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Mutation Research 628 (2007) 19–30
Genotoxic damage in field-collected three-spined sticklebacks(Gasterosteus aculeatus L.): A suitable biomonitoring tool?
Gertraud Wirzinger a,∗, Lennart Weltje b, Jens Gercken c, Holmer Sordyl c
a University of Applied Sciences Zittau/Gorlitz, Theodor-Korner-Allee 16, D-02763 Zittau, Germanyb J.W. Goethe University, Department of Ecology and Evolution–Ecotoxicology,
Siesmayerstrasse 70, D-60323 Frankfurt am Main, Germanyc Institute for Applied Ecology, Alte Dorfstrasse 11, D-18184 Neu Broderstorf, Germany
Received 25 November 2005; received in revised form 9 October 2006; accepted 12 November 2006Available online 8 January 2007
bstract
Three-spined sticklebacks (Gasterosteus aculeatus L.) were collected during different sampling trails from three locations inorthern Germany, which differ in the amount of sewage-treatment effluent that they receive. Due to natural population developments,
he size of the specimens caught decreased significantly from April to August. The fish were examined for DNA damage in theirlood cells by means of the comet (single-cell gel electrophoresis, SCGE) assay and the micronucleus test (MT). The suitabilityf stickleback erythrocytes as indicators for genotoxic substances in water was assessed. The median level of strand breakageanged from 5.23 to 9.67%, and decreased significantly from April to August. The difference between the locations was marginallyignificant. The amount of micronuclei was more variable (ranging from 0.40 to 4.35%), but appears to better reflect the pollutiontate of the sampling location. Significant differences between the locations were found. The relatively strong micronucleus inductionound in this study may be related to the fish species selected. Contrary to the SCGE results, a significant increase in the numberf micronuclei from April to August was observed. A significant negative correlation between strand breakage and micronucleias found for the sticklebacks from the most polluted location and for the pooled data of all locations. The length of the fish wasositively correlated with results of the SCGE and negatively with those of the MT, whereby males show a clearer relation between
ize and the amount of genotoxic damage. The test results are predominantly affected by seasonal impacts. This study indicates thathe outcome of the SCGE and MT applied to sticklebacks is determined by multiple factors, which need to be identified first beforehese tests can be applied routinely. Because of the profound negative correlation between SCGE and MT results, we recommendo apply both tests for the evaluation of the genotoxic potential of surface waters.s; Stick
2007 Elsevier B.V. All rights reserved.
eywords: Comet assay; Micronucleus test; Genotoxicity; Blood cell
∗ Corresponding author. Present address: J.W. Goethe University,epartment of Aquatic Ecotoxicology, Building A, Siesmayerstrasse0, D-60323 Frankfurt am Main, Germany. Tel.: +49 69 79824900;ax: +49 69 79824748.
E-mail address: [email protected] (G. Wirzinger).
383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mrgentox.2006.11.011
leback; Field monitoring
1. Introduction
As many chemicals with genotoxic potential areemitted to surface water through municipal and indus-trial waste water effluent, genotoxicity tests are gaining
importance. The detection of pollution is increas-ingly achieved by the use of bioindicators (species orbiocoenoses which react sensitively to stress in the envi-ronment) as they tend to accumulate pollutants, thereby
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20 G. Wirzinger et al. / Mutareflecting the site’s pollution history, and thus are suitablefor measurements of exposure and effects. Methods fordetecting DNA damage in organisms include the single-cell gel electrophoresis (SCGE) assay (comet assay)and the micronucleus test (MT). Both methods examinegenotoxicity by measuring the extent of DNA damage,albeit at different structural levels.
The SCGE is a method for the detection of single-and double-strand breaks as well as alkali-labile sites andexcision-repair events in DNA. The basis of all currentmethods was developed by Singh et al. [1]. The advan-tages of the SCGE are that it is a fast and simple methodthat can be applied to many cell types. Within a few hoursafter exposure, genotoxic damage may be measurablewith the SCGE [2]. In addition, it enables the exami-nation of single cells, and detection of minor genotoxicdamage is possible. Since the development and descrip-tion of the SCGE, it has been widely applied. Many ofthese studies concentrated on mammalian cells, but a fairnumber focused on marine and freshwater mussels andfish, examining the DNA damage in hemocytes, bloodcells or cells of gill, liver and gut (see reviews by Cotelleand Ferard [3] and by Lee and Steinert [4]). Some studiesinvestigated strand breaks in field-collected organisms,while others tested the impact of chemicals on the DNAin the laboratory [3,4].
