Genotoxic assessment on river water using different biological systems

Chemosphere 84 (2011) 47–53

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Genotoxic assessment on river water using different biological systems Emilene Arusievicz Nunes a,b, Clarice Torres de Lemos a,⇑, Léia Gavronski b, Tiago Nunes Moreira a, Nânci C.D. Oliveira a,b, Juliana da Silva b,⇑ a b

Foundation of the State Environmental Protection Henrique Luís Roessler/FEPAM – Cytogenetics Laboratory, Rio Grande do Sul, Brazil Laboratory of Genetic Toxicology, Lutheran University of Brazil, ULBRA, Rio Grande do Sul, Brazil

a r t i c l e

i n f o

Article history: Received 3 November 2010 Received in revised form 23 February 2011 Accepted 28 February 2011 Available online 23 March 2011 Keywords: Water pollution Mutagenesis Allium cepa Environmental biomonitoring V79 cells

a b s t r a c t This paper reports genotoxicity and toxicity data in water samples collected in Sinos River, an important water course in the hydrographic region of Guaíba Lake, Rio Grande do Sul State, south of Brazil. This river is exposed to intense anthropic influence by numerous shoes, leather, petrochemical, and metallurgy industries. Water samples were collected at two moments (winter 2006 and spring 2006) at five sites of Sinos River and evaluated using in vitro V79 Chinese hamster lung fibroblasts (cytotoxicity, comet assay and micronucleus test) and Allium cepa test (toxicity and micronucleus test). Comet and micronucleus tests revealed that water samples collected exerted cytotoxic, toxic, genotoxic and mutagenic effects. The results showed the toxic action of organic and inorganic agents found in the water samples in all sites of Sinos River, for both data collections. The main causes behind pollution were the domestic and industrial toxic discharges. The V79 and A. cepa tests were proved efficient to detect toxicity and genotoxicity caused by complex mixtures. This study also showed the need for constant monitoring in sites with strong environmental degradation caused by industrial discharges and urban sewages. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The current contamination of water resources, a consequence of anthropogenic discharges, is becoming a major problem in ever growing urban regions. In this scenario, changes in water composition will obviously have deleterious effects on the organisms inhabiting these areas, in addition to health hazards to humans (Ohe et al., 2004). Among the lethal and sub-lethal effects of these complex mixtures present in water, fertility disorders as well as cellular, metabolic, and DNA changes are also observed (Ohe et al., 2004; Villela et al., 2007). Epidemiological studies have failed to clearly characterize these effects, since the experimental procedure to demonstrate this influence is a time-consuming task. This scenario stresses the importance of efficiently and continuously monitoring possible impacted areas using screening assays. The Sinos River, in the hydrographic region of Guaíba Lake, Rio Grande do Sul (RS), southern Brazil, is exposed to intense anthropic influence by numerous shoes, leather, petrochemical, and metallurgy industries (Vargas et al., 1993, 1995; Lemos et al., 1994; Migliavacca et al., 2005; Terra et al., 2008). The region has the largest demographic density in RS, with 61% of the population, and is where the capital city of the state, Porto Alegre, is located. Because of the large amount of pollutants discharged in the Guaíba ⇑ Corresponding authors. Tel.: +55 51 3347 9239. E-mail addresses: [email protected] (C.T. de Lemos), [email protected] (J. da Silva). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.02.085

hydrographic region, and considering also the conflicting results published in literature, there is a lingering need for studies to characterize the main sites of pollutants discharge and identify the causal agents of damage to organisms and the ecosystem. Broadly speaking, the impact caused by the pollution discharged by the industries cited above in water bodies is quite varied and deserves to be studied in more detail (White and Rasmussen, 1998). Concerning the waste dumped by industrial or large industries, more specifically, not only volume but mainly its chemical composition plays an important role in terms of the risk posed to the environment (Nielsen and Rank, 1994; Smaka-Kincl et al., 1996). The hypothesis that waste of industrial and/or rural origin brings a higher risk – when compared to hazards brought about by urbanization – has been emphasized in several studies on the genotoxicity of samples collected in the natural environment. Nevertheless, it becomes increasingly clear that the greatest contribution to the overall genotoxic burden imposed on ecosystems derives mainly from urban waste (White and Rasmussen, 1998). However, the results of several studies that analyzed environmental samples influenced by urban waste are often varied and sometimes contradictory (Nielsen and Rank, 1994; Smaka-Kincl et al., 1996; Ralph and Petras, 1997; Lemos et al., 2006). Among the tests that are routinely recommended for the genotoxic evaluation of water, the Allium cepa test and the in vitro analysis using the Chinese hamster V79 cell line are widely employed (Boeira et al., 2001). The A. cepa genotoxicity test is an excellent plant-based test to study anaphase aberrations.

