A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters

Aquatic Toxicology 77 (2006) 1–10

A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters Claudia Pellacani, Annamaria Buschini, Mariangela Furlini, Paola Poli ∗ , Carlo Rossi Dipartimento di Genetica Antropologia Evoluzione, Universit`a di Parma, Parco Area delle Scienze 11A, 43100 Parma, Italy Received 21 July 2005; received in revised form 27 September 2005; accepted 21 October 2005

Abstract Since the 1980s, stricter water quality regulations have been promulgated in many countries throughout the world. We discuss the application of a battery of both in vivo and in vitro genotoxicity tests on lake water as a tool for a more complete assessment of surface water quality. The lake water concentrated by adsorption on C18 silica cartridges were used for the following in vitro biological assays: gene conversion, point mutation, mitochondrial DNA mutability assays on the diploid Saccharomices cerevisiae D7 strain, with or without endogenous P450 complex induction; DNA damage on fresh human leukocytes by the comet. Toxicity testing on yeast and human cells was also performed. In vivo genotoxicity was determined by the comet assay on two well-established bio-indicator organisms of water quality (Cyprinus carpio erythrocytes and Dreissena polymorpha haemocytes) exposed in situ. The in vivo experiments and the water samplings were carried out during different campaigns to detect seasonal variations of both the water contents and physiological state of the animals. Temperature and oxygen level seasonal variations and different pollutant contents in the lake water appeared to affect the DNA migration in carp and zebra mussel cells. Seasonal variability of lake water quality was also evident in the in vitro genotoxicity and cytotoxicity tests, with regards to water pollutant quantity and quality (direct-acting compounds or indirect-acting compounds on yeast cells). However, the measured biological effects did not appear clearly related to the physical–chemical characteristics of lake waters. Therefore, together with the conventional chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs: their use could permit the quantification of mutagenic hazard in surface waters. © 2005 Elsevier B.V. All rights reserved. Keywords: Comet assay; Carp; Zebra mussel; Leukocytes; Saccharomyces cerevisiae

1. Introduction Detection of potentially hazardous compounds in surface and ground waters is a complex task since many different sources (industrial drainages, agricultural irrigations and urban wastes) can contribute to water pollution. An increasing demand for cleaner rivers and lakes, groundwater and coastal beaches is evident. The 2000/60/EC Directive of the European Parliament, establishing a framework for Community action in the field of water policy, provides for progressively reducing the emission of hazardous substances into all ground and surface waters within the European Community (EC) and ensures stable, long-term planning of protective measures. Surface water health is commonly monitored by physical–chemical methods to detect the

∗

Corresponding author. Tel.: +39 0521 905608; fax: +39 0521 905604. E-mail address: [email protected] (P. Poli).

0166-445X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2005.10.010

presence of hazardous substances or priority pollutants, such as those from the 2000/60/EC European Water Framework Directive and the Convention for the Protection of the Marine Environment of the North-East Atlantic (Yearbook of International Co-operation on Environment and Development, 2003–2004) and the list of persistent organic pollutants drafted by the Stockholm Convention (Yearbook of International Co-operation on Environment and Development, 2003–2004). The pollutants on these lists have been selected because of their high toxicity to the environment and to aquatic organisms. The first step in monitoring water quality is to measure physical–chemical parameters. However, attention and alarm thresholds of these parameters only concern the toxic effects of the polluting substances studied and do not take into consideration the question of chronic exposure at low doses of noxious chemicals, frequently present in complex mixtures. On the other hand, biological monitoring assays could effectively define health risks for the environment and the man (Ohe et al., 2004).

2

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

In this study, we propose a methodological approach for better defining water quality and possibly directing efforts towards specific treatments for environmental retraining. A battery of in vitro and in vivo tests was set up to evaluate the genotoxic substances present in the water of a mesotrophic limnic lake located in central Italy. In vivo tests were performed in well-established bio-indicators of water quality: the fish Cyprinus carpio, stabled in artificial basins supplied by the current lake waters, and the zebra mussel Dreissena polymorpha, a filter-feeder organism, directly sampled from the lake. C. carpio, a teleost omnivorous feeder endemic of all freshwater bodies of Italy, is widely used as a sentinel organism (Gruber and Munn, 1998; de la Torre et al., 2002; Sumathi et al., 2001; Rajaguru et al., 2003; Buschini et al., 2004). D. polymorpha is an invasive species of freshwater mussels, which have become widely distributed throughout the world. The zebra mussel could prove to be an important organism from an ecological point of view because of their high contaminant uptake (Mersch et al., 1996a,b; Roper et al., 1996), high sensitivity to genotoxicants (Fent, 1996; Mersch et al., 1996a,b; Mersch and Beauvais, 1997; de Lafontaine et al., 2000; Pavlica et al., 2000), but few or no apparent toxic effects (Fent, 1996). In order to detect possible genotoxic effects on these aquatic organisms, we measured the DNA damage on fish erythrocytes (Pandragi et al., 1995; Sumathi et al., 2001; Rajaguru et al., 2003; Buschini et al., 2004; de Andrade et al., 2004; Winter et al., 2004) and molluscs haemocytes (Pavlica et al., 2001; Buschini et al., 2003a; Bolognesi et al., 2004). DNA damage was detected by the comet assay, a well-known micro electrophoretic assay (Singh et al., 1988; Fairbairn et al., 1995). This assay has been widely applied to aquatic environment both on vertebrate and invertebrate organisms (Belpaeme et al., 1996; Nacci et al., 1996; Mitchelmore and Chipman, 1998a,b; Kleinjans and van Schooten, 2002; Akcha et al., 2003). The genotoxicity of lake water has been also tested in vitro on human leukocytes for DNA damage by the comet assay (Singh et al., 1988; Fairbairn et al., 1995) and on Saccharomyces cerevisiae D7 strain for genetic recombination and nuclear/mitochondrial mutations (Zimmermann et al., 1975; Parry and Parry, 1984; Buschini et al., 2003b; Guzzella et al., 2004). The water sampling and the in vivo experiments were performed during different campaigns to detect seasonal variations of both the water contents and physiological state of the animals. 2. Materials and methods 2.1. Chemicals Reagents for electrophoresis, normal melting point (NMA) and low melting point agarose (LMA), dimethyl sulfoxide (DMSO), ethidium bromide (EtBr), fluorescine diacetate (FDA), ethyl methane sulfonate (EMS) and general laboratory chemicals were from Sigma; media and agar for S. cerevisiae cultures were from Difco.

