Considering the low urbandensity and the economic activities in the upper Sinos Riversite (Caraa) this region suffers relatively little influence fromdomestic and industrial sewage and genotoxic pollutantsmight come from small farms and livestock Contaminantsin this region threaten the sustainability of one of the mainbasins of southern Brazil responsible for water supply toapproximately 16 million inhabitants representing 17 ofthe population of the Rio Grande do Sul state [24] Furtherstudies in the upper Sinos River course should be conductedto identify sources of water contaminants so that measurescan be taken to reduce or eliminate their negative effects

5 Conclusion

Physicochemical parameters demonstrated decreasing waterquality from upstream to downstream of the Sinos Riverand comet assay in fish showed that the river water may becontaminated with genotoxic pollutants in the upper middleand lower courses Thus despite good water quality of theupper river course it should no longer be used as a referencesite in future genotoxicity studies Moreover the resultsshowed that complementing the physicochemical evaluationof river water with bioassays clearly permits acquisition ofinformation that cannot be obtained from the measurementof water quality parameters

Acknowledgments

This work was partially supported by a research Grantfrom Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq no 4771922007-6) and by a StudentFellowship from Fundacao de Amparo a Pesquisa do Estadodo Rio Grande do Sul (FAPERGS) to an undergraduatestudent (CR)

References

[1] G Frenzilli M Nigro and B P Lyons ldquoThe Comet assay forthe evaluation of genotoxic impact in aquatic environmentsrdquoMutation Research vol 681 no 1 pp 80ndash92 2009

[2] J P Sumpter ldquoProtecting aquatic organisms from chemicals theharsh realitiesrdquoPhilosophical Transactions of the Royal SocietyAvol 367 no 1904 pp 3877ndash3894 2009

[3] CWang YWang F Kiefer A Yediler ZWang andA KettrupldquoEcotoxicological and chemical characterization of selectedtreatment process effluents of municipal sewage treatmentplantrdquo Ecotoxicology and Environmental Safety vol 56 no 2pp 211ndash217 2003

[4] C Blasco and Y Pico ldquoProspects for combining chemical andbiological methods for integrated environmental assessmentrdquoTrends in Analytical Chemistry vol 28 no 6 pp 745ndash757 2009

[5] R Van der Oost J Beyer and N P E Vermeulen ldquoFish bioac-cumulation and biomarkers in environmental risk assessmenta reviewrdquo Environmental Toxicology and Pharmacology vol 13no 2 pp 57ndash149 2003

[6] J W Bickham S Sandhu P D N Hebert L Chikhi and RAthwal ldquoEffects of chemical contaminants on genetic diversityin natural populations implications for biomonitoring andecotoxicologyrdquo Mutation Research vol 463 no 1 pp 33ndash512000

[7] MHMedina J A Correa andC Barata ldquoMicro-evolution dueto pollution possible consequences for ecosystem responses totoxic stressrdquo Chemosphere vol 67 no 11 pp 2105ndash2114 2007

[8] A N Jha ldquoEcotoxicological applications and significance of thecomet assayrdquoMutagenesis vol 23 no 3 pp 207ndash221 2008

[9] A Dhawan M Bajpayee and D Parmar ldquoComet assay areliable tool for the assessment of DNA damage in differentmodelsrdquo Cell Biology and Toxicology vol 25 no 1 pp 5ndash322009

[10] FEPAM Fundacao Estadual de Protecao Ambiental Hen-rique LuizRoesslerQualidade ambientalmdashRegiaoHidrograficado Guaıba Qualidade das aguas da Bacia Hidrografica doRio dos Sinos 2009 httpwwwfepamrsgovbrqualidadequalidade sinossinosasp

[11] K K Blume J C Macedo A Meneguzzi L B Silva D MQuevedo andM A S Rodrigues ldquoWater quality assessment ofthe Sinos River Southern Brazilrdquo Brazilian Journal of Biologyvol 70 no 4 pp 1185ndash1193 2010

[12] A T Lemos D P Rosa J A V Rocha and V M F Var-gas ldquoMutagenicity assessment in a river basin influenced byagricultural urban and industrial sourcesrdquo Ecotoxicology andEnvironmental Safety vol 72 no 8 pp 2058ndash2065 2009

[13] M C S Scalon C Rechenmacher A M Siebel et al ldquoEvalua-tion of Sinos River water genotoxicity using the comet assay infishrdquo Brazilian Journal of Biology vol 70 no 4 pp 1217ndash12222010

[14] E A Nunes C T De Lemos L Gavronski T N Moreira NC D Oliveira and J da Silva ldquoGenotoxic assessment on riverwater using different biological systemsrdquo Chemosphere vol 84no 1 pp 47ndash53 2011