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Additionally, the test is very efficient and sensitive to detect a wide variety of pollutants in environmental monitoring approaches, namely heavy metals (Nielsen and Rank, 1994; Matsumoto et al., 2006), domestic and industrial sewage (Mitteregger et al., 2007), and water samples from rivers and lakes, which include the composition of complex mixtures of different substances (Smaka-Kincl et al., 1996). In turn, the Chinese hamster V79 cell line has been used as a test system to evaluate genotoxicity of environmental samples in several studies, as demonstrated in Terra et al. (2008) and Silva et al. (2002). In this scenario, the main objective of this study was to evaluate the genotoxic potential of the different pollution sources flowing into the Sinos River. Water samples were collected at two moments (winter 2006 and spring 2006), when an unusually high fish mortality was observed in some sites. Water samples were collected from five sites of Sinos River and evaluated using in vitro V79 Chinese hamster lung fibroblasts (cytotoxicity, comet assay and micronucleus test) and A. cepa test (toxicity and micronucleus test). 2. Materials and methods 2.1. Sampling sites Surface water samples were collected at five sites along the Sinos River and named according to their distance from the river mouth (Fig. 1). Table 1 describes each collection site. Samples were collected at two times, in June 2006 (T1) and October 2006 (T2), after a large-scale fish mortality event. The surface water samples were transported to the laboratory under refrigeration and stored at 4 °C for no longer than 4 d (APHA, 1998). Water samples were sterilized using 0.22-mm a cellulose

membrane filter for in vitro tests. For the A. cepa test samples were used in their original concentration. 2.2. V79 cell line 2.2.1. Culture and treatment Chinese hamster lung fibroblast cells (V79 cells) were cultured under standard conditions in MEM medium supplemented with 10% heat-inactivated FBS, 0.2 mg mL1 L-glutamine, 100 IU mL1 penicillin, and 100 mg mL1 streptomycin. Cells were kept in tissue-culture flasks at 37 °C and a 5% CO2 atmosphere, and were harvested by treatment with 0.15% trypsin-0.08% EDTA in PBS (Boeira et al., 2001). Cells (5  105 cells) were seeded in complete media and grown for 1 d prior to treatment with the river water samples for the comet assay (3 h) and micronucleus assay (24 h). To evaluate cell survival, 200 cells were grown overnight before treatment (24 h) with the water samples. 2.2.2. Cytotoxicity After treatment, cells were washed and incubated in complete medium at 37 °C and 5% CO2 for 7 d. Colonies were fixed with methanol and acetic acid (3:1), stained with 1% crystal violet and counted. Colony survival was expressed as a percentage of the negative control (200 lL of sterile distilled water). The concentration was considered cytotoxic when cell survival was <70% (Boeira et al., 2001; Cardozo et al., 2006). 2.2.3. Comet assay After a 3-h treatment with samples only from October 2006 (T2), cells were washed with ice-cold PBS, trypsinized, and resuspended in complete medium. Then, cell suspensions were dissolved in 0.75% low-melting point agarose, and immediately

Fig. 1. Location of the sampling sites at Sinos River Basin, Rio Grande do Sul, Brazil.

E.A. Nunes et al. / Chemosphere 84 (2011) 47–53

49

Table 1 Sampling sites and characterization. Code Si008 Si028 Si036 Si038 Si048 Si121

Coordinate 0

00

S29°52 36 S29°470 5300 S29°460 3400 S29°450 5000 S29°44’2100 S29°340 5300

0

00

W51°11 24 W51°110 2400 W51°110 3900 W51°100 3600 W51°070 2200 W50°280 0300

Location

Major contributions

Bridge Tabaí-Canoas; Canoas City Passo da Carioca; Sapucaia do Sul City Mouth of the Portão stream; Portão City João Correa waterway; São Leopoldo City Mouth of the Luis Rau; stream of Novo Hamburgo City Rolante river fountain; Rolante City