2.2. Monitoring of physical–chemical variables Lake water temperature, oxygen and pH were monitored once a day during each experiment using a Multiline P4 WTW probe. Chemical analyses were measured half way through each experiment. As previously reported (Monarca et al., 2004), nitrate (colorimetry, cadmium reduction method), ammonium (colorimetry, indophenol blue), phosphorus (colorimetry, ascorbic acid method), total organic carbon (TOC), biochemical oxygen demand (BOD), chemical oxygen demand (COD) and UV absorbance at 254 nm were determined according to the Standard Methods for Examination of Water and Wastewater (APHA, 1998). 2.3. In vitro experiments Genotoxicity and cytotoxicity were evaluated on extracts of lake water samples collected during the in vivo experiments (autumn, winter and summer). A further summer campaign was performed (summer* ). 2.3.1. Water concentration Water samples were filtered on a 50 ␮m inox filter and a 25 ␮m filter cloth to remove suspended solids. The samples were acidified with sulfuric acid at pH 7 and concentrated by adsorption on 10 g trifunctional silica cartridges C18 (SepPak Plus tC18 Environmental Cartridges, Waters Chromatography) according to Monarca et al. (1998) at a 30 ml/min flow rate (20 l/cartridge). Briefly, the C18 cartridges had previously been washed in sequence with ethyl acetate, dichloromethane, methanol and distilled water (40 ml per solvent). The water was sucked through a cartridge (20 l/cartridge) with a pump in a multisample concentration system (VAC ELUT SPS 24, Varian, Leini, Italy). The elution of the adsorbed cartridges was made using ethyl acetate, dichloromethane and methanol (40 ml per solvent). The eluates were reduced to a small volume by means of a rotating vacuum evaporator, dried under nitrogen flow, redissolved in DMSO (25 equivalent l/ml). The concentrates containing non-volatile organics were used for the in vitro assays (genotoxicity and cytotoxicity on S. cerevisiae D7 strain; comet assay and cytotoxicity in fresh human leukocytes). 2.3.2. S. cerevisiae Gene conversion, point mutation and mitochondrial DNA mutability assays were conducted using S. cerevisiae diploid D7 strain (Zimmermann et al., 1975) to determine the reversion frequencies at the ilvl-92 locus and the frequencies of mitotic gene conversion at the trp5 locus with or without activation. As an alternative to the assay with exogenous S9 activation, yeast cells were harvested during the logarithmic phase of growth in YEP 20% glucose (yeast extract and bacto peptone from Difco). In these conditions, the maximum cellular expression of P450 complex, i.e. endogenous metabolic activation was induced (Poli et al., 1992; Rossi et al., 1995); however, P450 activity was not detectable in cells collected during stationary growth phase. Both with and without endogenous metabolic activation, the cells (108 cells/ml) were inoculated in phosphate buffer 0.1 M,

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

pH 7.4, in the presence of different concentrations of testing samples (Guzzella et al., 2004), and kept in an alternating shaker (110 rpm) at 37 ◦ C for 2 h. The yeast cells were then plated on solid (agar from Difco) selective mineral medium to detect gene conversion and mutant reversion frequencies, respectively (Zimmermann et al., 1975). DMSO (50 ␮l/ml) was used as a negative control; 2-aminofluorene (2AF, 50 ␮g/ml) and EMS (100 ␮M) were used as positive controls when P450 was or was not induced, respectively. Mitochondrial DNA mutation induction was evaluated by determining the frequency of petite (respiratory deficient, RD) colonies in the D7 strain. The cells with or without metabolic activation were inoculated (108 cells/ml) in phosphate buffer 0.1 M, pH 7.4, at different sample concentrations, kept in an alternating shaker (110 rpm) at 37 ◦ C for 2 h and then plated on a solid complete medium with glucose as the sole carbon source. To detect petite mutants, the plates were overlaid with soft agar containing 2,3,5-triphenyl-tetrazolium chloride (Sigma) after 5 days of incubation (Ogur et al., 1957). After about 1 h, the respiratory proficient colonies turned red, whereas the respiratory deficient colonies remained white. DMSO (50 ␮l/ml) was used as a negative control; ethidium bromide (1 ␮g/ml) was used as positive control both when P450 was and was not induced. The data (three independent repeats for each dose) were considered positive if a significant dose–response relationship (p < 0.05, linear regression analysis) was found. S. cerevisiae diploid D7 strain was also used for toxicity evaluation by measuring the viability of the strain in the mutagenicity assays: the yeast cells, treated as previously described, were plated on a solid complete medium (2% glucose) to determine survival titre. 2.3.3. Leukocytes A whole blood sample of one healthy volunteer was centrifuged twice in an erylyse solution (155 mM NH4 Cl, 5 mM KHCO3 , 0.005 mM Na2 EDTA, pH 7.4) for leukocyte isolation, and then washed and resuspended at ∼106 cell/ml in phosphate buffered saline (PBS). The cell suspension was added to a microcentrifuge tube together with a water extract to produce the following doses: 0.25, 0.50, 0.75, 1.00 and 1.25 l equivalents per milliliter (leq/ml). The extract treatments, along with negative (DMSO, 50 ␮l/ml) and positive (ethyl methanesulfonate, 2 mM) controls (Maffei et al., 2005), were incubated for 1 h at 37 ◦ C. Following incubation, cell viability was determined using the fluorescein diacetate/ethidium bromide assay (FDA/EB-assay) (Merk and Speit, 1999), and the comet assay was performed only on cells having a viability >70%, as recommended by the International Workshop on Genotoxicity Test Procedures (Tice et al., 2000). 2.4. In vivo experiments The study, consisting of 20-day long experiments, was carried out in different seasons (autumn, winter and summer) in order to assess the basal DNA damage of two aquatic organisms (molluscs and fishes) in relation to different physical and chemical lake water conditions.