[15] V M F Vargas S B Migliavacca A C De Melo et alldquoGenotoxicity assessment in aquatic environments under theinfluence of heavy metals and organic contaminantsrdquoMutationResearch vol 490 no 2 pp 141ndash158 2001

[16] N R Terra I R Feiden J Fachel C T Lemos and E A NunesldquoEcotoxocological evaluation of sediment and water samplesfrom Sinos River Rio Grande do Sul Brazil using Daphniamagna and V79 cellsrdquoActa Limnologica Brasiliensia vol 20 no1 pp 63ndash72 2008

[17] L V Jacociunas R R Dihl M Lehmann M L Reguly andH H R de Andrade ldquoRecombinagenic activity of water andsediment from Sinos River and Araca and Garcas Streams(Canoas Brazil) in the Drosophila wing spot testrdquo Science ofthe Total Environment vol 408 no 3 pp 571ndash577 2010


[18] Conselho Nacional doMeio AmbientemdashCONAMA Resolucaon 3572005 Dispoe sobre a classificacao dos corpos de aguae diretrizes ambientais para o seu enquadramento bem comoestabelece as condicoes e padroes de lancamento de efluentes eda outras providencias Diario Oficial da Uniao Brasılia BrasilMars 2005

[19] R R Tice E Agurell D Anderson et al ldquoSingle cell gelcometassay guidelines for in vitro and in vivo genetic toxicologytestingrdquo Environmental and Molecular Mutagenesis vol 35 no3 pp 206ndash221 2000

[20] S B Nadin LM Vargas-Roig andD R Ciocca ldquoA silver stain-ing method for single-cell gel assayrdquo Journal of Histochemistryand Cytochemistry vol 49 no 9 pp 1183ndash1186 2001

[21] DAnderson T-WYu B J Phillips andP Schmezer ldquoThe effectof various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in theCOMET assayrdquo Mutation Research vol 307 no 1 pp 261ndash2711994

[22] M Pitarque A Creus R Marcos J A Hughes and D Ander-son ldquoExamination of various biomarkers measuring geno-toxic endpoints from Barcelona airport personnelrdquo MutationResearch vol 440 no 2 pp 195ndash204 1999

[23] APHA American Public Health Association StandardMethodsfor the Examination ofWater andWastewater Washington DCUSA 20th edition 1998

[24] Comitesinos ldquoComite de Gerenciamento da BaciaHidrograficado Rio dos Sinos Caracterizacao da Bacia Hidrografica do Riodos Sinosrdquo 2000 httpwwwcomitesinoscombr

[25] P R Kannel S Lee S R Kanel S P Khan and Y-S LeeldquoSpatial-temporal variation and comparative assessment ofwater qualities of urban river system a case study of the riverBagmati (Nepal)rdquo Environmental Monitoring and Assessmentvol 129 no 1ndash3 pp 433ndash459 2007

[26] T Ohe TWatanabe and KWakabayashi ldquoMutagens in surfacewaters a reviewrdquo Mutation Research vol 567 no 2-3 pp 109ndash149 2004

[27] A N Jha T H Hutchinson J M Mackay B M Elliott andD R Dixons ldquoEvaluation of the genotoxicity of municipalsewage effluent using the marine worm Platynereis dumerilii(Polychaeta Nereidae)rdquo Mutation Research vol 391 no 3 pp179ndash188 1997

[28] L D Claxton V S Houk and T J Hughes ldquoGenotoxicity ofindustrial wastes and effluentsrdquoMutation Research vol 410 no3 pp 237ndash243 1998

[29] P A White and J B Rasmussen ldquoThe genotoxic hazards ofdomestic wastes in surface watersrdquoMutation Research vol 410no 3 pp 223ndash236 1998

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MEDIATORSINFLAMMATION

of


Among the methods available to assess the many possibletoxic effects caused by the chemicals present in the environ-ment the analysis of DNA alterations in aquatic organismshas been shown to be a highly suitable method for evaluatingthe genotoxic contamination of environments being ableto detect toxicity at low concentrations of contaminants ina wide range of species [5] The pollution-induced geneticdamage might cause adverse effects to species and affect thestability of ecosystems [6ndash8]The comet assay or single cell gelelectrophoresis is a rapid sensitive and relatively inexpensivemethod to study DNA damage in different cell types andhas found wide application in genotoxicity evaluation in fishexposed to various xenobiotics in the aquatic environment[1 9] The comet assay is based on the ability of negativelycharged fragments of DNA to be drawn through an agarosegel in response to an electric field The extent of DNAmigration depends directly on the DNA damage present inthe cells [1]