Domestic sewage, oil refinery Domestic sewage, Leather, agriculture, paper recycling industry Domestic sewage, Tannery (over 40), Dredging of sand Domestic sewage, metallurgy, dredging of sand Sewage and footwear companies, leather finish Local reference, without anthropogenic contributions

spread onto a glass microscope slide pre-coated with a layer of 1% normal melting point agarose. Slides were then incubated in ice-cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, and 10% DMSO, pH 10.0) at 4 °C for at least 1 h. For the alkaline comet assay, comet slides were placed in a horizontal electrophoresis box containing freshly-prepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 13.0) at 4 °C for 20 min in order to facilitate DNA unwinding. A 300-mA and 25-V (0.9 V cm1) electric current was applied for 20 min to perform DNA electrophoresis (Silva et al., 2002; Collins, 2004). Slides were then neutralized (0.4 M Tris, pH 7.5) and stained using ethidium bromide 0.002%. For the analysis, images of 100 randomly selected cells (50 cells from each of two replicate slides) were observed per experimental culture. Cells were also visually scored into five classes, according to tail length: (1) class 0: undamaged, without a tail; (2) class 1: with tail shorter than the diameter of the head (nucleus); (3) class 2: with tail 1–2 times longer than the diameter of the head; (4) class 3: with a tail 2 times longer than the diameter of the head; (5) class 4: significant damage, with a long tail, measuring more than 3 times the diameter of the head. A value (damage index, DI) was assigned to each comet according to its class (Da Silva et al., 2000). Damage index ranged from 0 (completely undamaged: 100 cells  0) to 400 (with maximum damage: 100 cells  4). Damage frequency (DF) is the proportion of cells presenting tails after electrophoresis, and this was also considered in our analyses. Results are expressed as means and standard deviations. Bleomycin 2 lg mL1 (Blenoxane, Bristol) and sterile tridistilled water (200 lL) was used as positive and negative controls, respectively. 2.2.4. Micronucleus test To analyze micronuclei (MN), V79 cells were inoculated in 25cm2 culture flasks at a density of 5  104/flask with 5 mL of MEM medium and 200 lL of water samples, for 24 h at 37 °C and 5% of CO2. The negative control was sterile distilled water (200 lL). Bleomycin (Blenoxane from Bristol, 2 lg mL1) was used as positive control. The cultures were performed in duplicate and in parallel. At the end of the treatment, the cultures were washed twice with PBS and trypsinized. Cultures were then centrifuged, treated with hypotonic solution (1% sodium citrate) and fixed in methanol: acetic acid (3:1). Slides were stained with Giemsa and the MN analysis followed the criteria used by Fenech (1993). Two thousand cells were analyzed for each sample and controls. 2.3. A. cepa test The A. cepa test was performed as described by Fiskesjö (1993) with minor modifications (Mitteregger et al., 2007). Bulbs were transferred to 10 different test tubes and watered with cultivation water. Small, uniformly sized bulbs from a population of the common onion (A. cepa L.) of one same variety were chosen for the experiments. They were always purchased from one same farmer, who did not use pesticides. The old roots of onion bulbs were gently removed using a razor blade and onions were then placed directly in flasks containing different water samples and