3

2.4.1. D. polymorpha The study was carried out in three seasonal periods (autumn, winter and summer) on zebra mussels (30 specimens/ time/campaign, 2.8 ± 0.2 cm in length) directly collected within the lake population both at the start (time 0) and the end (time 20 days) of the study. As previously reported (Buschini et al., 2003a; Bolognesi et al., 2004), mussel haemolymph was gently aspirated from the posterior adductor muscle sinus with a hypodermic syringe containing 20 ␮l of phosphate buffered saline. The volume of haemolymph recovered per mussel was 10 ␮l with a final cell concentration of about 107 cell/ml. Haemocytes were immediately used for DNA damage assessment by the comet assay. 2.4.2. C. carpio In ecotoxicological studies, it is important to assess the toxic response of indigenous fauna as indicator species or sentinels of environmental contamination. However, field studies examining indigenous aquatic organisms can be hampered by the mobility of the sentinel organisms or by the absence of suitable indigenous animals. Transplantation method, i.e. using an in situ cage study, can present some advantages such as reduction of inter-individual variability (life history, genetic background and developmental stages) and control of geographical and temporal conditions of exposure. In this study, young specimens of C. carpio (age: less than 1 year; weight: 20–30 g), supplied by the “Centro Ittiogenico di Sant’Arcangelo” (Perugia, Italy), were maintained (30 fishes/campaign) in a 1 m3 stainless steel basin that received water flowing from the lake for 10 days before the start of experimentation to allow acclimatization. Erythrocyte samplings were performed at the end of the acclimatization period (time 0) and after a further 20 days of exposure in the lake water. As previously reported (Buschini et al., 2004), blood samples were taken through intracardiac puncture with heparinized syringes from each fish anaesthetized with 0.1 g/l of MS-222 Finquel (SCUBLA S.n.c); after 5 recovery in filtered lake water, the fishes were replaced in their basin. After dilution (about 107 cells/ml) in 0.1 M phosphate buffer containing 0.2% citric acid, 0.1 M NaCl, 1 mM EDTA, pH 7.8, the cells were started off immediately on comet assay for DNA damage assessment. The study was carried out in the same seasonal periods of zebra mussel experiments (autumn, winter and summer). 2.5. Single cell gel electrophoresis assay Single cell gel electrophoresis (SCGE) was basically performed according to Singh et al. (1988). Directly withdrawn cells (fish erythrocytes or mollusc haemocytes, about 105 cells) or treated leukocytes (about 105 cells) were added to 80 ␮l of 0.65% low melting agarose (LMA) in PBS and then transferred onto degreased microscope slides previously dipped in 1% normal melting agarose for the first layer. The agarose was allowed to set for 5 min at 4 ◦ C before adding a final layer of LMA. After agarose solidification, the slides were placed in a lysing solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, 1% Triton

4

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

X-100 and 10% DMSO, pH 10) in a Coplin jar at 4 ◦ C over-night in the dark. Alkaline DNA unwinding was carried out in a gel electrophoresis chamber containing a freshly prepared buffer (1 mM Na2 EDTA, 300 mM NaOH, pH > 13) and electrophoresis was performed in the same buffer at 0.78 V cm−1 and 300 mA. The times of DNA unwinding and electrophoresis were chosen in relation to the cell type: 10 and 20 for fish erythrocytes (Buschini et al., 2004), 5 and 10 for mollusc haemocytes (Buschini et al., 2003a; Bolognesi et al., 2004), and 20 and 20 for human leukocytes, respectively. DNA unwinding and electrophoresis were performed in an ice–water bath. Once the electrophoresis had been carried out, the slides were washed in a neutralisation buffer (0.4 M Tris–HCl, pH 7.5). After staining with 100 ␮l ethidium bromide (10 ␮l/ml), observations were made under a fluorescence microscope (Leica DMLS, 400×) equipped with an excitation filter BP 515–560 nm and a barrier filter LP 580 nm, using an image analysis system (Cometa Release® 2.1 Sarin, Florence, Italy). Fifty randomly selected cells per slide (two slides per sample) were analyzed. The samples were coded and evaluated blind. DNA damage was measured as the total length of migration (TL). In vitro tests (leukocytes) were repeated independently three times; in vivo experiments were performed on 30 specimens (fishes or molluscs) per point. A SPSS 11 package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The median values of DNA migration of each in vitro experiment, or each specimen for in vivo tests, were used for variance analysis. Data were also compared using Dunnett’s version of t-test. Specific activity, i.e. TL increase/leq of water (linear regression analysis), was calculated for the in vitro experiments. 3. Results and discussion 3.1. Physical–chemical parameters The data (Table 1) indicate a typical seasonal temperature fluctuation. According to the concentrations of nitrates and Table 1 Physico-chemical characteristics of lake water in the different seasonal sampling periods Parameters

Autumn

Winter

Summer

pH (at 20 ◦ C) Temperature (◦ C) O2 (ppm) O2 (% saturation) UV 254 nm (abs/cm) TOC (mg/l) COD (mg/l) BOD (mg/l) Nitrates (mg/l) Ammonium (mg/l) Phosphates (mg/l)

7.5 ± 0.2 18.8 ± 0.5 7.6 ± 0.3 80.4 ± 3.7 0.092 6.4 19 1.3 0.11 <0.04 0.03

7.8 ± 0.1 8.1 ± 2.2 10.5 ± 0.8 90.1 ± 2.5 0.163 9.4 35 2.1 0.16 <0.04 <0.02

7.4 ± 0.1 23.6 ± 1.2 5.6 ± 1.0 67.3 ± 10.0 0.670 5.8 17 0.9 <0.10 0.06 <0.02

TOC, total organic carbon; COD, chemical organic demand; BOD, biological organic demand.