The subtropical Sinos River is one of the main riverslocated in Brazilrsquos southernmost state Rio Grande do Sul andis an example of an impacted watercourse The Sinos Riverbasin presents an area of approximately 4000 km2 whichcovers 32 municipalities and provides drinking water for12 million inhabitants and for one of the most importantindustrial centers in Brazil [10] In the downriver directionthe river is divided into upper middle and lower coursesfollowing a gradient of pollution The Sinos River in theupper course has low demographic density and suffers mod-erate impacts from small amounts of agricultural waste andrelatively little influence from domestic sewage The middlecourse has a major impact with large areas used for ricecultivation and cattle ranching and has a larger number ofinhabitants In the lower course the demographic density andconcentration of industries are high [10] The most denselypopulated urban areas include three cities with more than200000 inhabitants (Novo Hamburgo Sao Leopoldo andCanoas) which are located in the lower course of the river

Studies by the State Environmental Protection Agency(Fundacao Estadual de Protecao Ambiental FEPAM)revealed high loads of domestic and industrial sewage inthe lower course of the Sinos River [10] Moreover somechemical parameters in the middle course revealed that itswater quality index was similar to that of the lower region ofthe basin [11] Studies investigating the genotoxicity of watersamples collected from the Sinos River have yielded bothpositive [12ndash14] and negative [14ndash17] results

In our previous investigation of genotoxicity of SinosRiver the results suggested that the sampling site nearthe river spring is not appropriate as reference site to fishgenotoxicity bioassays [13] Therefore the aim of the presentstudy was to evaluate the water quality of Sinos River throughphysicochemical parameters and genotoxic assay using thefish Hyphessobrycon luetkenii as organism test

2 Materials and Methods

21 Sampling Sites Water samples were collected in Marchand July 2008 (summer and winter resp) at three sampling

sites in the upper Sinos River course near the river spring(town of Caraa S 29∘4310158402610158401015840 and W 50∘16101584046) in the middlecourse (town of Parobe S 29∘4110158400510158401015840 andW50∘5010158405210158401015840) and inthe lower river course (town of Sapucaia do Sul S 29∘4710158405310158401015840and W 51∘1110158402410158401015840)

22 Fish Bioassay Healthy juvenile specimens of indige-nous fish species Hyphessobrycon luetkenii (Boulenger 1887)(Characidae) were obtained from a local fish farm Ani-mals were randomly divided into four groups of about12 specimens and placed in a 10 L aquarium with well-oxygenated dechlorinated tap water at 21∘C plusmn 2∘C for a96 h acclimatization period they were then released into10 L aquariums with undiluted water samples from each sitewithin 4 h after sampling Tap water was used as negativecontrol group The exposure period was 48 h with no foodsupply

23 Comet Assay After the exposure period the comet assaywas performed on peripheral erythrocytes according to Ticeet al [19] Slides were precoated with normal melting pointagarose A mixture of 5 120583L of blood sample collected fromcaudal veins of fish with 95120583L low melting point agarose(07) was added to the slide and immediately covered with acoverslip and then kept for 10min in a refrigerator to solidifyAfter solidification of the gel coverslips were gently removedand the slides were immersed in cold freshly made lysingsolution (25MNaCl 100mM EDTA and 10mM Tris pH102 to which 1 Triton X-100 and 10 DMSO had beenadded) and refrigerated at 4∘C for 6ndash24 h After the lysisthe slides were placed in a horizontal electrophoresis boxside by side The tank was filled with fresh electrophoresisbuffer (300mMNaOH and 1mMEDTA pH gt 13) at 4∘CTheliquid covered the slides which were then left in the solutionfor 20min before the power was turned on Electrophoresiswas performed at 25V and 300mA (sim095Vcm) for 20minThe steps above were carried out under red light to avoidinduction of DNA damage After electrophoresis the slideswere gently removed from the tank and neutralizing buffer(04M Tris pH 75) was added to the slides dropwise threetimes letting it sit for 5min each timeThe slides were rinsedthree times with distilled water air-dried for at least 24 h andthen fixed and stained with silver stain according to Nadin etal [20]

For evaluation of DNA damage 100 cells per individualwere analyzed under an optical microscope at 400x magni-fication The analysis of the slides was conducted using thequality assurance criteria suggested by Tice et al [19] Allslides were coded and examined blind by the same observerThe cells were visually scored according to tail length intofive classes from undamaged (class 0) to complete damage(class 4) [21] Value was assigned to the different categories(from class 0 = 0 to class 4 = 4) and a damage index wascalculated [(No of class 0 cells times 0) + (No of class 1 cells times 1)+ (No of class 2 cells times 2) + (No of class 3 cells times 3) + (No ofclass 4 cells times 4)] for each fish [22] Therefore the total scoreper individual ranged from 0 (all undamaged) to 400 (allmaximally damaged) The damage frequency was calculated




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5 Conclusion


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Volume 2014

ToxinsJournal of

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AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




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3 Results





4 Discussion


5








Upper courseParobe

















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5 Conclusion


Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Upper courseParobe

















a


a


a






5 Conclusion


Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




5 Conclusion


Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Genotoxic Potential and Physicochemical ...

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