The MT detects general disruptions in the chro-mosomal distribution. Micronuclei are small cellularchromatin bodies, separated from the main nucleusbut with similar coloring (achieved by the Pappen-heim dye, for example) [5]. They are generated in twoways: through aneugenic substances (e.g. mitomycinC), which damage the spindle apparatus, and by clas-togenic substances (e.g. benzo(a)pyrene), which inducechromosomal breaks. The resulting chromosomes orchromosomal fragments form micronuclei. The firstroutine test to quantify micronuclei was developed bySchmid [6]. It is applied with light or fluorescencemicroscopy or by using a cytometer. A premise for thismethod is the occurrence of cell division. Consequently,the MT takes a longer response time than the SCGE. Anumber of in vitro studies with the MT concentrated oneffects of known toxicants on mammalian cells [7–9],but the MT has also been applied to (field-collected) fish[10–12] and mussels [5,13–15].
Three-spined sticklebacks (Gasterosteus aculeatusL.) are commonly used as biomonitor for toxic sub-stances in small freshwater bodies [16–18]. In this work,
the erythrocytes of sticklebacks were analysed by SCGEand MT to investigate if this fish species may serve as abioindicator for genotoxic effects. To this end, three sam-pling locations were selected that differ in the amountssearch 628 (2007) 19–30
of sewage and agricultural run-off they receive. In addi-tion, the influence of age and gender of the fish on theoutcome of SCGE and MT were studied. Finally, theoutcome of the MT was correlated with the results of theSCGE to study the relation between the different typesof DNA damage measured by these assays.
2. Materials and methods
2.1. Field sampling
Field sampling was conducted at seven sites in three creeks(Ableitergraben, Kraaker Muhlenbach and Haubach), locatedin the north-eastern part of Germany (Fig. 1). The first samplingtrail was in April/May 2002, the second in June/July 2002. Thesites sampled are a good representative of the course of thecreek and its ecosystem.
The Ableitergraben (with sampling sites NS, upstream,and VB, downstream) is considered to be the most pollutedlocation, since it receives all the effluent of a large sewage-treatment plant (of 200,000 population equivalents; the dailywaste-water amount (domestic and possibly industrial) wason average 21,309 m3/day during this study) and is influencedby the surrounding farming areas. The location KraakerMuhlenbach (with sites OR, upstream, UR, K and Mo,downstream) is known as a comparatively clean water body.However, it does receive discharges of a small communalsewage-treatment plant (population equivalent of 4800) andis possibly also influenced by the surrounding agriculturalactivities. The water quality was determined with the clas-sification system of the German Working Party on Water(LAWA).
As our results of the first two sampling trails did not meetthe expectations stated above, and the Kraaker Muhlenbachcould not be considered as a reference site due to higher strandbreakage rates, additional sampling of the Ableitergraben andHaubach was conducted in August 2002. We suspected to findlittle or no genotoxicity in the Haubach sticklebacks, sincethe Haubach is located in a pristine and almost undisturbedenvironment. Therefore, this water body served as a referencelocation.
The choice of sampling locations should enable theappraisal if genotoxic damage is related to water pollution, inthis case the amount of sewage effluent in the water. Thereby,it was assumed that the fish from the Ableitergraben wouldshow higher rates of strand breakage and micronuclei thanthe fish from the Kraaker Muhlenbach, because many chemi-cals and pharmaceuticals in waste water are not eliminated bysewage treatment. For all creeks, detailed information on thecompounds present is lacking. To our knowledge no pollutionhot spots exist.
The three-spined stickleback (G. aculeatus L.) was the onlyfish species occurring naturally in all three water bodies. Thisrelatively stationary fish species is quite adaptable with regardto water quality [18]. At each site, 10–20 adult sticklebackswith a minimal length of 3.0 cm were caught with a fishing
G. Wirzinger et al. / Mutation Research 628 (2007) 19–30 21
F erania (c
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ig. 1. (A and B) Map of the German state Mecklenburg-Western Pomircles) and the sewage-treatment plants (closed squares).
et. In total, 150 fish were caught and analysed in this study.he fish were transported to the institute in the water of the
espective sampling site and kept in the laboratory at 16 ± 2 ◦Cor no longer than 48 h. The water was constantly aerated andacrophytes were placed in the aquaria to minimize social
tress. Prior to the analysis of their blood, fish were sexed,nd their fresh weight and length was determined. The lengthf the fish was used as a surrogate parameter for age (andonsequently as a substitute for time-of-exposure). The SCGEas carried out on the 2 days following sampling. The blood
mears for the MT (see below) were evaluated in between theampling trails.
.2. Blood-cell preparation
First, the SCGE and MT procedures were tested with ery-hrocytes of laboratory-cultured rainbow trout (Oncorhynchusykiss) and the known genotoxicant H2O2. These primary
aboratory experiments served as a positive control for DNAamage and to check if the methods were properly establishedn our laboratory.
The procedure for field-collected sticklebacks was as fol-ows. After narcotization by placing the animals in a bowl
ith a solution of tricaine methanesulfonate (160 mg/L waterf the sampling site), the pygostyle of the sticklebacks wasevered. The blood drop, emerging from the caudal vein,as collected with a pipette and added to Eppendorf tubesith 0.5 mL cooled Heparin-HBSS (Hank’s Balanced Saltinset), showing the three water bodies with their sampling sites (closed
Solution) to prevent clotting. Depending on cell density,50–100 �L of this blood suspension was added to 200–1000 �LHBSS.
