Environ Monit Assess (2008) 147:75–81 DOI 10.1007/s10661-007-0099-z

Genotoxicity screening of the river Rasina in Serbia using the Allium anaphase–telophase test Mladen Vujošević & Snežana Anđelković & Gojko Savić & Jelena Blagojević

Received: 31 July 2007 / Accepted: 14 November 2007 / Published online: 15 December 2007 # Springer Science + Business Media B.V. 2007

Abstract Evaluation of the presence of genotoxic substances is especially important in rivers that serve as a source of drinking water. Nine water samples collected along the river Rasina in Serbia were analyzed for potential toxic and genotoxic effects using the Allium anaphase–telophase test. Inhibition of root growth relative to the negative control (synthetic water) was observed in all samples. Analysis of the genotoxic potential, through scoring anaphase and telophase aberrations, showed that in seven of the nine samples the level of aberrations was significantly increased relative to the negative control but was lower than that obtained for the positive control (methyl methanesulfonate). Changes in the relation between spindle and chromosome types of aberrations were found in some samples, indicating differences in the potential genotoxic substances present at the analyzed sites. The data, which were obtained from samples collected at the highest level M. Vujošević (*) : J. Blagojević Department of Genetic Research, Institute for Biological Research “Siniša Stanković”, Bulevar despota Stefana 142, 11060 Belgrade, Serbia e-mail: [email protected] S. Anđelković : G. Savić Faculty of Natural Sciences and Mathematics, Department of Biology, Lole Ribara 29, Kosovska Mitrovica, Serbia

of river water, warn that during periods of low flow the values could reach genotoxic activity. The Allium anaphase–telophase test can be recommended as an monitoring system, that can serve as the first alert for the presence of genotoxic environmental pollutants. Keywords Allium test . Bioassay . Pollution . Genotoxicity . Water quality

Introduction The world’s rivers have never been under more pressure from pollution. Although water management is evolving in countries facing transition, a water crisis has not yet come into focus. Thus, rivers are often used as recipients for waste from different industries and communes. Increased use of surface water as a source of drinking water is producing growing concern about the presence of genotoxic and/ or carcinogenic substances in rivers and lakes. In addition, other important water uses, such as irrigation, livestock watering, recreation and industrial water supplies, must be further addressed in the management of water resources. Consequences of the presence of genotoxic substances in water are numerous and include possible cancer induction, acceleration of ageing processes (e.g. arteriosclerosis) and the appearance of heritable diseases in the offspring or reduction in fertility (Majer et al. 2005).

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An elevated incidence of cancer is well documented in organisms living in areas where genotoxins were detected (Silberhorn et al. 1990; Tchounwou et al. 1996; Van Noorden et al. 1997). Moreover, recent studies demonstrate a growing number of species loss in freshwater ecosystems (Ricciardi and Rasmussen 1999). Although waters are regularly analyzed almost everywhere, they do not include screening for the presence of substances suspected of producing genotoxic effects. Most waters are polluted from different scattered sources and complex mixtures are formed making it impossible to assess the hazard only from the chemical indicators of water quality (Nielsen and Rank 1994). Analysis of chemical data does not allow the prediction of biological assay results. A significant number of tests has been developed for screening for genotoxicity (Fiskesjö 1987, 1988; Grant 1999). Among those that are routinely recommended for the genotoxic evaluation of water, the Allium test occupies a prominent position (Ma 1999). This test is especially suitable because no condensation or purification of the samples is necessary. Moreover, plant bioassays are more sensitive to environmental stresses than other systems (Fiskesjö 1985a; Grant 1994) and mutagenic effects can be studied under a wide range of environmental conditions. Furthermore, plants are easy to grow and handle, while low cost is an additional advantage for using the Allium test. Since its first introduction (Levan 1938), this test has been adapted and successfully used many times for examining wastewater, natural waters from rivers and lakes, drinking water and different compounds soluble or insoluble in water (Fiskesjö 1981, 1985b; Metcalfe et al. 1985; Van der Gaag et al. 1990; Houk 1992; Rank and Nielsen 1997; Grover and Kaur 1999; Vujošević et al. 2001; Yüzbaşioğlu 2003; Monarca et al. 2003, 2005; Obute et al. 2004; Kuraś et al. 2006). The objective of this study was to screen for the presence of genotoxic substances in water from the river Rasina using the Allium anaphase–telophase test. The Rasina is a 92 km long river in south central Serbia. It drains an area of 994 km2 and belongs to the Black Sea drainage basin. After passing through the Rasina region, it flows into the river Zapadna Morava near the city of Kruševac. The lower Rasina region is densely populated and uses water from an artificial lake made in the upper

