Ecotoxicology (2010) 19:77–84 DOI 10.1007/s10646-009-0390-6
Genotoxicity monitoring of freshwater environments using caged carp (Cyprinus carpio) Go¨ran I. V. Klobucˇar Æ Anamaria Sˇtambuk Æ Mirjana Pavlica Æ Mirela Sertic´ Peric´ Æ Branimir Kutuzovic´ Hackenberger Æ Ketil Hylland
Accepted: 4 July 2009 / Published online: 21 July 2009 Ó Springer Science+Business Media, LLC 2009
Abstract The present study deals with genotoxicity assessment of freshwaters using caged carp (Cyprinus carpio). Carps were transplanted from a fish-farm to three differently polluted sites in eastern Croatia. Two polluted sites were situated in the river Drava, downstream from the cities of Belisˇc´e and Osijek, while the reference site was in the Nature Park Kopacˇki rit, a preserved wetland area with limited anthropogenic influence. Exposure lasted for 3 weeks and was repeated for 3 years (2002–2004). DNA damage was assessed in erythrocytes of the exposed animals by the Comet assay and micronucleus test (MNT). In order to evaluate possible differences in stress responses to polluted water in situ and in aquaria a laboratory exposure was performed with water from the studied location in the second year of the study. Carp from the sites with high anthropogenic influence (Belisˇc´e and Osijek) had higher
G. I. V. Klobucˇar (&) A. Sˇtambuk M. Sertic´ Peric´ Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia e-mail:
[email protected] M. Pavlica Department of Molecular Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia B. Kutuzovic´ Hackenberger Department of Biology, University of Josip Juraj Strossmayer, Trg Ljudevita Gaja 6, 31000 Osijek, Croatia K. Hylland Department of Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway K. Hylland Norwegian Institute for Water Research, CIENS, Gaustadalleen 21, 0349 Oslo, Norway
average DNA damage as expressed in both the MNT and Comet assay. Of the two, the Comet assay appeared to be more sensitive following both caging and aquaria exposures. The results from this study suggest that 3 weeks caging exposure of C. carpio may be a useful strategy to monitor for genotoxic agents in freshwater ecosystems. Keywords Comet assay Micronucleus test Fish Cyprinus carpio Biomonitoring
Introduction Exposure of aquatic organisms to environmental pollution often results in genotoxic insult, either through direct genotoxicity, or through the induction of cellular stress. Bearing in mind the importance of DNA in maintaining homeostasis of all organisms and in the transfer of information to the offspring, an assessment of the integrity of DNA is important when determining pollution-related stress in living organisms. An alteration of genetic diversity patterns, a phenomenon referred to as ‘‘evolutionary toxicology’’ (Bickham and Smolen 1994), has been observed in some fish populations exposed to environmental contaminants (Murdoch and Herbert 1994; Theodorakis and Shugart 1997; Medina et al. 2007). Fish are commonly used in pollution monitoring as they are widely distributed aquatic vertebrates that bioaccumulate toxic substances and respond to low concentrations of environmental pollutants and mutagens (Al-Sabti and Metcalfe 1995; Russo et al. 2004). Fish respond to chemicals in a manner similar to that of higher vertebrates and there is an increasing interest in developing in vivo genotoxicity assays with fish as model systems (Powers 1989; Al-Sabti 1991).