the controls (10 onions per water sample), and then left to germinate at 18–22 °C. The negative control was well water (Viamão/RS) and the positive control was paracetamol solution 0.5%. After 2 d, when the roots reached 1.5–2.0 cm in length, three roots from each bulb were harvested during the second mitotic cycle to analyze microscopic parameters. The roots were immediately fixed in acetic acid and ethanol (1:3; v/v) for 24 h, transferred to 70% ethyl alcohol, and stored in a refrigerator. The onions were germinated for another 5 d and afterwards the root length of the three largest roots was measured and used as an index of toxicity. Only one experiment was performed for each sample. Two fixed root tips from each bulb were rinsed in distilled water, hydrolyzed in 1 N HCl at 60 °C for 8 min and rinsed again to prepare the slides. A double staining method combining the Feulgen squash and the Fast Green reaction technique was used to stain both the nuclear contents and the contour of the cell wall. Microscopic slides were prepared by squashing the root tips in acetic acid 45% (Fiskesjö, 1993). The following microscopic parameters were observed: (a) the mitotic index (MI) (1000 cells per slide); (b) Micronuclei (MN). MN were scored under an oil immersion lens (100) and 2000 cells from each onion bulb were examined. 2.4. Physicochemical analysis Physicochemical analyses were performed by the Division of Chemical Analysis of FEPAM, a certified laboratory, according to standard methods. The following parameters were analyzed: air and water temperature, conductivity, pH, dissolved oxygen (DO), biological oxygen demand (BOD5), chemical oxygen demand (COD), ammoniacal nitrogen, organic nitrogen, total phosphates, total suspended solids, phenols, surfactants, copper, chlorides, total chromium, trivalent chromium, hexavalent chromium, aluminum, iron, mercury, and zinc. All analyses followed the methodology described in Standard Methods, 20th edition (APHA, 1998). 2.5. Statistical analysis The correlation of significance between the sites sampled and the negative control was determined by analysis of variance (ANOVA), Dunnett’s multiple comparisons of means; program Graphpad Prism, version 3.0. Micronuclei frequency in V79 cells induced by samples and controls was analyzed by the Z-test, comparing two Poisson distributions (Zar, 1996). Significance was considered to be P < 0.05. The response obtained in the cultures exposed to the samples in which there were twice as many micronuclei as compared to the parallel negative control, without reaching statistical significance was considered a sign of genotoxicity. 3. Results The exposure of samples collected in sites Si008, Si038 and Si048 on T1 inhibited the growth of V79 cells, which remained as less than 70% of the number of colonies observed in the negative control (Fig. 2). However, the treatment of these cells with samples collected in sites Si008 (T1) and Si038 (T2) significantly induced the

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E.A. Nunes et al. / Chemosphere 84 (2011) 47–53

Colony-forming ability (%)

120 100 80 60 40 20 0 Negative

Si008

Si028

Si036

Si038

Si048

Si121

control

T1

T2

Fig. 2. Cytotoxicity evaluated by V79 colony forming ability. The line represents the accepted toxicity limit. Two samplings: June (T1) and October (T2) 2006. Si036, T1, were not conducted.

10

Micronucleus frequency

9

a

8

a

7 6

b

5

b

4 3 2 1

T1

Si121

Si048

Si038

Si036

Si028

Si008

Negative control

Si188

Si121

Si048

Si038

Si028

Si008

Negative control

0

T2

Fig. 3. Number of micronucleus events in V79 cells in the study area. (a) P < 0.05; (b) signs of mutagenicity tendency; two samplings in June (T1) and October (T2) 2006.

increase in micronuclei, in comparison to the negative control. Apart from this, signs of mutagenicity tendency were observed in V79 cells exposed to samples collected in sites Si028 (T1) and Si036 (T2) (Fig. 3). The comet assay revealed a significant increase in index and frequency of DNA damage induced by exposure to samples collected in all sites on T2, in comparison to the negative control (P < 0.01, one-way ANOVA, Dunnett test) (Table 2). The mean values of the parameters analyzed in the A. cepa test (mitotic index, presence of micronuclei and root growth) measured

Table 2 Damage and Frequency index (mean ± standard deviation) of water samples from October 2006 (T2).

** a b

T2

Damage index

Frequency damage

Negative controla Si008 Si028 Si036 Si038 Positive controlb

31.17 ± 8.52 116.71 ± 16.44** 115.67 ± 8.52** 160.17 ± 37.13** 161.17 ± 14.37** 206.50 ± 18.33**

20.17 ± 3.75 66.17 ± 9.92** 63.50 ± 8.79** 75.33 ± 11.15** 77.33 ± 10.26** 86.83 ± 23.58**

Significant at P < 0.01 in relation to negative control; Dunnett’s-test. Tridistilled sterile water. Bleomycin (Blenoxane from Bristol – 2 lg mL1).

in samples collected in different sites of the Sinos River at two sampling times are shown in Table 3. No increase in micronuclei frequency was observed in the test, for any sample collected at the two collection times (Table 3). In the A. cepa test, cytotoxicity is measured by the occurrence of changes in mitotic index. Except for the sample collected in site Si121 on T1, all samples collected in the Sinos River were cytotoxic in the A. cepa test. The measurement of root length, which evaluates cytotoxicity, all samples collected inhibited the growth of the plant’s structure, in comparison to the negative control, once again except for the sample collected in site Si121, on T1. Table 4 shows the physicochemical parameters measured for all collection sites. Also for the same site (Si121), no alterations in physicochemical parameters was observed as established in CONAMA resolution 357/05 (Table 4). 4. Discussion The area studied in the present study represents one of the regions of the state of Rio Grande do Sul with the most concentrated urban occupation and most intense level of industrial activity in the state (metallurgy, shoe manufacturing, chemical industries, among others). Due to the region’s high exposure to toxic pollutants and widespread use of its waters for human consumption,