Environ Monit Assess (2008) 147:75–81

Rasina as a source of drinking water. Pollution from domestic, industrial and agricultural wastewaters is present. Different industrial objects, including two chemical plants and a tyre factory are located in the vicinity of the river banks.

Materials and methods Water samples were taken at nine sites along the river from January to March 2006 (Fig. 1) and analyzed by the procedure of Fiskesjö (1985a, 1993), as modified by Rank and Nielsen (1993) and known as the Allium anaphase–telophase genotoxicity assay. Care was taken to cover all parts of the river including unpolluted and polluted areas. Although this test operates over a wide pH range (3.5–11.0), we checked the pH value for each river sample. All values were in the range 7.1–7.8. Description of the sites where samples of water were taken (Fig. 1): Sample 1 near to the river spring, no obvious pollution exists; Sample 2 after the small town of Brus, (population about 5,000) with municipal wastewater, no industry is functioning; Sample 3 before the artificial lake (a facility providing a source of water), municipal wastewater from a not densely populated urban region; Sample 4 after the artificial lake, no contaminants; Sample 5 municipal wastewater, runoff from soil contaminated by agricultural practices, unlined open dumps of different wastes; Sample 6 waste water from the dairy industry; Sample 7 municipal wastewater from the river Gaglovska, leaching from soil contaminated by agricultural use, unlined open dumps of different wastes, tyre industry (pneumatics, industrial explosives and pyrotechnics), gasoline pump near the river; Sample 8 two chemical factories (soaps, detergents and cosmetics; plant protection products, chemicals used in ore flotation, paints for construction industry), tyre industry, gasoline pump; Sample 9 near outflow of river, dumps and railroad.

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Fig. 1 Study area location in Serbia, including sites along the river Rasina where samples were taken

The first four sites were situated in the upper flow of the Rasina and were considered as a relatively clean area. From sites 6 to 9 the Rasina flows though the industrial region of the city of Kruševac. Commercial onion bulbs of Allium cepa obtained from local companies. Onions weighing between 2 and 4 g were selected for the assay. The yellow shallows and the dry center of the primordial were removed before the onions were placed in the water samples. The test procedure measures both general toxicity and genotoxicity. For each sample, as well as the positive and negative controls, twelve onions were placed on test tubes filled with test liquids. Methyl methanesulfonate – MMS (final concentration 10 μg/l) was used as the positive control and synthetic water as the negative one. Synthetic water was made of MgSO4 (60 mg/l), CaSO4 (60 mg/l) and KCl (4 mg/l). Test solutions were changed each day with fresh one. For the first 24 h the onions were grown in freshly made synthetic water and afterwards exposed for 2 days to the samples of river water. The two onions with poorest growth were eliminated from each group after 48 h. To assess toxicity and genotoxicity, roots from each bundle were cut off on the fourth day and the length of each root, was measured to the nearest mm in all groups. For evaluating genotoxicity, roots tips were hydrolyzed in 1 N HCl at 60°C for 12 min. Five apical parts of the root tips from each onion were placed on a slide, stained with 2% orcein and squashed in 45% acetic acid. The slides were coded and examined blind.

After calculating the mitotic index, chromosome aberrations were scored on slides with a mitotic index higher than 1%. About one hundred mitoses per slide (only anaphase and telophase stages) were examined for five slides in each group. The following aberrations were scored: bridges, fragments, vagrant chromosomes, multipolarity and c-mitoses. The standard χ2-test and Two by two frequency tables were employed for statistical analysis.