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In order to obtain information on adverse genotoxic effects of environmental pollutants, marine and freshwater fish have been exposed to water from polluted areas (Belpaeme et al. 1998; Sumathi et al. 2001) or intraperitoneal injection (Pandrangi et al. 1995) under controlled laboratory conditions. Field based research has included sampling of native fish populations (Pandrangi et al. 1995; Devaux et al. 1998; Brown and Steinert 2004; Russo et al. 2004) and cage exposure in situ (Whitehead et al. 2004; Winter et al. 2004; Guilherme et al. 2008). The Comet assay is a widely established biomarker of genotoxicity. A lack of standardisation of the method is still considered an important issue (Lee and Steinert 2003; Frenzilli et al. 2009) although its adaptability can also be viewed as an advantage since the Comet assay can be tailored for different species and endpoints. The Comet assay has been applied to fish cells in vitro (Nacci et al. 1996; Devaux et al. 1997; Avishai et al. 2002) and for environmental genotoxicity monitoring using various fish species (Pandrangi et al. 1995; Flammarion et al. 2002; Frenzilli et al. 2009). The micronucleus test (MNT) in circulating erythrocytes of fish has been widely utilized for both laboratory treatments in vivo and for in situ genotoxicity surveys. For a review of piscine MNT, see Udroiu (2006). Any assessment of the pollution impact in aquatic ecosystems using indigenous fish fauna can be made more difficult by unknown migrations of the fish species under study for e.g., feeding and breeding activity. This will create an uncertainty on how well any observed effects represent the water quality at or close to the site of capture. Also, certain fish species can be absent from the particular site of interest or difficult to catch. Transplantation, i.e., in situ cage studies, offers advantages such as the precise knowledge of the place and duration of exposure, both difficult to establish in surveys of native populations (Oikari 2006; Pellacani et al. 2006). Using transplanted animals from the same non-polluted source will also potentially reduce inter-individual variability (common genetic background, life history and developmental stages) and minimizes the influence of adaptive mechanisms (Klobucˇar et al. 2003; Pellacani et al. 2006). There have not been many peer reviewed articles published over the last 30 years describing the use of fish caging as a field research technique in aquatic toxicology (see Oikari 2006). So far there have been only a few studies using transplanted (caged) fish for genotoxicity assessment of freshwater pollution (Whitehead et al. 2004; Winter et al. 2004; Barbee et al. 2008; Bony et al. 2008). Common carp (Cyprinus carpio) has been proposed as a good species for cage exposure since it can endure 4 week exposure with minimal stress (Van der Oost et al. 1998). It has also been considered as a hardy species which will
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tolerate handling (Oikari 2006). Common carp has previously been successfully used in genotoxicity assessment studies (Sumathi et al. 2001; Buschini et al. 2004; Pellacani et al. 2006). This study describes the application of two genotoxicity biomarkers (MNT and Comet assay) for transplanted carp (C. carpio) caged at two polluted sites situated in the river Drava (Belisˇc´e and Osijek) and at a reference site in the Nature Park (NP) Kopacˇki rit, in eastern Croatia over three subsequent years (2002–2004). In order to evaluate possible differences in stress response to polluted water in natural and artificial surroundings, laboratory exposure of carp in aquaria containing water from the studied locations was also conducted in 2003.