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E.A. Nunes et al. / Chemosphere 84 (2011) 47–53 Table 3 Micronuclei, mitotic index and root length (mean ± standard deviation) of Allium cepa exposed to water from different collecting sites. T1 and T2 means two different sampling times (June and October, 2006, respectively).

* **

Site

n

Micronuclei cells (MN/2000)

Mitotic index (dividing cells/2000)

Length of roots (cm and %)

T1 Negative control Si008 Si028 Si036 Si038 Si048 Si121 Positive control

10 10 10 10 10 10 10 10

0.7 ± 1.06 0.3 ± 0.67 0.9 ± 1.10 0.4 ± 0.70 1.3 ± 1.25 0.6 ± 0.84 0.8 ± 1.03 9.4 ± 3.78*

43.0 ± 10.74 3.2 ± 5.99* 5.5 ± 4.09* 1.2 ± 1.47* 1.2 ± 1.23* 1.1 ± 1.37* 31.1 ± 11.97 1.4 ± 1.65*

11.40 ± 0.65 (100%) 7.53 ± 0.76* (66%) 7.78 ± 0.43* (68%) 5.98 ± 0.57* (52%) 5.20 ± 0.49* (46%) 6.06 ± 0.76* (53%) 11.03 ± 0.46 (97%) 0.8 ± 0.25** (7%)

T2 Negative control Si008 Si028 Si036 Si038 Si048 Si121 Positive control

10 10 10 10 10 10 10 10

0.4 ± 0.69 0.0 ± 0.00 0.6 ± 1.07 1.0 ± 1.25 0.5 ± 0.71 0.3 ± 0.67 0.5 ± 0.71 8.6 ± 1.96*

41.5 ± 5.60 1.3 ± 1.49* 1.9 ± 1.66* 0.7 ± 0.82* 0.4 ± 0.70* 0.7 ± 1.06* 12.0 ± 5.44* 1.1 ± 1.29*

11.28 ± 0.56 (100%) 6.00 ± 0.64* (53%) 6.14 ± 0.63* (54%) 4.90 ± 0.41* (43%) 5.04 ± 0.61* (44%) 5.48 ± 0.50* (49%) 9.02 ± 0.59* (80%) 0.93 ± 0.19** (8%)

P < 0.05. Significant at P < 0.01 in relation to negative control; Dunnett’s-test. Negative control = water; Positive control = paracetamol.

Table 4 Physicochemical analysis of samples collected in different sites of the study area. Parameter

Air temperature Water temperature Conductivity pH Dissolved oxygen (mg L1) BOD5a (mg L1) CODb (mg L1) Ammoniacal nitrogen (mg L1) Organic nitrogen (mg L1) Total phosphates (mg L1) Total solids (mg L1) Phenols (mg L1) Surfactants (mg L1) Copper (mg L1) Chlorides (mg L1) Total chromium (mg L1) Trivalent chromium (mg L1) Hexavalent chromium (mg L1) Aluminum (mg L1) Iron (mg L1) Mercury (mg L1) Zinc (mg L1)