Prior to conducting the SCGE, the vitality of the erythro-cytes was assessed with trypan blue, a quality control test tocheck the cell preparation. In the event of cell damage, trypanblue penetrates the cell membrane, and dead or damaged cellsappear blue. The premise for applying the SCGE (at least 70%cell vitality) was achieved in all our analyses.
2.3. Comet assay (SCGE)
The SCGE was based on the protocols of Singh et al. [1] andZorn [13]. The cells were embedded in two layers of agaroseon fully frosted slides. The agarose solutions used for theblood cells were dissolved in PBS (phosphate-buffered saline;0.2 g KCl, 8 g NaCl, 0.2 g KH2PO4, 1.442 g Na2HPO4·2H2O,adjusted to pH 7.5 with 1.0N HCl). The slides were pre-treatedwith 600 �L of 1% NMA (normal melting agarose) solution,which was scraped off after solidification to erase all uneven-ness. For the first layer, a 0.5% NMA solution was melted and200 �L was transferred to the slides and allowed to solidify onice for 5 min. Of the above-mentioned cell suspension, 30 �L
was added to 120 �L of 0.7% LMA (low melting agarose),which was previously melted. One hundred and twenty micro-liters of this mixture was transferred to the slides precoatedwith NMA. A cover glass was placed on the gel and the slideswere cooled on ice for 5 min, then transferred to a warming
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22 G. Wirzinger et al. / Mutatray at 37 ◦C for 10 min. After removing the cover glass, afinal layer of 120 �L of 0.7% LMA was added to the slideswhich were again covered with a cover glass, cooled on ice for15 min to solidify and then placed on a warming tray at 37 ◦C.After removing the cover glass, the slides were placed in thelysis solution (stock solution: 293 g NaCl, 74.4 g EDTA, 2.4 gTris, adjusted to pH 10 with NaOH, working solution: 178 mLstock solution, 2 mL Triton and 20 mL DMSO, pH 9.6) for atleast 1 h in the refrigerator. Hereafter, all work was carried outin the semi-dark to avoid UV-light damage to the DNA. Theexcess salt was removed by rinsing the slides in distilled water.The slides were then placed in the electrophoresis chamberand the cold electrophoresis solution (24 g NaOH and 0.74 gEDTA in 2 L destilled water, pH > 13) was added. Alkalineunwinding was allowed for 20 min, followed by 20 min ofelectrophoresis at 300 mA and 25 V. Finally, the slides wereneutralized for 5 min in 0.4 M Tris (48.5 g Tris and pH 7.5with 25% HCl). To allow scoring, the slides were stainedwith 50 �L ethidium bromide (20 mg/L) and examined undera fluorescence microscope (Olympus, 400× magnification).On each slide, 80 randomly chosen erythrocyte nuclei wereexamined using Komet 4.0 software (Kinetic Imaging Ltd.,Liverpool, England). Highly damaged nuclei (>75% DNA inthe comet’s tail) were not scored. The level of DNA damage,i.e. strand breaks, is expressed as the percentage of DNA in thecomet’s tail.
2.4. Micronucleus test (MT)
A drop of blood was placed on a fat-free slide. A coverslip was set on the slide at a sharp angle until it touched theblood drop. The blood spread along the side of the cover slipwhich was then pulled rapidly and evenly over the slide. Twoslides per animal were prepared. The smears were air-dried andstained immediately according to the Pappenheim-dye proce-dure [19]. First, the slides are stained for 3 min with 20–30drops of May-Grunwald, then with the same amount of a 1:1May-Grunwald:distilled water mixture for 1 min, followed byGiemsa (0.3 mL per 10 mL distilled water, pH 7) for 15–20 min.Finally, the slides are thoroughly rinsed with distilled water andallowed to dry. If staining could not be carried out immediatelythe slides were fixed with 99% methanol for 5 min and stored ina dry place at room temperature and stained later. For each ani-mal 1000 erythrocytes were scored at 1000× magnification todetermine the percentage of micronucleated cells. Our criteriafor micronuclei are as described by Dopp et al. [5]. Micronu-clei have a round or ovoid shape and a maximum diameter ofone third of that of the cell nucleus. Their staining is similaror brighter and they are separated from the main nucleus (nochromosomal bridge).
2.5. Statistical analysis
First, the data were checked for normality by means ofthe D’Agostino and Pearson omnibus normality test. Sinceboth SCGE and MT data did not pass the normality test
search 628 (2007) 19–30
(p < 0.0001), non-parametric tests were applied to analyse theresults. The Kruskal–Wallis test was used for comparing morethan two samples, followed by Dunn’s multiple comparisonpost-test. For comparing two samples, the Mann–Whitney’stest was used. Correlation was calculated according toSpearman. For all tests, the level of significance was set at95% (α = 0.05). All calculations were performed with thesoftware programme Prism, version 4.02 (GraphPad Software,San Diego, CA, USA).