Materials and methods Study area The investigated area is situated in the eastern Croatia. The two selected polluted sites were in the Drava River, downstream from the cities of Belisˇc´e and Osijek, while the reference site was in the Nature Park Kopacˇki rit, a preserved wetland area of low anthropogenic influence (Fig. 1). The Belisˇc´e site is under pressure from municipal wastewaters (*12,000 inhabitants) and effluents from a paper and pulp mill industry. This area is also a region of intensive agriculture ([4,500 ha). Water at the Osijek site is polluted with municipal wastewaters (*130,000 inhabitants), chemical (detergent and soap, textile) and metal industries. Furthermore, autumn is a period of intensive beet processing in the Osijek sugar production factory whose effluents greatly degrade water quality due to the high concentrations of nitrogen and phosphorous. These locations are known to be polluted since they have already been used in previous ecotoxicological studies by Jaric´ and Stepic´ (2005), Zˇaja et al. (2006) and Sˇtambuk et al. (2009). Water quality assessment Water quality of the chosen polluted sites is regularly monitored by the Croatian public institution for water resources management ‘‘Hrvatske vode’’. Sampling and assessment of water quality is done in accordance with Croatian (HRN) or international standards (ISO-EN) by authorized laboratories. Data on the selected polluted sites are shown in Table 1. The values for dissolved oxygen and oxygen saturation were calculated as the 10th percentile of the measured values through the year (12–26 measurements) and for all other physicochemical and microbial parameters the values were calculated as the 90th percentile of the measured values throughout the year. For the
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79
Fig. 1 Map showing study area and location of exposure sites: 1. Kopacˇki rit—reference site, 2. Belisˇc´e, 3. Osijek
biological parameters the values were calculated as a median of two yearly samplings. Experimental design Caging exposure In order to minimize the possible influence of adaptive mechanisms of native populations and to have as much uniformity as possible in the stock of individuals, transplanted caged organisms were used from the same nonpolluted source and exposed in situ for the same period of time. Juvenile specimens of common carp C. carpio (age: \1 year; 9.7 ± 1.8 cm standard length; 18.1 ± 10.4 g wet weight) were collected from the fish-farm ‘‘Grudnjak’’ (Osijek, Croatia). Animals were transplanted to the selected monitoring sites in freshwaters of eastern Croatia (Fig. 1) and placed in mesh cages (50 cm in diameter and 120 cm height, app. 235.5 l) 1.5 m above the river bottom using a buoy-anchor system. During autumn (from September to October) of 2002, 2003, and 2004, caging exposure (*25 animals per cage, (1 fish per 9.42 l) was conducted for 3 weeks. At particular exposure sites a place with a slow current was preferred in order not to exhaust the fish by constant swimming against the current. During cage exposures, daily water temperatures at investigated sites ranged from 13 to 15.4°C in 2002, from 17 to 20.6°C in 2003, and from 12.4 to 15.4°C in 2004. Aquaria exposure During the caging exposure in 2003, a group of 15 farmreared fish was held over the same period of 3 weeks in 40 l glass aquaria (1 fish per 2.67 l) with natural
photoperiod in the facilities of the Department of Biology at J. J. Strossmayer University in Osijek. Water obtained from the studied locations was renewed every 12 h by removing 2/3 of the water from aquaria. The fish were fed daily with commercial fish food. Water temperature was 19 ± 1°C and dissolved oxygen was maintained above 60% of the saturation level by continuous aeration of the water in aquaria. Blood sampling Peripheral blood was collected from the caudal vein with heparinized syringes from each fish (6–8 specimens for Comet assay and 10–23 for MNT). After blood samples had been taken, the carp’s spinal cord was immediately severed and the fish was weighed and length measured. Blood samples were kept on ice and immediately processed for genotoxicity. The micronucleus test Smears were prepared from 10 ll heparinized blood and left to dry. Smears were fixed with 1% glutaraldehyde in phosphate-buffered saline (PBS; 145 mM NaCl, 6 mM Na2HPO4; 4 mM KH2PO4; pH 7.4) for 5 min and afterwards stained with bisbenzimide 33258 (Hoechst) at final concentration 1 lg/ml for 5 min. The slides were scored under the Zeiss Axioplan epifluorescence microscope at 10009 magnification. On each slide 2000 cells were counted. Micronuclei (MN) were identified according to criteria described by Kirsch-Volders et al. (2000, 2003). Micronuclei were defined as small round structures in the cytoplasm smaller than 1/3 of the nucleus diameter. Also, MN has to be in the same optical plane as the main