phosphates, Lake Trasimeno can be defined mesotrophic. TOC, COD, BOD and nitrates reached their highest concentration values during winter. The UV absorbance, which is considered by Chin et al. (1994) as an index of the presence of aromatic compounds and could be related to the water content of humic/fulvic acids, was lower for the autumn and winter samples than for the summer sample. 3.2. In vitro tests We tested in vitro, together with the extracts of lake water collected at the same time as in vivo experiments (autumn, winter and summer), also a further extract (summer* ). The summer* water had some similar physical–chemical characteristics (23 ◦ C; COD: 14 mg/l; BOD: 0.8 mg/l; nitrates: <0.1 mg/l and TTHM: <0.1) to summer value (see Table 1) with higher values of TOC (8.5 mg/l versus 5.8 mg/l) and AOX (35 ␮g/l versus 8 ␮g/l) and lower values of UV 254 nm (0.071 abs/cm versus 0.670 abs/cm) and TTHMFP (350 ␮g/l versus 475 ␮g/l). 3.2.1. S. cerevisiae Cytotoxic effects were detected in cells both with or without induction of cytochrome P450 (Fig. 1). We did not find any significant cytotoxic effects in yeast cells without P450, irrespective of the samples (Fig. 1A), thus showing the lack of direct-action compounds. On the other hand, some significant effects were observed in the presence of P450 complex (Fig. 1B) in relation to the dose (p < 0.001, Fisher’s F) and the sampling period (p < 0.001, Fisher’s F) with interaction dose–response (p = 0.001, Fisher’s F). “Winter” (cell survival marginal mean: 76.9%) resulted significantly (p < 0.001) more toxic than “autumn” (84.3%), “summer* ” (88.1%, p = 0.004 respect to “autumn”, p < 0.001 respect to “summer”), and “summer” (92.9%). The first toxic dose was 0.75 leq/ml for the two summer samples (p < 0.05) and 0.25 leq/ml for autumn (p < 0.05) and winter (p = 0.001) samples. The data obtained in the mutagenicity assays in the yeast after treatment with water extracts is reported in Table 2. The yeast system allows the assessment of both direct mutagens (in stationary growth phase cells, i.e. without P450 expression) and indirect-acting compounds, which are detected in logarithmic growth phase cells expressing high levels of cytochrome P450. The data specifically indicate: • Summer* : Both direct (for nuclear revertants) and indirect (for gene conversion and RD induction) genotoxic compounds were detected. • Autumn: The quality of active pollutants differed completely from the summer* sample, i.e. direct induction of gene conversion and mitochondrial mutation and nuclear mutation only induced in P450 producing cells. • Winter: Significant effects (convertants and RDs but not revertants) were only present in metabolically active cells (i.e. presence of pro-mutagenic agents). This extract induced the highest gene conversion frequency. • Summer: The extract was able to increase significantly the frequencies of gene convertants in stationary growth phase

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

5

Fig. 1. Saccharomyces cerevisiae D7 strain: cell survival after treatment with the extracts of lake water sampled during four seasonal periods (summer* , autumn, winter and summer). Mean (±S.D.) of three independent experiments. STAT, cells collected during the stationary growth phase (i.e. without induction of P450 complex) and LOG, cells collected during logarithmic growth phase (i.e. with induction of P450 complex). Filled symbols, p < 0.05 with respect to dose 0.

Table 2 Different genetic effects, reported as frequencies of convertants at the trp5 locus (conv), revertants at the ilvl-92 locus (rev) and respiratory deficients (RD), induced in S. cerevisiae D7 strain by lake water extracts of four different sampling periods (summer* , autumn, winter and summer) and negative (DMSO) and positive controls (ethyl methanesulfonate, EMS; 2-aminofluorene, 2AF; ethidium bromide, EtBr) Dose (leq/ml)

STAT

LOG

trp5 locus (conv/106 cells)

ilvl locus (rev/107 cells)

Petite (RD/103 cells)

5.0 ± 0.7

2.3 ± 0.5

0

6.8 ± 0.6

0.25 0.50 0.75 1.00 1.25

5.1 9.1 5.6 4.3 4.7

± ± ± ± ±

0.5 0.6 0.4 0.4 0.4

5.5 6.6 7.0 9.6 10.4

± ± ± ± ±

0.3 0.4 0.5 0.6 0.9

1.6 0.9 2.3 2.5 1.2

± ± ± ± ±

0.5 0.4 0.9 1.0 0.6

19.5 20.4 23.9 24.8 29.5

± ± ± ± ±

1.5 1.5 0.9 1.7 1.7

6.1 4.8 7.7 7.7 7.5

± ± ± ± ±

0.6 0.6 0.5 0.7 0.5

3.1 3.4 3.4 3.9 5.0

± ± ± ± ±

0.5 0.6 0.4 0.8 l.l

Autumn

0.25 0.50 0.75 1.00 1.25

11.6 9.3 15.6 17.4 19.0

± ± ± ± ±

1.3 1.1 0.9 1.3 1.3

3.5 4.6 3.3 3.5 5.3

± ± ± ± ±

0.5 0.5 0.4 0.6 0.5

3.9 3.8 4.4 4.8 5.6

± ± ± ± ±

0.5 0.5 0.4 0.6 0.6

17.5 13.4 17.1 18.2 13.8

± ± ± ± ±

1.2 0.9 1.0 1.1 1.1

5.6 6.0 6.5 9.4 18.3

± ± ± ± ±

0.4 0.5 0.5 0.6 1.2

2.5 2.6 2.5 1.6 1.8

± ± ± ± ±

0.5 0.6 0.5 0.4 0.6

Winter

0.25 0.50 0.75 1.00 1.25

6.2 8.5 5.9 4.9 11.0

± ± ± ± ±

0.4 0.7 0.5 0.4 0.4

4.0 3.4 5.3 3.8 5.9

± ± ± ± ±

0.5 0.5 0.4 0.4 0.6

1.3 2.1 2.1 1.4 1.6

± ± ± ± ±

0.5 0.5 0.4 0.4 0.5

28.9 44.9 55.4 26.7 29.1

± ± ± ± ±

1.7 2.5 3.0 2.9 2.8

3.7 7.3 3.6 3.9 3.7

± ± ± ± ±

0.4 0.6 0.5 0.5 0.6

2.4 2.5 3.0 3.6 4.2

± ± ± ± ±

0.3 0.5 0.5 0.4 0.4

Summer

0.25 0.50 0.75 1.00 1.25

6.5 8.4 10.1 10.3 13.9

± ± ± ± ±

0.5 0.5 0.7 0.5 0.6

3.6 5.0 4.5 4.8 3.6

± ± ± ± ±

0.3 0.4 0.5 0.5 0.4

0.9 l.l 1.0 1.0 1.3

± ± ± ± ±

0.6 0.4 0.4 0.5 0.6

13.4 17.3 16.9 18.6 14.3

± ± ± ± ±

1.2 1.4 1.6 1.6 1.2

5.1 4.1 4.5 4.5 4.7

± ± ± ± ±

0.6 0.4 0.5 0.4 0.6

7.1 9.0 11.3 16.8 19.1

± ± ± ± ±

0.4 0.4 0.6 0.7 0.7

1659 ± 172 4.0 ± 0.7 –

– – 130 ± 15

15.2 ± 1.1

ilvl locus (rev/107 cells)

DMSO (50 ␮g/ml)

765 ± 181 7.3 ± 0.5 –

1.6 ± 0.4

trp5 locus (conv/106 cells)

Summer*

EMS (100 ␮g/ml) 2AF (5 ␮g/ml) EtBr (1 ␮g/ml)

4.3 ± 0.5

Petite (RD/103 cells)

– 286 ± 40 –

– 317 ± 50 –

– – 347 ± 33

STAT, cells collected during the stationary growth phase (i.e. without induction of P450 complex) and LOG, cells collected during logarithmic growth phase (i.e. with induction of P450 complex). Mean ± S.D. of at least three independent experiments. Significant dose–response relationships are reported in bold type (p < 0.05, linear regression analysis).