Results The degree of toxicity of the analyzed samples was assessed from the mean root lengths expressed as a percentage of the mean root length of the negative control. Inhibition of growth was observed for all samples, compared to the negative control (Fig. 2). The greatest inhibition (~40%) of root growth occurred with sample 8. The relative number of total chromosome aberrations was the highest for sample 7 (17.69%) and the lowest for sample 4 (Table 1). In studied samples the most frequent single aberrations were cells with bridges (39.01%) or multipolarity (38.70%), while vagrant chromosomes were present in 9.44% of aberrant cells. Fragments and c-mitosis were found in slightly less than 0.7% of aberrant cells. Statistical analysis of aberration frequencies scored in anaphase and telophase indicated that all samples differed significantly from the positive control, but only two of them (1 and 4) did not differ significantly from the negative control (Table 2).

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Environ Monit Assess (2008) 147:75–81

90 85 Mean root length (% of negative controle)

Fig. 2 Average mean root length in analyzed samples (10 bulbs per sample) expressed as percentage of negative control (average root length in the negative control was 26.4 mm)

80 75 70 65 60 55 50 1

2

3

4

5

6

7

8

9

Samples

Comparison of the results for toxicity and genotoxicity showed that samples 2, 5, 6 and 7, which showed inhibition of growth of less than 25%, had genotoxic potential. The pollution in these samples could be prevalently of organic origin. In contrast, sample 4, which exhibited a much higher inhibition of root growth was not genotoxic. This inhibition of root growth might have come from a deficiency of microelements in the water. However, the greatest

inhibition of root growth, found in sample 8, was associated with a high genotoxic potential. Two groups of aberrations were detected in the analyses. One type is produced by spindle disturbance and it embraces vagrant chromosomes, multipolar configurations and c-mitoses, while the other type is produced by action on the chromosomes and it includes bridges and fragments. Both types of aberrations occurred at the same time in some cells. Relations

Table 1 Presence of different types of aberrations in samples of the river Rasina water Sample

BR

MP

Vch

FR

Cm

MA

Total aberrant cells

Normal cells

Aberrant cells %

NC PC 1 2 3 4 5 6 7 8 9 ∑ (1–9) (%)

16 54 22 41 26 6 34 21 45 22 35 252 (39.01)

13 17 12 33 25 14 20 48 26 26 46 250 (38.70)

2 13 5 7 6 5 9 12 4 11 2 61 (9.44)

0 10 0 0 1 0 1 0 2 0 0 4 (0.62)

0 2 0 0 0 0 0 0 1 0 2 3 (0.46)

1 35 6 12 4 10 6 3 11 15 9 76 (11.76)

32 131 45 93 62 35 70 84 89 74 94 646

514 361 476 476 482 488 465 415 414 419 460 4,095

5.86 26.62 8.64 16.34 11.39 6.69 13.08 16.83 17.69 15.01 16.96 13.63

BR bridge, MP multipolarity, Vch vagrant chromosomes, FR fragments, Cm C-mitosis, MA number of cells with multiple aberrations, NC negative control, PC positive control

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Table 2 Comparison of frequency of aberrant cells among controls (NC – negative control; PC – positive control) and river samples (two by two tables) Samples

PC

1

2

3

4

5

6

7

8

9

NC PC 1 2 3 4 5 6 7 8

84.3***

3.07 n.s. 57.04***

30.77*** 16.75*** 14.61***

10.60** 39.53*** 2.24 n.s. 5.68*

0.31 n.s. 73.64*** 1.40 n.s. 24.54*** 7.14**

16.50*** 29.86*** 5.38* 2.33 n.s. 0.72 n.s. 12.68***

31.81*** 13.98*** 15.5*** 0.05 n.s. 3.14 n.s. 25.53*** 2.86 n.s.

35.92*** 11.52*** 18.46*** 0.34 n.s. 4.71* 29.21*** 4.25* 0.13 n.s.

23.67*** 20.16*** 9.93** 0.35 n.s. 2.96 n.s. 18.33*** 0.79 n.s. 0.62 n.s. 1.31 n.s.

33.45*** 14.40*** 11.57*** 0.08 n.s. 6.89** 26.94*** 3.21 n.s. 0.00 n.s. 0.10 n.s. 0.74 n.s.

*p<0.05; **p<0.01; ***p<0.001; n.s. not significant

between the spindle and chromosome types of aberrations (Table 3) were analyzed between successive samples (Two-by-two tables) and a significant difference was observed between samples from sites 4 to 8.