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80 Table 1 Water quality data of the chosen polluted sites Analyses
Physico-chemical analyses
Oxygen levels
Nutrients
Microbial analyses
Belisˇc´e
Measured parameter
Osijek
2002
2003
2004
2002
2003
2004 8.18
pH
8.05
8.36
8.38
8.06
8.36
Conductivity (lS/cm)
355.5
383.0
437.5
400.0
360.0
410.0
Alkalinity (mg CaCO3/l)
191.5
170.0
171.5
199.5
178
167.0
Dissolved oxygen (mg O2/l)
9.95
8.2
6.80
8.90
7.3
6.70
Chemical oxygen demand (mg O2/l)
4.55
4.7
3.90
5.75
5.45
4.55
Biological oxygen demand (mg O2/l)
6.85
7.4
4.15
7.75
6.75
5.20
Ammonical nitrogen (mg N/l) Nitrite nitrogen (mg N/l)
0.29 0.03
0.29 0.021
0.23 0.02
0.34 0.03
0.29 0.02
0.29 0.02
Nitrate nitrogen (mg N/l)
2.71
2.94
3.16
3.16
2.83
4.07
Total nitrogen (mg N/l)
3.34
3.47
3.66
3.78
3.45
4.54
Total phosphorous (mg P/l)
0.35
0.31
0.13
0.31
0.27
0.48
Total coliform count (TFC/100 ml)
1,600
863
521
4,870
24,000
88,200
Fecal coliform count (FCC/100 ml)
30
339
510
1,044
24,000
88,200
Total aerobic bacteria count (TBA/ml 22°C)
8,075
3,316
3,100
38,330
38,618
52,074
Biological analyses
P–B saprobic index
2.01
2.11
2.14
2.11
2.15
1.98
Trace metals
Cu (lg/l)
–
–
–
2.487
3.576
1.728
Cd (lg/l)
–
–
–
0.077
0.088
0.078
Cr (lg/l)
–
–
–
2.406
1.139
0.328
Pb (lg/l)
–
–
–
1.138
2.422
1.005
Hg (lg/l)
–
–
–
0.099
0.167
0.07
Mineral oils (mg/l)
–
–
–
0.071
0.009
0.034
Phenols (mg/l) DDT (lg/l)
– –
– –
– –
0.006 0
0.009 0.001
0.004 0.001
Organic contaminants
Given values represent the authentic values calculated as a 90th percentile of the annual measurements (12–26 measurements a year), with the exception of dissolved oxygen and oxygen saturation which are calculated as 10th percentile. For P–B saprobic index authentic value is calculated as a median of two yearly samplings. Values for 2002 and 2004 are given in Sˇtambuk et al. (2009) ‘‘–’’ Not determined
nucleus and its boundary should be distinguishable from that of the main nucleus. Only intact cells with distinct nuclear and cellular membranes were scored. The Comet assay The alkaline Comet assay (single cell gel electrophoresis assay) was performed according to the basic procedure of Singh et al. (1988) with slight modifications. Five micro litre of blood diluted in PBS (1:200) was mixed with 95 ll of 0.5% low melting point (LMP) agarose and placed on a normal 1% agarose precoated microscope slides. After solidifying for 2.5 min at 0°C, a third layer of 0.5% LMP agarose was added and left to solidify as described. The cells were lysed in freshly made lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris–HCl, 10% DMSO, 1% Triton X-100, pH 10), for 1 h at 4°C. After rinsing with redistilled water, the slides were placed on the horizontal gel box, covered with cold alkaline buffer (0.3 M NaOH,
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1 mM EDTA) pH [ 13 and left for 20 min. Electrophoresis was run in the same buffer at 25 V (0.83 V/cm) and 300 mA for 20 min at 4°C. After electrophoresis the slides were neutralised in a cold neutralisation buffer pH 7.5 (0.4 M Tris–HCl), 2 9 5 min, fixed in methanol:acetic acid (3:1) for 5 min and stored in the dark at room temperature. Prior to examination, the slides were rehydrated and stained with 10 lg/ml ethidium bromide and examined using a Zeiss Axioplan epifluorescence microscope. For every slide (per animal) 50 cells were examined, and the extent of DNA migration was determined as a percentage of the tail DNA using an image analysis system Komet 5, Kinetic Ltd. Cells with 50% or more of tail DNA were excluded from the analysis. Such cells probably represent dead or dying cells; therefore measurement of the increase in DNA migration in their absence is more important for the evaluation of genotoxicity than an increase that depends on them (Hartmann and Speit 1997; Rank et al. 2005).