6

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

cells and mitochondrial mutants in P450-induced cells. The highest induction of respiratory deficient mutants was found in this sample. Seasonal variability in the genotoxic and cytotoxic activity of lake water was evident with regards both to water pollutant quantity and quality (direct-acting compounds or indirect-acting compounds). Cell survival appeared to be related to the seasonal sampling: the highest cytotoxicity was observed with the winter water sample. On the other hand, the various genetic endpoints we considered were able to detect other kinds of pollutants present in all the water extracts even though they changed in relation to the sampling period. 3.2.2. Human leukocytes Means of cell survivals and median TL values for fresh human leukocytes treated with extracts of lake waters sampled during the four collection periods (summer* , autumn, winter and summer) are reported in Fig. 2. Different responses were produced by the samples from each of the study periods. • Summer* : Cell toxicity with the FDA/EDB assays was not detected. On the other hand, the extract produced increases in DNA damage even if only the highest concentration (1.25 leq/ml) was able to induce a significant increase in DNA migration (p < 0.01). • Autumn: The sample induced weak, even if significant, cytotoxic effects (9% cell survival decrease/leq, R2 = 0.87). The same extract induced a significant increase in DNA migration in relation to the administered dose: DNA damage was produced by the lowest concentration of raw water extract that was tested (0.25 leq/ml; p < 0.05) with 15.5 ␮m DNA migration increase/leq (R2 = 0.89). • Winter: The water extract showed a clear dose–response relationship (43.5 ␮m DNA migration increase/leq, R2 = 0.80) with high levels of genotoxicity: the TL values produced by concentrations ≥1.00 leq/ml were comparable with those produced by 100 ␮M styrene oxide (positive control; median TL = 57.38 ± 1.69 ␮m). In addition, high levels of cytotoxicity were detected (22% cell survival decrease/leq, R2 = 0.95). • Summer: A significant increase (p < 0.05) in DNA damage with respect to the negative control (DMSO) was observed only at the highest concentration (1.25 leq/ml) with no cytotoxic effect. The cytotoxic effects of water extracts depended greatly on the collection period with summer ≈ summer* < autumn (p < 0.001) and winter as the highest (p < 0.001), and with a trend similar to that found in S. cerevisiae. The findings also suggest that the comet assay is sensitive to seasonal environmental differences in surface water quality. The data sets from the four collection periods showed major variations in the responses. The DNA migration values had a trend similar to that found for the cytotoxicity (summer ≈ summer* < autumn < winter; p < 0.001). These findings may, at least partially, be due to the different chemical compositions of the water collected at the different sampling

periods during the study. The winter water appears to contain more levels of agents able to induce both cytotoxic and genotoxic effects in human leukocytes. 3.3. In vivo test 3.3.1. D. polymorpha Some previous studies have reported that mussel tolerance to pollutants is regulated by seasonal changes (Van Benschoten et al., 1995) and by temperature (Harrington et al., 1997; Buschini et al., 2003a), since temperature-dependent metabolic and filtration rates (Quigley et al., 1993; Kilgour and Baker, 1994) can modulate exposure to toxicants. In this study, the data obtained by the comet assay on haemocytes of zebra mussels directly collected in the lake water during the three different seasonal periods (Table 3I) were not significantly different. Comparable results were obtained for all the samplings in different seasons, both at the start of observation period and 20 days after, when the total length of DNA migration in the comet assay was considered. The great interindividual variability could affect the results and mask eventual seasonal differences. The use of the ratio between the migration length and the diameter of the comet head (LDR, see Tice et al., 2000) as a parameter to represent the data of the genotoxic effects in D. polymorpha was found (Bolognesi et al., 2004) to be able to reduce the inter-individual variability of the data (from 20–30% for TL to 5–10% for LDR). When considering LDR (Table 3II), our results are partially in agreement with literature data (Quigley et al., 1993; Kilgour and Baker, 1994; Van Benschoten et al., 1995; Harrington et al., 1997; Buschini et al., 2003a). A lower DNA damage was found during the coldest period (time 0; p = 0.02, winter versus both autumn and summer). However, in winter, a significant increase (p < 0.001) of baseline DNA breakage is detected in the second sampling (20 days later). Table 3 Comet assay in vivo: DNA damage measured during three seasonal experimentations (autumn, winter and summer, 20 days) in haemocytes of mussels (Dreissena polymorpha, 30 specimens/sampling) directly collected within the lake population Autumn

Winter

Summer

I: TL (␮m) Seasonal mean 0 day 20 days

12.78 ± 3.56 12.94 ± 4.20 12.62 ± 2.85

12.01 ± 3.92 11.07 ± 3.53 15.26 ± 4.12

13.84 ± 3.29 13.94 ± 3.88 13.74 ± 2.64

II: LDR Seasonal mean 0 day 20 days

2.63 ± 0.22 2.73 ± 0.25 2.53 ± 0.19

2.40 ± 0.15 1.98 ± 0.17 2.82 ± 0.12

2.66 ± 0.14 2.60 ± 0.17 2.72 ± 0.11

I: TL, total length, median of the value distribution of 100 cells/specimen, mean ± S.D. of all the specimens considered; II: LDR, length–diameter ratio, median of the value distribution of 100 cells/specimen; mean ± S.D. of all the specimens considered. Together to the seasonal means (60 molluscs), there are reported the values detected at the beginning (0 day) and the ending (20 days) of each experimentation. Significant differences between the first (0 day) and the last (20 days) sampling are pointed out as underlined values (p < 0.001).

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

7

Fig. 2. Human leukocytes: cytotoxic and genotoxic effects induced by 1 h in vitro treatment with the extracts of lake waters sampled during four seasonal periods (summer* , autumn, winter and summer). (A) Cell survival (mean ± S.D. of three independent experiments). (B) DNA migration detected the comet assay (TL, total length, median of the value distribution of 100 cells/experiment; mean ± S.D. of three independent experiments). Filled symbols, p < 0.05 with respect to dose 0.