Si008

Si028

Si036

Si038

Si048

Si121

T1

T2

T1

T2

T1

T2

T1

T2

T1

T2

T1

T2

26 17.2 112.5 6.8 3.2* 2 22 1.15 1.38 0.21* NR ND 0.14 0.009 NR 0.041 NR NR NR NR NR 0.025

25 23.4 109.2 7.3 3.3* 3 16 1.54 1.85 0.29* 81 NR NR 0.010* 7.0 0.032 0.032 ND 1.86* 2.19* ND 0.034

25 16.8 129.8 6.9 2.0* 3 22 1.28 1.20 0.20* 164 ND 0.23 0.009 18.0 0.010 NR NR NR NR NR 0.031

34 24.4 156.2 7.1 0.3* 3 20 2.33 2.71 0.26* 123 NR ND ND 12.2 0.030 0.030 ND 0.52* 1.47* ND ND

25 16.5 113.5 7.0 0.2* 3 85 14.3* 5.00 0.40* 637* 0.035* 0.56 0.008 130.0 0.105* NR NR NR NR NR 0.102

26 26.4 116.2 6.5 2.2* 3 15 2.19 2.14 0.34* 101 NR NR 0.008 7.4 0.034 0.034 ND 4.78* 2.66* ND NR

25 18.1 92.1 7.1 1.4* 2 18 0.81 0.60 1.17* 110 ND 0.12 0.010* 8.5 ND NR NR NR NR NR 0.023

34 23.2 145.2 6.7 2.0* 19* 2 1.91 0.30 0.30* 100 NR NR 0.006 11.0 0.033 0.033 ND 1.29* 3.19* ND ND

24 18.3 482.0 7.0 0.5* 17* 89 10.9* 5.30 1.46* 291 0.008* 2.52 0.150* 39.0 ND NR NR NR NR NR 0.184*

30 23.4 471.0 6.7 0.5* 8* 85 12.3* 6.9 1.04* 300 0.008* 1.10 ND 26.0 0.058* NR NR NR NR NR 0.310*

17 15.0 45.8 6.8 7.1 <1 5 0.08 0.50 0.02 44 NR 2.40 0.006 3.0 0.020 NR NR NR NR NR ND

30 23.1 33.9 8.2 5.7 <1 7 0.09 0.50 0.02 47 NR NR ND 1.9 0.006 NR NR NR NR NR ND

ND: not determined. ND = not detected. NR = not realized. * Values at odds with the CONAMA resolution n.357/05. a BOD: biochemical oxygen demand. b COD: chemical oxygen demand.

industries, irrigation, recreation, fishing and navigation, this river basin has been the object of environmental monitoring (Vargas et al., 1993, 1995; Lemos et al., 1994; Migliavacca et al., 2005; Villela et al., 2007; Terra et al., 2008). In the present study, the two test systems utilized (V79 cell line and A. cepa) reveal that the samples collected at the different sites in Sinos River exert toxic and cytotoxic effects, apart from genotoxic and mutagenic action. The samples collected in sites Si008, Si038 and Si048 were cytotoxic to V79 cell line and toxic to A. cepa. Similar results have been previously obtained for toxicity in microcrustaceans (Terra et al., 2008) using samples of water collected in the same sites as in the present study. The responses observed in the micronucleus test for V79 cell line (increase in frequency of MN in cells treated with samples

Si008 and Si038) suggest chromosome loss or break caused by substances generated by the different anthropic activities in the study area, affecting V79 cells DNA directly, since in our methodology these tests were conducted with no metabolic activation. These effects may be caused by compounds characteristically present in areas around cities, virtually exposed to complex mixtures of pollutants that may be found in the area investigated in the present study. More specifically, almost all of these sites are under the influence of domestic sewage and a vast demand of industrial waste, and one site is near of agricultural areas (FEPAM, 2010). The tanneries located near sites Si028, Si036 and Si048 could explain the presence of compounds like chromium, a well-known clastogenic agent that may induce the formation of micronuclei in several test systems, both in vitro and in vivo (Lemos et al.,