Genotoxicity monitoring of freshwater environments
Statistical analysis Mean values of DNA damage for each group were calculated based on the mean of each individual within a group and data presented as mean and corresponding SEM for both Comet assay and MNT. Pairwise comparisons were performed using the Mann–Whitney U-test. Two levels of significance are reported: P B 0.05; P B 0.01.
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indicated Belisˇc´e as the most polluted (even in comparison to Osijek, P \ 0.02), opposed to the aquaria experiment where the site Osijek appeared to have the highest genotoxic potential as well as in the 2004 caging exposure. The Comet assay was not conducted in 2002. The frequency of cells with 50 and more percent of tail DNA varied but was overall lowest at the reference site while the most elevated percentage of tail DNA was observed at polluted location Osijek (Table 2).
Results
Cell viability
Micronucleus test data
Cell viability, evaluated by trypan-blue exclusion test, was consistently above 89% in all exposed animals and above 95% in animals from the reference site.
There was an apparent increase in the frequency of micronuclei in the blood cells of carp caged at the polluted sites, but it was not statistically significant in either the cage or the aquarium experiments (Fig. 2). The incidence of micronucleated erythrocytes after exposure of carp to water from the reference site varied from 0.08% (aquaria) to 0.33% (cage) while at the site Belisˇc´e it ranged from 0.15% (aquaria) to 0.38% (cage). The third site, Osijek, exhibited highest frequencies of micronucleated erythrocytes in caged carps (0.55%), while the lower values were again observed in individuals exposed in laboratory conditions (0.13%). Comet assay data Statistically significant increase in DNA damage of carp erythrocytes as measured by the Comet assay was evident at polluted sites compared to the reference site (Fig. 3). This increase was observed in both years of caging and in the aquaria experiment. Interestingly, in 2003, caged carp
Fig. 3 The level of the DNA damage measured by Comet assay (percentage of tail DNA, mean ? SEM) in erythrocytes of carp after 3 weeks in situ caging exposure to freshwater environments in years 2003 and 2004, and after 3 weeks laboratory exposure to fieldcollected water from the selected locations in year 2003. * P B 0.05, ** P B 0.01—denotes statistical difference from the respective reference value at the site Kopacˇki rit
Table 2 Percentage of carp erythrocytes with more than 50% tDNA at selected locations in year 2003 and 2004 Site
Year
Cells with C50% tDNA (%) Cage
Kopacˇki rit Belisˇc´e Fig. 2 Number of micronuclei in carp erythrocytes (mean ? SEM) after 3 weeks in situ caging exposure to freshwater environments in years 2002, 2003, and 2004 and after 3 weeks laboratory exposure to field-collected water from the selected locations in year 2003
Osijek Kopacˇki rit Belisˇc´e Osijek
2003
2004
Aquaria
0.26
1.12
5.33
0
0
4.61
2.49
–
2.61
–
14.87
–
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Discussion The results of the MNT and the alkaline Comet assay, conducted on common carp erythrocytes, have indicated the same sites as being polluted (Figs. 2, 3). These sites have already been identified as polluted using ethoxyresorufin O-deethylase (EROD) and acetylcholinesterase (AChE) activity measurements in caged carp (Jaric´ and Stepic´ 2005) and multixenobiotic defence mechanisms (MXR) activity, Comet assay and MNT in caged painter’s mussels (Zˇaja et al. 2006; Sˇtambuk et al. 2009). Red blood cells from carp held at the two sites with substantial anthropogenic influence (Belisˇc´e and Osijek) had higher averages of both the MN and % tDNA. In general, the Comet assay displayed greater sensitivity than the MNT, both in caging and aquaria exposures. This was also observed in the study of de Andrade et al. (2004) in which they used native gray mullet (Mugil sp.) and catfish (Netuma sp.) in assessing genotoxicity in the marine environment using the Comet assay and MNT. A lower sensitivity of MNT for various natural fish species was also indicated by Lee and Steinert (2003). In contrast, Bombail et al. (2001) detected elevated MN frequencies in butterfish (Pholis gunnellus) erythrocytes from contaminated sites while Comet assay did not indicate any significant differences between fish from polluted and