3. Results
3.1. General
The methods applied here were tested first with bloodof rainbow trout exposed to the known genotoxicantH2O2 (positive control). The fact that significantpercentages of tail DNA and micronuclei could bedetected in fish blood cells demonstrated that bothmethods were properly established in our laboratory(results not shown).
The relation between length (L) and fresh weight(FW) of the sticklebacks can be described with the equa-tion FW = (aL)3, in which a is a dimensionless shapecoefficient [20]. As an example, for the pooled data(n = 150), a = 0.2177 ± 0.0009 and r2 = 0.94. Length andfresh weight of the sticklebacks was highly correlatedat all locations and also for the pooled data (n = 10–150,in all cases r > 0.97 and p < 0.0001). Therefore, we onlyused stickleback length as an indicator of age of the fish,and therefore as an indicator of the time of exposure,in assessing correlations with the outcome of the SCGEand MT.
The sticklebacks caught later in the year (July andAugust) were significantly smaller (Kruskal–Wallis andDunn’s multiple comparison post-test, p < 0.001) thanthose caught in April (Fig. 2). The fish caught in Aprilhad a mean length of 6.11 cm and those of July andAugust 4.23 and 4.17 cm, respectively.
The sex of four fish at the Haubach location could notbe determined. Consequently, these fish were excludedfrom the gender-specific statistical analyses.
3.2. Comet assay (SCGE)
Results for the SCGE at the subsampling sites withinthe locations Ableitergraben and Kraaker Muhlenbachwere not significantly different (Mann–Whitney U,
p = 0.749 and Kruskal–Wallis, p = 0.941, respectively).Hence, no relation between the distance of the subsam-pling site to the point of discharge could be established.Therefore, subsampling site data were pooled for
G. Wirzinger et al. / Mutation Research 628 (2007) 19–30 23
F ngth inf f the len
ebbt
DpameM
a7(osers
mwt(
Table 1). Data for the males of the Ableitergraben arepresented in Fig. 4. These highly significant correla-tions were most pronounced for males, which showed
Table 1Correlation between fish body length and SCGE results (asterisksindicate significance)
Data n r p
Total 150 0.268 0.0009*Ableitergraben 64 0.424 0.0005*Kraaker Muhlenbach 76 −0.009 0.9393Haubach 10 −0.430 0.2182
Males 51 0.480 0.0004*Ableitergraben 26 0.678 <0.0001*Kraaker Muhlenbach 24 0.313 0.1367Haubach 1 Too few data
F2A
ig. 2. Size of the sampled three-spined stickleback. (A) Average fish lerom April fish (***p < 0.001, n = 14–62). (B) Frequency distribution o
ach location. Medians differed marginally significantlyetween the three locations (Kruskal–Wallis, p = 0.038),ut Dunn’s multiple comparison post-test could not iden-ify between which locations.
At the Ableitergraben, the median percentage of tailNA decreased significantly in time (Kruskal–Wallis,= 0.0001) from 9.67% in May to 6.75% in July,nd further to 5.23% in August (Fig. 3). Dunn’sultiple comparison post-test showed significant differ-
nces between all sampling times (May–July, p < 0.05,ay–August, p < 0.001, July–August, p < 0.05).At the Kraaker Muhlenbach, the median percent-
ge of tail DNA decreased from 9.16% in April to.23% in June (Fig. 3), which was significantly differentMann–Whitney U, p = 0.012). The median percentagef tail DNA of the Haubach sticklebacks, 6.33%, was theecond lowest found in this study. At the locations Ableit-rgraben and Kraaker Muhlenbach, the strand breakageate decreased in time (Fig. 3), which is also true for allampling sites.
The median percentage of tail DNA of the pooled
ales and females was not significantly different,hich was also the case for males and females athe locations Ableitergraben and Kraaker MuhlenbachMann–Whitney, p > 0.05).
ig. 3. Results of the SCGE, expressed as the percentage tail DNA, for all s5 and 75% percentile values, respectively, with median values within the bsterisks indicate a significant difference from the first sampling result at the
cm against sampling month. Asterisks indicate a significant differencegth specified for April (closed symbols) and August (open symbols).
The fish body length was significantly and positivelycorrelated with results of the SCGE for all fish pooled,for pooled fish from the Ableitergraben, all pooled malesand for the males of the Ableitergraben (p ≤ 0.0009, see
Females 95 0.190 0.0658Ableitergraben 38 0.312 0.0567Kraaker Muhlenbach 52 −0.100 0.4794Haubach 5 Too few data
ampling locations and months. Bottom and top of the box representox (n = 10–62). Error bars indicate minimum and maximum values.same location (*p < 0.05, ***p < 0.001).