3.3.2. C. carpio Literature data have stated that environmental parameters and physiological factors affect DNA damage baseline. Xenobiotic metabolising enzymes are known to be strongly modulated in fish, in response to environmental conditions, such as temperature, stress, diet, reproductive activity and presence of pollutants (Huuskonen et al., 1995; Mitchelmore and Chipman, 1998a,b). Water temperature can alter the baseline of DNA damage (Anitha et al., 2000; Buschini et al., 2003a). In carp, while low environmental temperatures induce a lower RNA trasduction activity

(Goolish et al., 1984), increasing temperatures directly stimulate the protein synthesis (de la Higuera et al., 1998). Repair mechanisms could also be increased with a consequent great quantity of repair-intermediates, i.e. strand breaks. In actual fact, hyperthermia was shown to be able to modulate DNA repair processes, with a shortening of the time required for DNA repair, as assessed by the comet assay (Blasiak et al., 2003). Moreover, acclimation response, which involves the regulation of gene expression (Kinoshita et al., 2001), is considered to be markedly significant for eurythermal temperature fish, such as carp, which

Fig. 3. Comet assay in vivo: DNA damage measured during three seasonal experimentations (autumn, winter and summer, 20 days) in erythrocytes of fishes (Cyprinus carpio, 30 specimens/sampling) exposed to lake water (TL, total length, median of the value distribution of 100 cells/specimen; mean ± S.D. of all the specimens considered). (I) Seasonal mean of the fish population and (II) mean TL at the start (0 day) and the end (20 days) of each seasonal period. Significant differences among the seasonal means are indicated by different letters (p < 0.001). Significant differences between the first (0 day) and the last (20 days) sampling of each seasonal period are reported: * p < 0.05, *** p < 0.001.

8

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

can survive under a wide temperature range spanning from near 0 to over 30 ◦ C. The results that we obtained with the comet assay applied to carps, previously acclimated (10 days) after transplantation in the experimental stainless box supplemented with flowing lake water, are reported in Fig. 3 as means of each seasonal period and as values measured at the start (0 day) and end (20 days) of each experimental session. The mean seasonal DNA migration resulted significantly different for the three periods with autumn < winter < summer (p < 0.001). Furthermore, blood resampling in the same fish populations at the end (20 days) of experimental sessions shows that, in autumn, DNA migration value remained similar to time 0 value, in winter, TL value significantly increased with respect to time 0 sampling (p < 0.001), in summer, DNA migration was significantly (p < 0.05) less than time 0 value. The biological response was modulated by many factors, such as fish full acclimation and their biochemical/physiological conditions together with seasonal changes in environmental parameters. The highest seasonal mean of DNA migration detected during the summer session could suggest a relationship among DNA damage baseline, faster fish metabolism at higher temperature and low O2 concentration of lake water as stressing agent. However, the acclimation (20 days after) appeared to overcome the effects of lake water exposure. The seasonal mean of DNA damage baseline higher during the winter experimentation (the lowest temperature) than in autumn could be related to the presence of a biologically active mixture in the lake water as also shown by the high cytotoxicity and DNA damage observed in human leukocytes. The significant increase of DNA migration at the end of winter experimentation period (20 days) with respect to the start (0 day), as also found for LDR in D. polymorpha, could indicate an acclimation of animals to the worst quality of the winter water. 4. Conclusions Surface waters, such as rivers, lakes and seas, receive large quantities of wastewater from industrial, agricultural and domestic sources. These waters, which contain many unknown compounds, are used as a source of drinking water, as well as for agricultural and recreational activities. Consequently, water pollution can be a serious problem for public health and the aquatic ecosystem. Known and unknown mutagenic/genotoxic compounds become the components of complex environmental mixtures that can have adverse health effects on humans and indigenous biota. Reports on the occurrence of malignancies and other pathophysiological conditions in aquatic organisms following exposures to suspected genotoxins have increased (Depledge, 1994, 1996, 1998). It was pointed out (Depledge, 1994) that when pollutants give rise to altered metabolism as a consequence of damage to genetic material, the effects on individual organisms are likely to result in changes in Darwinian fitness, altered growth rates, reproductive output and viability of offspring, with significant implications for the long-term survival of exposed populations. It is expected that genotoxic effects may dis-

turb the ecosystems one way or another (Depledge, 1998; Jha, 1998). In this study, a battery of in vitro and in vivo tests, together to physical–chemical water analysis, were performed to better define the surface water quality. Taking the in vitro findings into consideration, it is possible to claim that the comet assay (which detects DNA damage) on human leukocytes and the S. cerevisiae test (which detects nuclear and mitochondrial mutations) are very sensitive assays for studying the water extract. The yeast test can provide some information on the cytotoxic and genotoxic load of surface water in relation to the effects on the microorganisms naturally present in surface water. The in vitro test with human cells may have a theoretical value for human risk assessment. In fact, the comet assay appears to provide useful information in terms of DNA damage on the potential genotoxicity of a complex mixture, such as surface waters that could be employed as drinking water supplies. The results obtained in the present study applying the comet assay in D. polymorpha collected during three different seasonal periods in the lake were comparable. Only relevant results may be considered as an early warning for a long-term hazard in indigenous animals. However, a significant increase of DNA damage in mussel erythrocytes was observed at the end of experimentation in the same seasonal period (winter) whose water extract resulted the most cytotoxic and genotoxic in human cells. An increased baseline of DNA damage (mean of experimental session) was found in erythrocytes of transplanted C. carpio exposed in flowing lake water in relation to higher temperature and lower O2 concentration (summer). When temperature did not affect DNA damage baseline (winter), C. carpio detected the presence of genotoxic effectors in the lake water and confirmed the data obtained in the comet assay on human leukocytes. The data obtained at the end of experimentation indicated that the length of acclimation period significantly affected the level of DNA damage in relation to both winter and summer. However, the comet assay on the two bio-indicators did not result completely suitable in monitoring of surface water genotoxicity. A disadvantage in applying the comet assay to D. polymorpha and C. carpio has been the high inter-individual variability and the difficulty to distinguish the influence of physiological and environmental fluctuation from anthropogenic stressors/pollutants. The suitability of in vitro tests (comet assay on human cells and yeast tests) to detect surface water chemicals that may produce genetic damage was confirmed, although we cannot establish a clear relationship among biological effects and water physical–chemical characteristics. The use of S. cerevisiae, eukaryotic organism, is of particular interest because it allows to also evaluate genetic endpoints (mitotic gene conversion and mitochondrial DNA mutability) not detectable by Salmonella typhimurium test. In addition, the application of the comet assay to human cells represents a further test in the evaluation of the potential risk for human populations. On the other hand, the chemical analysis, the most direct method to prove the existence of specified substances in surface waters, cannot evaluate