cleaner areas. The amount of micronucleated erythrocytes of field-collected three-spined sticklebacks (Gasterosteus aculeatus L.) also appeared to better reflect the pollution at the locations investigated in that study than the Comet assay (Wirzinger et al. 2007). Regardless of occasional inconsistency in response to pollution in fish erythrocytes between these two parameters, polluted sites have generally been indicated by positive results of at least one of the two used genotoxicity biomarkers. Differences between results for the two in any given study are probably caused by the nature of these two assays thus strengthening the rationale of using them both since they complement each other by providing different aspects of DNA damage (Klobucˇar et al. 2003). Results for carp from the reference site, at both investigated years, as well as from the control aquaria containing water from the reference site, were similar for the Comet assay indicating low levels of DNA damage (5–7% tDNA). At the polluted sites DNA damage was up to three and a half times higher (11–18% tDNA) what can be considered as a significant induction of pollution-related genotoxic effect. Similar increase in the amount of the DNA damage (up to 3.5 fold) was noted in painter’s mussels (Unio pictorum) caged at the same locations (Belisˇc´e, Osijek) and at the same time, compared to reference location at Kopacˇki rit Nature Park (Sˇtambuk et al. 2009). Direct comparisons of the results of the Comet assay carried out by different research groups are sometimes
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hampered by differences in protocols. For that reason, the results of different studies are compared using an induction ratio. Using this approach our results are comparable with those of Whitehead et al. (2004) that observed 1–5 fold induction of DNA strand breakage in the erythrocytes of caged Sacramento sucker (Catostomus occidentalis) after 1 week of exposure in waters receiving agricultural chemical runoff. Similar results were observed by Bony et al. (2008), i.e., a 3–5 fold increase in the DNA damage in European minnow (Phoxinus phoxinus) exposed to chronic environmental concentrations of a mixture of two pesticides (diuron and azoxystrobin) in a 4-week mesocosm experiment. The same authors found 4–6 fold higher values of DNA damage in erythrocytes of brown trout (Salmo trutta fario) larvae exposed in situ to a river receiving pesticides from surrounding vineyards. The DNA damage of liver cells of caged chub (Leuciscus cephalus) was only up to twofold increased after 4 week exposure to contaminated river water, slightly higher than values measured in native chub from the same locations (Winter et al. 2004). Approximately threefold increase in the amount of DNA strand breaks was measured in erythrocytes of native chub (L. cephalus) from a polluted river in France (Flammarion et al. 2002). Similar results have been observed for native populations of chub from River Sava in Croatia (Pavlica et al. submitted). Though it is difficult to compare studies in view of the varying durations of exposure, different developmental stages of fish, the choice of tissue used in the evaluation etc., it should be noted that values of DNA damage measured by the Comet assay in caged fish tend to be in the upper range of those measured in wild-caught fish from polluted freshwaters, as already observed by Bony et al. (2008) and Winter et al. (2004). Although Comet assay results for carp erythrocytes correctly indicated polluted sites in both caging and laboratory treatments, there were some differences between caged fish and those exposed in the aquaria to the water from the same location. Laboratory exposed carp to the field-collected water from the reference site (Kopacˇki rit) and the most polluted site, Osijek, had slightly higher % tDNA compared to carp caged at the same locations, while DNA damage in carp exposed in the laboratory to the water from Belisˇc´e was lower than in cage exposed animals. This could be attributed to pollution input into the river that would not be detected in the aquaria exposure. Our results indicated that stress possibly caused by the cage exposure, which can be a source of additional DNA damage, may be considered negligible or certainly similar to the stress caused by the aquaria exposure. Furthermore, similar values of the DNA damage obtained in control individuals, either exposed in situ at the Kopacˇki rit or in the laboratory to the water from the same reference