24 G. Wirzinger et al. / Mutation Re
Fig. 4. Correlation of the SCGE results with the length of the malesticklebacks of the Ableitergraben (n = 26, r = 0.678 and p < 0.0001).
the highest correlation coefficients in combination withthe highest significance (i.e. lowest p values).
3.3. Micronucleus test (MT)
Overall, the MT displayed more potential to differ-entiate genotoxic effects than the SCGE (the maximumdifference was a factor of 11, against a factor of 2 for tailDNA).
The medians of the subsampling sites within thelocations Ableitergraben (Mann–Whitney U, p = 0.694)and Kraaker Muhlenbach (Kruskal–Wallis, p = 0.887)were not significantly different, which was also observedfor the SCGE results. Therefore, subsampling site datawere pooled for each location. Medians differed signif-icantly between locations (Kruskal–Wallis, p < 0.0001).Dunn’s multiple comparison post-test found signifi-cant differences between the locations Ableitergraben
and Kraaker Muhlenbach (p < 0.001) and between theKraaker Muhlenbach and Haubach (p < 0.05), while thelocations Ableitergraben and Haubach were not signifi-cantly different.Fig. 5. Results of the MT, as micronuclei rate (%), for all sampling locations avalues, respectively, with median values within the box (n = 9–60). Error bars indifference from the first sampling result at the same location (**p < 0.01, ***p
search 628 (2007) 19–30
The micronuclei values of the Ableitergraben were0.95% in May, 2.20% in July and 4.15% in August. TheJuly and August the values were significantly higher thanthose of May (Kruskal–Wallis, p = 0.0001) and Dunn’smultiple comparison post-test showed significant differ-ences between May and July (p < 0.01) and May andAugust (p < 0.001). July and August values were notsignificantly different. At the Kraaker Muhlenbach thelevels of micronuclei were the lowest, with median val-ues of 0.40% in April and 0.70% in June (Fig. 5).These values differed significantly from each other(Mann–Whitney U, p = 0.0019). The median micronu-clei rate at the Haubach was 1.30% and thus higher thanobserved in the sticklebacks at the Kraaker Muhlenbach,but lower than that of the Ableitergraben sticklebacks.At the Ableitergraben and the Kraaker Muhlenbach, themicronuclei rate increased with time (Fig. 5).
Next to micronuclei, we found the related phe-nomenon of budding cell nuclei and binucleated cells,which was quite common in the fish sampled at theAbleitergraben and at the Kraaker Muhlenbach. Buddingcell nuclei were also found in the fish of the Haubach,but their blood had no binucleated erythrocytes.
The fish body length was significantly and negativelycorrelated with results of the MT (Table 2). The onlynon-significant correlations were found for the fish fromthe Haubach location (which produced the only positivecorrelation coefficient) and the females at the KraakerMuhlenbach (which yielded the smallest negative corre-lation coefficient).
3.4. Correlation between SCGE and MT results
Since length was positively correlated with SCGEresults (Table 1) and negatively with MT results(Table 2), it is to be expected that the correlationbetween SCGE and MT will turn out negative. Indeed,
nd months. Bottom and top of the box represent 25 and 75% percentiledicate minimum and maximum values. Asterisks indicate a significant< 0.001).
G. Wirzinger et al. / Mutation Re
Table 2Correlation between fish body length and MT results (asterisks indicatesignificance)
Data n r p
Total 144 −0.468 <0.0001*Ableitergraben 61 −0.490 <0.0001*Kraaker Muhlenbach 74 −0.299 0.0102*Haubach 9 0.250 0.5206
Males 51 −0.563 <0.0001*Ableitergraben 26 −0.500 0.0093*Kraaker Muhlenbach 24 −0.445 0.0332*Haubach 1 Too few data
Females 90 −0.438 <0.0001*Ableitergraben 35 −0.504 0.0020*Kraaker Muhlenbach 50 −0.271 0.0573Haubach 5 Too few data
Table 3Correlation between SCGE and MT results (asterisks indicatesignificance)
Data n r p
Total 144 −0.299 0.0003*Ableitergraben 61 −0.364 0.0039*Kraaker Muhlenbach 74 −0.194 0.0975Haubach 9 −0.033 0.948
Males 51 −0.351 0.0117*Ableitergraben 26 −0.438 0.0254*Kraaker Muhlenbach 24 −0.141 0.5115Haubach 1 Too few data
Females 90 −0.248 0.0187*
ppA(
FD
Ableitergraben 35 −0.321 0.0598Kraaker Muhlenbach 50 −0.179 0.2135Haubach 5 Too few data
ooled data for all fish, pooled males, pooled females,ooled fish from the Ableitergraben and males from thebleitergraben showed significant negative correlations
Table 3). Data for pooled fish are presented in Fig. 6.
ig. 6. Relation between the micronuclei rate and percentage of tailNA for pooled data (n = 144, r = −0.299, p = 0.0003).
search 628 (2007) 19–30 25
4. Discussion
4.1. General
The three-spined stickleback is known to be quiteadaptable to water quality [18]. Because of its widedistribution in European freshwater and estuarineecosystems [18], this species may be suitable for envi-ronmental monitoring. A study by van den Dikkenberget al. [21] recommended the stickleback as a suitable fishfor laboratory and field experiments, as this species wasequally sensitive or even more sensitive than other, non-native standard fish species (e.g. Danio rerio and Oryziaslatipes) and is rather easy to culture in the laboratory.