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

either the adverse effects of low level pollutants, moreover very difficult to determine, or possible additive, synergistic or antagonistic events. Therefore, in vitro mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs, together to the conventional chemical analysis, in order to efficiently assess the presence of genotoxins and evaluate the mutagenic hazard in surface waters. Acknowledgements Supported by the Italian Ministry of University and Research (MURST 1999). We would like to thank Gillian Mansfield (Director of the Language Centre, University of Parma) for the English revision of the manuscript. References Akcha, F., Vincent Hubert, F., Pfhol-Leszkowicz, A., 2003. Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds. Mutat. Res. 534, 21–32. Anitha, B., Chandra, N., Gopinath, P.M., Durairaj, G., 2000. Genotoxicity evaluation of heat shock in gold fish (Carassius auratus). Mutat. Res. 496, 1–8. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington DC, pp. 8/29–8/34. Belpaeme, K., Delbeke, K., Zhu, L., Kirsh-Volders, M., 1996. Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay. Mutagenesis 11, 485–492. Blasiak, J., Widera, K., Pertynski, T., 2003. Hyperthermia can differentially modulate the repair of doxorubicin-damaged DNA in normal and cancer cells. Acta Biochim. Polon. 50, 191–195. Bolognesi, C., Buschini, A., Branchi, E., Carboni, P., Furlini, M., Martino, A., Monteverde, M., Poli, P., Rossi, C., 2004. Comet and micronucleus assays in zebra mussel cells for genotoxicity assessment of surface drinking water treated with three different disinfectants. Sci. Total Environ. 333, 127–136. Buschini, A., Carboni, P., Martino, A., Poli, P., Rossi, C., 2003a. Effects of temperature on baseline and genotoxicants-induced DNA damage in haemocytes of Dreissena polymorpha. Mutat. Res. 537, 81–92. Buschini, A., Carboni, P., Furlini, M., Poli, P., Rossi, C., 2003b. Sodium hypochlorite-, chlorine dioxide- and peracetic acid-induced genotoxicity detected by the comet assay and Saccharomyces cerevisiae D7 tests. Mutagenesis 19, 157–162. Buschini, A., Martino, A., Gustavino, B., Monfrinotti, M., Poli, P., Rossi, C., Santoro, M., D¨orr, A.J.M., Rizzoni, M., 2004. Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization. Mutat. Res. 557, 119–129. Chin, Y., Aiken, G., Loughlin, E., 1994. Molecular weight, polydispersity and spectroscopic properties of aquatic humic substances. Environ. Sci. Technol. 28, 1853–1858. de Andrade, V.M., de Freitas, T.R.O., da Silva, J., 2004. Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment. Mutat. Res. 560, 57–67. de la Higuera, M., Garz´on, A., Hidalgo, M.C., Perag´on, J., Cardenete, G., Lupi´an˜ iez, J.A., 1998. Influence of temperature and dietary-protein supplementation either with free or coated lysine on the fractional proteinturnover rates in the white muscle of carp. Fish Physiol. Biochem. 18, 85–95. de la Torre, F.R., Ferrari, L., Salibian, A., 2002. Freshwater pollution marker: response of brain acetylcholinesterase activity in two fish species. Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol. 131, 271–280.

9

de Lafontaine, Y., Gagn´e, F., Blaise, C., Costan, G., Gagnon, P., Chan, H.M., 2000. Biomarkers in zebra mussels (Dreissena polymorpha) for the assessment and monitoring of water quality of the St. Lawrence River (Canada). Aquat. Toxicol. 50, 51–71. Depledge, M.H., 1996. Genetic ecotoxicology: an overview. J. Exp. Mar. Biol. Ecol. 200, 57–66. Depledge, M.H., 1994. Genotypic toxicity: implications for individuals and populations. Environ. Health Perspect. 102, 101–104. Depledge, M.H., 1998. The ecotoxicological significance of genotoxicity in marine invertebrates. Mutat. Res. 399, 109–122. Fairbairn, D.W., Olive, P.L., O’Neill, K.L., 1995. The comet assay: a comprehensive review. Mutat. Res. 339, 37–59. Fent, K., 1996. Ecotoxicology of organotin compounds. Crit. Rev. Toxicol. 26, 1–117. Goolish, E.M., Barron, M.G., Adelman, I.R., 1984. Thermo-acclimation response of nucleic and protein content of carp muscle tissue: influence of growth rate and relationship to glycine uptake by scales. Can. J. Zool. 62, 2164–2170. Gruber, S.J., Munn, M.D., 1998. Organophosphate and carbamate insecticides in agricultural waters and cholinesterase (ChE) inhibition in common carp (Cyprinus carpio). Arch. Environ. Contam. Toxicol. 35, 391–396. Guzzella, L., Monarca, S., Zani, C., Feretti, D., Zerbini, I., Buschini, A., Poli, P., Rossi, C., Richardson, S.D., 2004. In vitro potential genotoxic effects of surface drinking water treated with chlorine and alternative disinfectants. Mutat. Res. 564, 179–193. Harrington, D.K., Van Benschoten, J.E., Jensen, J.N., Lewis, D.P., Neuhauser, E.F., 1997. Combined use of heat and oxidants for controlling adult zebra mussels. Water Res. 31, 2783–2791. Huuskonen, S., R¨as¨anen, T., Koponen, K., Lindstr¨om-Sepp¨a, P., 1995. Timecourse studies of the biotrasformation enzymes in control rainbow trout when adjusting to new habitats. Marine Environ. Res. 39, 79–83. Jha, A.N., 1998. Use of aquatic invertebrates in genotoxicological studies. Mutat. Res. 399, 1–2. Kilgour, B.W., Baker, M.A., 1994. Effects of season, stock, and laboratory protocols on survival of zebra mussels (Dreissena polymorpha) in bioassays. Arch. Environ. Contam. Toxicol. 27, 29–35. Kinoshita, S., Itoi, S., Watanabe, S., 2001. cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio. Fish Physiol. Biochem. 24, 125–134. Kleinjans, J.C.S., van Schooten, F.J., 2002. Ecogenotoxicology: the evolving field. Environ. Toxicol. Pharmacol. 11, 173–179. Maffei, F., Buschini, A., Rossi, C., Poli, P., Cantelli Forti, G., Hrelia, P., 2005. Use of the comet test and micronucleus assay on human white blood cells for in vitro assessment of genotoxicity induced by different drinking water disinfection protocols. Environ. Mol. Mutagen. 46, 116–125. Merk, O., Speit, G., 1999. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. Environ. Mol. Mutagen. 33, 167–172. Mersch, J., Beauvais, M.-N., Nagel, P., 1996a. Induction of micronuclei in haemocytes and gill cells of zebra mussels Dreissena polymorpha, exposed to clastogens. Mutat. Res. 371, 47–55. Mersch, J., Wagner, P., Pihan, J.C., 1996b. Copper in indigenous and transplanted zebra mussels in relation to changing water concentration and body weight. Environ. Toxicol. Chem. 15, 886–893. Mersch, J., Beauvais, M.-N., 1997. The micronucleus assay in the zebra mussel Dreissena polymorpha, to in situ monitor genotoxicity in freshwater environments. Mutat. Res. 393, 141–149. Mitchelmore, C.L., Chipman, J.K., 1998a. Detection of DNA strand breaks in brown trout (Salmo trutta) hepatocytes and blood cells using the single cell gel electrophoresis (comet) assay. Aquat. Toxicol. 41, 161– 182. Mitchelmore, C.L., Chipman, J.K., 1998b. DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutat. Res. 399, 135–147. Monarca, S., Zanardini, A., Feretti, D., Dalmiglio, A., Falistocco, E., Manica, P., Nardi, G., 1998. Mutagenicity of extracts of lake drinking water treated with different disinfectants in bacterial and plant test. Water Res. 32, 2689–2695.