Genotoxicity monitoring of freshwater environments
location, indicated minimal impact of the stocking density in the applied density range (1 fish per 2.67 l in aquaria and 1fish per 9.42 l in cage). The highest number of erythrocytes with more than 50% tDNA, probably indicating an ongoing process of cell death, was always present, in cage or aquaria exposure, at the polluted sites. The observed frequency of MN in fish erythrocytes from NP Kopacˇki rit are in accordance with the basal levels of micronucleated erythrocytes in carp reported by other authors (Gustavino et al. 2001; Grisolia and Starling 2001; Buschini et al. 2004; Zhu et al. 2004: Sapone et al. 2007), thus confirming the relatively pristine nature of the environment. The absence of statistical significance in the observed increase in the number of micronucleated erythrocytes at polluted sites (Belisˇc´e and Osijek) could be explained by either a lack of severe genotoxic pollution or low sensitivity of the method. Gustavino et al. (2001) observed statistically significant increase of MN frequencies in carp irradiated with X-rays (0.1–2 Gy), but no significant induction was observed after intraperitonal injection with colchicine (1.6 9 10-2 mg/kg). Zhu et al. (2004) found significantly elevated frequencies of MN in erythrocytes of carp exposed to cadmium, chromium and copper. Carp treated with higher concentrations of disinfectants for potabilization (sodium hypochlorite, chlorine dioxide but not peracetic acid) gave statistically significant increases after 10 and 20 days of exposure (Buschini et al. 2004; Sapone et al. 2007). These are all experiments where specific chemical or physical agents were used. No significant differences in the frequency of micronucleated erythrocytes were observed for native carp, but also not for two other fish species (Tilapia rendalli and Oreochromis niloticus), collected at sites of different pollution levels (Grisolia and Starling 2001). When the same authors used intraperitoneal injections of cyclophosphamide and mitomycine in the positive control experiments, a statistically significant induction of micronucleated erythrocytes was observed. It is also worth mentioning that C. carpio had the smallest increase of MN frequencies among all three species used in that experiment. However, a statistically significant increase in micronuclei frequencies was observed in natural populations of carp from polluted areas of river using flow cytometry (Llorente et al. 2002). Udroiu (2006) states that fish species with fewer but larger chromosomes are recommended for MNT. As many teleosts have an elevated number of chromosomes, carp is no exception with its 98 minute chromosomes (Al-Sabti 1986), which can be considered a disadvantage for its use in MNT. Indeed, several papers reported good correlation of MN induction with pollution intensity or chemical concentrations in fish species having lower number of chromosomes, such as sticklebacks (2n = 42) and loach
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(Misgurnus anguillicaudatus) (2n = 50) (Al-Sabti 1991; Chu et al. 1996; Peichel et al. 2001; Wirzinger et al. 2007). The frequencies of micronucleated erythrocytes were lower in the carp from aquaria when compared to caged individuals in the first 2 years of the research. Interestingly, in 2004 the frequencies of MN in erythrocytes of caged carp were lower than in previous 2 years, and more similar to those from the aquaria experiment. Since the Comet assay showed elevated DNA damage both in 2003 and 2004 at Belisˇc´e and Osijek, this result could be attributed to a decrease of an unknown aneugenic stressor in the river water. This study suggests that 3 weeks caging exposure of C. carpio may be successfully applied in freshwater genotoxicity biomonitoring and that carp erythrocytes can be used for Comet assay in detecting pollution-related genotoxicity. MNT on carp erythrocytes is also a good indicator of pollution although not as sensitive as the Comet assay. Acknowledgments We acknowledge support by the Research Council of Norway, for the project no. 150463 with Norwegian Institute for Water Research (NIVA) and by the Croatian Scientific Research Council for the project no. 119-0982934-3110.
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