The fish caught later in the year were significantlysmaller than those caught in April. It seems that thisdecrease in size is due to natural population develop-ments. In spring, we mostly find adult animals (largerfish) but during the summer months the juveniles makeup a larger proportion of the total fish population.
Detailed information on the chemicals present in eachof the investigated creeks is not available and a chemicalcharacterization is beyond the scope of this paper, whichaimed at establishing a qualitative relation betweenthe amount of sewage (Ableitergraben > KraakerMuhlenbach > Haubach) and the occurrence of geno-toxic damage. A characterization of the KraakerMuhlenbach showed that the sewage discharge has onlylimited influence on the water quality, as determinedwith the classification system of the German WorkingParty on Water (LAWA) [22]. Chemical analyses of theAbleitergraben, conducted in 1999–2001, showed thatvalues of adsorbable organic halogens (AOX) and heavymetals were below the limits of the German DrinkingWater Ordinance (personal communication, municipalsewage-treatment plant, Schwerin, Germany). However,AOX and heavy metals are not necessarily the maincontributors to the genotoxic damage in the fish. Othercomponents, e.g. PAHs and their metabolites, may playa significantly greater role [23,24]. For the Haubachno information is available, but due to its locationin a pristine environment, no pollution whatsoever isanticipated.
4.2. Comet assay
The SCGE is known to be a sensitive and gentlemethod, eliminating “artificial” (e.g. stress-induced)
DNA damage [25]. The assay can be used in all isolatedcell types, which makes the SCGE an appropriate testsystem for monitoring genotoxic effects in wildlifeorganisms [26].
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26 G. Wirzinger et al. / MutaApparently, the amount of sewage in the water hasno influence on the amount of strand breaks, as thefish at the more distant site (VB) of the Ableitergrabenshow the same percentage of tail DNA as those sam-pled close to the sewage-treatment plant (NS) (resultsnot shown). Also, the fish at the Kraaker Muhlenbachgenerally had higher percentages of tail DNA than thoseof the Ableitergraben, which receives more sewage efflu-ent. However, Moraes de Andrade et al. [27] found apositive correlation between the size of the town closeto the examined water body and the amount of DNAdamage in fish, which was probably due to the increasedeffluent and municipal wastewater discharge. It has beenargued that the test results may depend on other fac-tors, e.g. the input from agriculture [28,29], which mayhave played a role at our location Ableitergraben, andthe age of the fish (see below). The lack of correla-tion between exposure to pollutants and the results ofthe SCGE may be attributed to rapid DNA repair pro-cesses, increased metabolic breakdown, interaction andincreased genotoxic excretion [30].
The decrease of the percentage tail DNA during thesampling period (Fig. 3) is probably related to sea-sonal or endogenous effects (e.g. growth, developmentand reproduction). Although some studies found noseason-dependent effects [31], a large number of studiesdid indicate seasonal variation in DNA damage (strandbreaks) in fish, with the DNA damage increasing dur-ing warmer months [32,33]. Bolognesi et al. [34] andAndrade et al. [35] found an increase in DNA damageduring the year, with the lowest values in winter, which isin contrast to our SCGE results (Fig. 3), but in agreementwith our MT results (Fig. 5). Many studies also showthe influence of higher temperatures, which enhance thelevel of DNA damage [29,32–35].
Age can be another factor, since the fish caught laterin the year (July and August) were significantly smallerthan those collected in the previous months (Fig. 2).According to a study with rodents [31], the SCGEshows age- and gender-dependent differences, wherebyfemales showed higher levels of DNA damage. How-ever, in our study, the correlations of fish body length(as an indicator of age) and the DNA damage (Table 1)were significant for males, but not for females. A vari-ation in sensitivity with age is also supported by Lindeet al. [36], who found that young brown trout (Salmotrutta) were more sensitive than older specimens. Thestrong correlation between length and our results with
the SCGE for pooled fish indicates that older, i.e. bigger,fish have more DNA damage.The percentage tail DNA at the reference locationHaubach was similar to the August value of the Ablei-
search 628 (2007) 19–30
tergraben sticklebacks (Fig. 3), both derived fromsmall fish (Fig. 2). Hence, the significant tail DNA inthe Haubach fish is either caused by natural turnoverrates and repair mechanisms in the cell nuclei, or maysimply represent a background value caused by diffusepollution. These assumptions are supported by variousstudies documenting significant percentages of strandbreaks in field-collected animals from unpolluted areas[13,36,37]. Comparing our baseline value (Haubach)with other studies is difficult, as they only mention thatthere are differences, but do not present specific values.In aquaculture sticklebacks, the baseline value was14–17% tail DNA while most cultured cell lines fre-quently showed background levels of 5–10% (personalcommunication, Dr Timothy Williams, BirminghamUniversity, UK). In addition, Buschini et al. [32,33]report a strong seasonal influence, as well as significantinter-individual variability in the level of baselineDNA damage in hemocytes of zebra mussels anderythrocytes of carp, respectively. Bolognesi et al. [34]observed seasonal differences in baseline levels of DNAmigration in zebra mussel cells, which were related tothe water temperature.