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2001). These results come as no surprise, given the strong and varied anthropic presence observed in these sites. A. cepa did not show any increase in MN frequency, maybe due to the fact that mutagenicity signs cannot be properly compared between both MN tests (V79 and A. cepa), since the mitotic index observed in A. cepa is very low, and therefore, it can be speculated that MN formation would be diminished. The high DNA damage indexes and frequencies observed in the present study using the comet assay are expected, in the light of the poor hydrological quality as observed in the measurements of the physicochemical parameters assessed. Some metals like aluminum, zinc, iron and chromium have been proved to induce genotoxicity (Godet et al., 1996). Although all samples induced genotoxicity in V79 cells, only those samples collected in sites Si008 and Si038 induced also mutagenicity in these cells. This difference may be attributed to the distinctive nature of the two assays, since both, in addition to genotoxicity, can reflect different forms of environmental stress. While the MN test detects nonrepairable damages, such as clastogenic and aneugenic lesions, the comet assay detects recent lesions that can be repaired, such as breaks and alkali-labile sites (Silva et al., 2002; Matsumoto et al., 2006; Villela et al., 2007). Metal is a kind of agent that causes damage that is quickly repaired and it is detected positive in the comet assay and negative in the MN test, in different studies (Andrade et al., 2004; Villela et al., 2007). Therefore, it is possible to suggest that the cells damages caused by agents present in samples are being repaired. Although the A. cepa test did not show mutagenicity in the sites studied, the analysis of root growth and mitotic index had revealed toxicity in all Sinos River sites. Therefore, the inhibition of root growth in fact is a measure of the inhibition of cell division, measured as a decrease in mitotic index (Liu et al., 1992; Marcano et al., 2004). In this sense, it has been reported that values below 22% of the negative control value may be lethal to the test organism (Antonsiewicz, 1990). Palácio et al. (2005) investigated the sensitivity of A. cepa to specific sulphonated heavy metals, like copper and zinc, in Toledo River, and concluded that these metals were responsible for the toxicity observed. Concerning total chromium, values above those described in the CONAMA 357 resolution were observed in sites Si036 and Si048, where tanneries and shoe factories represent the main pollution sources. The samples collected in these sites were genotoxic for V79 cells, apart from the toxicity and cytotoxicity for A. cepa. Toxicity but not mutagenicity was observed in A. cepa also by Mitteregger et al. (2007), in a study that analyzed samples collected in a stream that is an affluent of Sinos River and polluted by tanneries. Other authors have also detected toxicity instead of mutagenicity in the yeast test (microscreen) and for Salmonella microsome in almost all sites of the Sinos River, relating to the organic components and metals, demonstrating the complexity of polluting agents in that river basin (Vargas et al., 2001; Lemos et al., 2009). In a study with amphibians, Ralph and Petras (1997) also demonstrated the association between toxicity and influence of different industries. Apart from the industrial waste, formed mainly by organic compounds, solids and grease, chromium, sodium, sulfides, sulfates, chlorides, nitrogen and other metals, the Sinos River also receives polluting loads from untreated domestic sewage of the various municipalities in the region, which have been reported as a source of toxic and mutagenic effects to the environment (White and Rasmussen, 1998). As a whole, no difference was observed between collections (T1 and T2), which demonstrates that the effect of mortality of fish observed in Sinos River shortly time before the collection made on T2 did not present any definitive correlation with any physicochemical parameter evaluated. However, it should be noted that, as a whole, dissolved oxygen values were quite low

in 2006, in comparison to the reference values defined by CONAMA 357 (2005). The values observed were below 4.0 mg L1. Therefore, dissolved oxygen is a very important parameter in the classification of natural waters, as well as the definition of water quality indices (WQI). The WQI adopted was defined by the National Sanitation Foundation and adapted by the authorities of the state of Rio Grande do Sul in 1990 (FEPAM, 2010), excluding the parameter temperature and replacing nitrate content for ammonium nitrogen content. The following parameters are considered in the calculation of WQI: dissolved oxygen, fecal coliforms, BOD, pH, ammonium nitrogen, total phosphate, turbidity, and total solids. The measurements of WQI data for the Sinos River made in 2006 reveal that water quality was very poor for sites Si036 and Si048, poor for sites Si028 and Si038, and regular for site Si008, which as a whole may be observed in physicochemical parameters. The complex distribution of toxic agents in the Sinos River basin, with more critically affected sites observed in the cities of Portão and Novo Hamburgo, demonstrates the need for constant local monitoring based on biological and chemical diagnoses, which are essential to the implementation of cleaning and local environmental control measurements. Comet and micronucleus tests using the two test organisms (V79 and A. cepa) revealed that water samples collected exerted cytotoxic, toxic, genotoxic and mutagenic effects. The results showed the toxic action of organic and inorganic agents found in the water samples in all sites of Sinos River, for both data collections. The main causes for pollution were the toxic discharges of domestic and industrial waste. Although chemical analysis showed increased values for certain environmental contaminants, results did not show any direct correlation between genotoxic and cytotoxic data and specific contaminants detected by the chemical analysis. The damage observed may be due to complex mixtures formed by de river water and pollutants. The impact of anthropogenic influence and of the excessive and wide-ranging utilization of the hydrological resources has become the subject of discussions and concerns as to the water quality in rivers and effluents. The isolation and identification of all the pollutants present natural waters are difficult tasks, an obstacle that shows the importance of using bioindicators instead of complex analytical techniques to evaluate the quality of polluted water resources. References

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