10

C. Pellacani et al. / Aquatic Toxicology 77 (2006) 1–10

Monarca, S., Zani, C., Richardson, S.D., Thruston Jr., A.D., Moretti, M., Feretti, D., Villarini, M., 2004. A new approach to evaluating the toxicity and genotoxicity of disinfected drinking water. Water Res. 38, 3809–3819. Nacci, D.E., Cayula, S., Jackim, E., 1996. Detection of DNA damage in individual cells from marine organisms using the single cell gel assay. Aquat. Toxicol. 35, 197–210. Ogur, M., St John, R., Nagai, S., 1957. Tetrazolium overlay technique for population studies of respiration deficiency in yeast. Science 125, 928–929. Ohe, T., Watanabe, T., Wakabayashi, K., 2004. Mutagens in surface waters: a review. Mutat. Res. 567, 109–149. Pandragi, R., Petras, M., Ralph, S., Vrzoc, M., 1995. Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp. Environ. Mol. Mutagen. 26, 345–356. Parry, E.M., Parry, J.M., 1984. The assay of genotoxicity of chemicals using the budding yeast Saccharomyces cerevisiae. In: Venitt, S., Parry, J.M. (Eds.), Mutagenicity Testing. A practical Approach. IRL Press Limited, Oxford, England, pp. 119–147. Pavlica, M., Klobucar, G.I.V., Moja, N., Erben, R., Papes, D., 2001. Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat. Res. 490, 209–214. Pavlica, M., Klobucar, G.I.V., Vetma, N., Erben, R., Papes, D., 2000. Detection of micronuclei in haemocytes of zebra mussel and great ramshorn snail exposed to pentachlorophenol. Mutat. Res. 465, 145–150. Poli, P., Buschini, A., Campanini, N., Vettori, M.V., Cassoni, F., Cattani, S., Rossi, C., 1992. Urban air pollution: use of different mutagenicity assays to evaluate environmental genetic hazard. Mutat. Res. 298, 113–123. Quigley, M.A., Gardner, W.S., Gordon, W.M., 1993. Metabolism of the zebra mussel (Dreissena polymorpha) in Lake St. Clair of the Great Lakes. In: Nalepa, T.F., Schloesser, D.W. (Eds.), Zebra Mussels: Biology, Impacts, and Control. Lewis Publishers, Boca Raton, pp. 295–306. Rajaguru, P., Suba, S., Palanivel, M., Kalaiselvi, K., 2003. Genotoxicity of a polluted river system measured using the alkaline comet assay on fish and earthworm tissues. Environ. Mol. Mutagen. 41, 85–91.

Roper, J.M., Cherry, D.S., Simmers, J.W., Tatem, H.E., 1996. Bioaccumulation of toxicants in the zebra mussel Dreissena polymorpha, at the Times Beach Confined Disposal facility, Buffalo, New York. Environ. Pollut. 94, 117–129. Rossi, C., Poli, P., Buschini, A., Cassoni, F., Cattani, S., deMunari, E., 1995. Comparative investigations among meteorological conditions, air chemical–physical pollutants and airborne particulate mutagenicity: a long-term study (1990–1994) from a northern Italian town. Chemosphere 30, 1829–1845. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.X., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Sumathi, M., Kalaiselvi, K., Palanivel, M., Rajaguru, P., 2001. Genotoxicity of textile dye effluent on fish (Cyprinus carpio) measured using the comet assay. Bull. Environ. Contam. Toxicol. 66, 407–414. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C, Sasaki, Y.F., 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. Van Benschoten, J.E., Jensen, J.N., Harrington, D.K., DeGirolamo, D., 1995. Zebra mussel mortality with chlorine. J. Am. Waterworks Assoc. 87, 101–108. Winter, M.J., Daya, N., Hayes, R.A., Taylor, E.W., Butler, P.J., Chipman, J.K., 2004. DNA strand breaks and adducts determined in feral and caged chub (Leuciscus cephalus) exposed to rivers exhibiting variable water quality around Birmingham, UK. Mutat. Res. 552, 163– 175. Yearbook of International Co-operation on Environment and Development, 2003–2004. From the Fridtjof Nansen Institute. Published by http:// www.earthscan.co.uk/ http://www.earthscan.co.uk/, 11th volume. Zimmermann, F.K., Kern, R., Rasenberg, H., 1975. A yeast for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation. Mutat. Res. 28, 381–388.