A chronic exposure to pollutants can lead to an accu-mulation of DNA strand breaks, as the DNA-repaircapacity of fish cells, an important mechanism to pro-tect the DNA integrity, is rather low compared to that ofother species [38]. However, several studies presented adecrease of DNA damage when fish from polluted areaswere transferred to clean water [3,39]. This may be due tothe instability of the genotoxic agents, the metabolic con-version of these agents to non-genotoxic metabolites orthe activation of DNA-repair processes. Extensive frag-mentation of DNA, which could lead to very small DNAfragments that are difficult to detect in alkaline SCGE,may be another factor.
Lee and Steinert [4] stated that different cell typesmay have different background levels of DNA single-strand breaks, due to a variation in excision repair andmetabolic activity, as well as, e.g. anti-oxidant concen-trations, and that the normal DNA damage can be highlyvariable, due to cell type heterogeneity, cell cycle, cellturnover frequency, etc. Russo et al. [10] and Buschini etal. [33] determined the turnover rate of fish erythrocytesto be approximately 100 days.
4.3. Micronucleus test
The selection of cell type is a critical issue in rou-tine application of the MT for biomonitoring purposes.According to Masuda et al. [2] and Sanchez-Galan et al.[12], the fact that they found higher levels of micronuclei
tion Re
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G. Wirzinger et al. / Muta
n brown trout gill cells than is reported for erythrocytesf other fish species, can be attributed to the cell typenvestigated. We propose that in addition to cell type, theifferences may also depend on the selected fish speciess the micronucleus levels in the present study wereuch higher than those observed in other fish species
15,16,40–42]. This is supported by Rodriguez-Cea etl. [11], who noted that some fish species (e.g. brownrout) are more sensitive to genotoxic pollutants thanther species, such as eel (Anguilla anguilla) or minnowPhoxinus phoxinus).
Izquierdo et al. [15] found a clearly negative asso-iation between micronucleus levels and the distancerom effluent inlets. Indeed, the micronucleus inductiont the Ableitergraben was significantly higher than thatf the Kraaker Muhlenbach, which confirms the find-ngs of this and other studies concerning the influence ofollution [15,43]. However, the findings at the Haubachre not explained by these studies as we found micronu-lei in fish from an environment that was assumed to benpolluted.
Baseline micronucleus levels in hemocytes ofivalves ranged from 0.5 to 2‰ [14,44]. In situ investi-ations of Rodriguez-Cea et al. [11] with different fishpecies resulted in levels between 0.8 and 2.76‰. Thesealues are lower than our lowest value, 0.4%, in April athe Kraaker Muhlenbach (Fig. 5) and much lower thant the Haubach, which may be an indication of somenknown stressor at the Haubach. The high variabilityf our values for micronuclei at each location is a phe-omenon that seems to be specific for the micronucleusest and is also described by Mersch and Beauvais [44].
The occurrence of seasonal variation in micronucleusrequencies is reported in several studies [11,33,34,44]ut there are also studies that did not find a seasonalnfluence [35,45,46]. Rodriguez-Cea et al. [11] suspecthat the sampling of the fish, which was done in summerhigher water temperature and lower water quality due toow-flow conditions) was the cause for the higher in situ
icronucleus levels than in the laboratory experiments,hich were done in autumn. In spite of their first study,resler et al. [47] did find significantly higher numbers oficronuclei in samples collected during rainy seasons,
nduced by municipal sludge, garbage and chemicalsashed off from soil and plants. Therefore, the increase
n micronuclei during our study may be the expression ofeasonal effects, but it could also be related to the run-offrom agricultural fields.
In the study of Heuser et al. [31], the micronucleusest with rodents showed no significant gender- and age-elated differences. In our study, the fish body lengthas significantly and negatively correlated with results
search 628 (2007) 19–30 27
of the MT in most cases, regardless of gender (Table 2),which implies that smaller, i.e. younger fish are moresensitive to micronucleus-inducing substances. This is inline with the results of Zuniga-Gonzalez et al. [48] whoreported the same effect (higher numbers of micronucleiin younger animals) with other species (birds, mam-mals, reptiles), explaining this effect by assuming aslower maturation of the reticulo-endothelial system.Rodriguez-Cea et al. [11] could not rule out an influ-ence of the age of the fish on the results of the MT, withthe sensitivity to the test decreasing with fish age. Theoccurrence of increased micronucleus induction due toage, among cancer and other environmental influencesis also described [8].