Discussion The River Rasina, which was chosen for screening for the presence of genotoxic substances, is officially a river of third class quality according to Republic Hydrometeorological Service of Serbia. This classification is based on water from a station near the river outflow (sample 9). Water from the artificial lake, formed in upper part of the river, is used as a source of drinking water and for recreation activities. This river represents the average situation of water management and care in Serbia. Although water pollution legislation exists, control of industrial activities is far from strict, and no attention is paid to overuse of pesticides in agriculture. In addition, many unofficial dumps are formed on the banks of rivers. Municipal wastewater also produces additional contamination. Simultaneous use of toxicity and genotoxicity tests is necessary for adequate assessment of the risk from different contaminants in the environment (Evseeva et al. 2003). It is clear that there are several sources of water pollution that work together to reduce overall river water quality. Use of the Allium test, which includes both points, as a screening tool clearly showed the places where pollution of the river starts. The first increase in frequency of aberrations was found at site 2 after small town of Brus, demonstrat-

ing that pollution due to uncontrolled municipal activities could be as serious as pollution from agriculture or industry. The frequency then decreased due to the large quantity of water in the accumulation lake in sample 4. From that point the frequency of aberrations in the lower part of the river rose to a degree which showed clear genotoxic potential. A significant change in the ratio of different types of

Table 3 Frequency of two different types of changes in samples of the river Rasina water (χ2 values from comparison of successive sample) Sample

Changes at the level of spindle

Changes at the level of chromosomes

NC PC 1

15 (46.9%) 32 (31.68%) 17 (43.59%)

17 (53.1%) 69 (68.32%) 22 (56.41%)

2

40 (43.01%)

41 (44.09%)

3

31 (53.45%)

27 (46.55%)

4

19 (76.00%)

6 (24.00%)

5

29 (44.62%)

36 (55.38%)

6

60 (74.07%)

21 (25.93%)

7

31 (38.75%)

49 (61.25%)

8

37 (61.67%)

23 (38.33%)

9

50 (58.82%)

35 (41.18%)

χ2

0.35 0.22 3.71 7.15* 13.15* 20.44** 7.21* 0.12

*p<0.01; **p<0.001

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aberrations was found between four successive samples (from sites 4 to 8). Most of the industry in the area around the city of Kruševac is located in the region covered by these samples. In addition, pollution from agriculture practice, from unofficial dumps and other sources is also present. It is obvious that the type of pollutants changed in successive samples. Thus, in sample 5 alterations at the level of the spindle made up 44.62% of the aberrations, while in sample 6 they accounted for 74.07%. In sample 6 pollution from wastewater coming from the dairy industry is prevalent. It is well known that estrogen, otherwise present in cow milk, can produce changes connected with spindle disturbance (Metzler et al. 1998). The sample from site 7, where pollution from a wider range of sources is present, showed a similarly high proportion of chromosome type aberrations (61.25%) as the known mutagen MMS (68.32%), indicating the presence of clastogenic substances. Anyhow, it is clear that genotoxicity was not caused by single compound but reflected the presence of cumulative and/or synergistic effects. Considering that the samples of water were taken when the river was in full spate, it is obvious that during periods of low flow the frequencies will be near values obtained for known mutagen (positive control – MMS). The results obtained confirm the advantage of using the Allium test for the evaluation of the genotoxic potential of river water. This assay, when used as an monitoring system, can serve as the first alert for the presence of hazardous environmental pollutants. However, much attention should be given to interpretation of the results of toxic effects expressed simply through root growth. Thus, inhibition of growth can result from the absence of necessary nutrients in the water or the presence of insoluble compounds, which prevent the uptake of nutrients. In some cases the presence of organic pollution can even stimulate root growth. Therefore, the final decision about the biological quality of water samples must rely mainly on the results of analysis for genotoxicity. Finally, the presence of genotoxic pollutants in river water raises the question of their significance for human health. The answer to this question, as well as any further risk assessment, awaits the results of a similar biological survey of drinking water samples taken from this source and from an epidemiological survey of cancer incidence in the surrounding area.

Environ Monit Assess (2008) 147:75–81 Acknowledgments This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia, Grant no. 143011.

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