The phenomena of budding cell nuclei and binucle-ated cells have a similar origin as micronuclei and aresupposed to be genotoxic occurrences [7]. Erythrocytesof fish in polluted waters often show micronuclei, butbinucleated cells are more common [49]. Sanchez-Galanet al. [12], who examined brown trout, reported that bin-ucleated cells are induced by unspecific pollution alongwith micronuclei. Ayllon and Garcia-Vasquez [50] sug-gest that nuclear abnormalities (e.g. budding cell nucleiand binucleated cells) are potential indicators of geno-toxicity.
4.4. Correlation between SCGE and MT results
The reason for using the two tests in this study isthat they both are non-specific biomarkers which reflectdifferent forms of environmental stress. An analysis ofpooled data demonstrated a strongly negative correla-tion between the outcome of both tests. Contrary to ourobservations, many studies found a positive correlationbetween the SCGE and the MT [10,35,45]. Klobucar etal. [14] mentioned that in some studies positive resultsof the MT are accompanied with negative results in theSCGE. The negative correlation may depend on the fishspecies chosen and/or on the nature of both assays.
The SCGE detects primary DNA lesions that are thenet result of DNA damage (strand breaks, alkali-labilesites, DNA cross-links) and repair mechanisms, imply-ing that DNA strand breaks only have a temporal nature[3,14,34]. The detected DNA damage such as alkali-labile sites and single-strand breaks may be removedafterwards by the DNA-repair system [3,14,51]. It is alsoknown that aneugenic activity can cause negative resultsin the SCGE but may induce micronuclei as well as DNA
crosslinks, which retard migration in the SCGE [14].In fish, the SCGE directly detects DNA strand breaksin circulating erythrocytes, which may be induced veryearly after exposure to genotoxins [33]. The lack of
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correlation between the SCGE results and pollution isprobably due to DNA-repair processes that conceal thepreviously inflicted but repairable DNA damage [30].
The MT measures persistent DNA damage that can-not be repaired [14,30]. This consists of small subsetsof unrepaired double-strand breaks [3], breaks at thelevel of chromatids [10], unrepairable aneugenic effects[34] or DNA lesions after their fixation into chromo-some mutations [51]. According to Buschini et al. [33],micronuclei are the result of chromosome breaks thatoccur in the stem cells in the cephalic kidney. The even-tually micronucleated erythrocytes require a passagethrough mitosis to be recognizable. Chromosome dam-age is a major change that cannot be repaired by the cell[30].
Length and weight were negatively correlated withthe results of the MT, but positively correlated with theoutcome of the SCGE, although the latter correlationswere more often not significant. The results indicatethat pollutants may have multiple effects on the DNA,expressed as strand breaks or micronuclei. Sturm etal. [16] found that body weight and length of stick-lebacks were inversely related with the cholinesterase(ChE) activity, which was influenced by the presence oforganophosphorus pesticides. However, the sex of thefish showed no correlation with the ChE activity.
5. Conclusions
Three-spined sticklebacks from three water bodieswith different levels of sewage and agricultural dischargewere examined for DNA damage using the comet assayand the micronucleus test. The sampling time medi-ans were significantly different at all locations for bothSCGE and MT. Sampling time (seasonality) may havean influence on the results, but it is more likely that thedifference between the respective sampling times is dueto the size (i.e. age) of the fish. For the MT a more pro-nounced relation between the amount of sewage and thelevel of genotoxic damage was detected.
The detection of significant strand breaks andmicronuclei indicate that a SCGE assay and/or MT offish blood cells may generally be suitable for environ-mental monitoring to gather information. DNA damagemeasured by the SCGE appears earlier than do micronu-clei and is rather short-lived, indicating recent pollutionevents. Micronuclei require cell division and exist aslong as the cell itself does. The persistence of micronu-
clei in the cell is longer than that of single-strand breaksand alkali-labile sites, which are more or less constantlyrepaired. By combining both tests in field monitoringand laboratory research, both short-term genotoxic[
search 628 (2007) 19–30
damage as well as more persistent pollutant effects canbe detected. The routine use of the three-spined stickle-back as a biomonitor has great potential, but can only berecommended under the premise of further research intothe negative correlation between the results of the twotests, the impact of the sampling time, and the age ofthe fish.
Acknowledgements
We thank B. Bernau and B. Gobel (Institute forApplied Ecology) for their help with sampling andassistance in the laboratory. M. Lupke and J. Rollwitz(University of Rostock) are acknowledged for their helpwith the evaluation of the MT. We also thank two anony-mous reviewers for their helpful comments. This paperoriginates from the B.Sc. thesis of G.W., under supervi-sion of Prof. W. Ramm (University of Applied Sciences,Zittau, Germany).
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