Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.)

Mutation Research 538 (2003) 31–39

Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.) Darius Stonˇcius a , Juozas R. Lazutka b,∗ ˇ Centre for Environmental Studies, Vilnius University, 21 Ciurlionis St., Vilnius LT-2009, Lithuania ˇ Department of Botany and Genetics, Vilnius University, 21 Ciurlionis St., Vilnius LT-2009, Lithuania

a b

Received 2 December 2002; received in revised form 6 March 2003; accepted 25 March 2003

Abstract The spontaneous levels of micronuclei in erythrocytes were established in embryos of the black-headed gull of two natural populations. In total 216 blood samples from the same number of individuals were examined. A statistically significant decrease in the number of spontaneous micronucleated erythrocytes was found after 13 days of incubation. We found no statistically significant difference in the spontaneous frequencies of micronucleated erythrocytes in the embryos of the two colonies studied, although they differed in anthropogenic load. Results of analysis of variance indicated that egg incubation time was the only variable significantly (P = 0.0001) affecting spontaneous frequency of micronucleated erythrocytes in the embryos of black-headed gulls. We also took 78 eggs of different developmental stages from both colonies and exposed them for a further 24 h to a dose of benzo[a]pyrene (30 ␮g per egg). After exposure to benzo[a]pyrene, the frequency of micronucleated erythrocytes was not increased in the embryos incubated for a total period of 13 days. A statistically significant increase in the number of micronucleated erythrocytes was recorded in the benzo[a]pyrene-treated embryos incubated for a total period of 14 days. Decrease in numbers of spontaneous micronucleated erythrocytes after the 13 day of incubation and increased levels of benzo[a]pyrene-induced micronuclei after the 13 day of incubation were discussed to be caused by changes in spleen and liver function in advanced developmental stages of the embryo. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Black-headed gull; Embryo; Micronucleus test; Benzo[a]pyrene; Genotoxicity; Monitoring

1. Introduction Global distribution of various pollutants is affecting not only humans in urbanised areas but also wildlife occupying distant regions. As a result, greater emphasis is now being placed on studying wild populations to relate various effects of pollutants with processes in these populations [1].

∗ Corresponding author. Tel.: +370-5-239-8256; fax: +370-5-239-8204. E-mail address: [email protected] (J.R. Lazutka).

Birds are suitable indicator organisms of environmental pollution for several reasons. To ensure mass-specific amounts of oxygen required for flight, they take up large volumes of air along with any other gases or particles present, so that they may be a sensitive indicator of environmental contamination by gases or airborne particles at low concentrations [2]. Birds also belong to the highest levels of trophic chains in ecosystems, therefore able to accumulate or biomagnify various contaminants. Despite of these advantages, birds have been quite rarely used in genotoxicological monitoring as test animals [3–8]. The most common avian laboratory

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system for genotoxicity testing is the incubated egg of the domestic hen (Gallus domesticus) [9–17]. The avian embryo in early stages of embryonic development provides a wide range of metabolic activities enabling activation of promutagens, their detoxication and excretion [15]. Moreover, laying female birds excrete xenobiotic compounds into the eggs [18,19]. Overall patterns of chemicals accumulated in eggs indicate the local pollution of breeding sites [20]. Sensitive reaction of the developing embryo to accumulated toxic chemicals and the possibility to study relationships between contaminants, eggshell quality and hatching success could be mentioned as additional advantages [20]. Consequently, birds and their embryos in particular can effectively be used in pollution monitoring and testing of environmental contaminants for their genotoxicity. The early presence of a metabolic competency in the embryonic stage allows the examination of pollutants that require metabolic activation before exerting their mutagenic effects, as is the case for PAHs, for example [21]. A further advantage is that erythrocytes constitute the major fraction of blood of the embryos in advanced incubation stages [22,23]. These characteristics allow application of the micronucleus test to the chicken embryo [15,17]. This test is a simple and rapid method for measuring chromosome breaks and aneuploidy [24,25]. The black-headed gull (Larus ridibundus L., Charadriiformes, Laridae) is one of the most common colonial bird species not only in Lithuania, but also in other European countries [26]. In studies of colonial birds, the use of eggs allows fast and cheap collection of statistically relevant samples without compromising breeding success. First, females of the black-headed gull can produce replacement clutches [27] in case the first clutch was lost. Second, use of incubated bird eggs enables to reduce significantly the disturbance of breeding birds caused by blood sampling from adult individuals. Such disturbance may be quite significant—it was found that breeding females of the black-headed gulls caught on nests tended to change colony [28]. Attempts to collect blood samples from nestlings may even be more destructive because frightened chicks often leave their nests and are then killed by adult birds from other breeding pairs defending their territory. For these reasons, collecting eggs from colonial bird species and using them in toxicology studies is quite usual practice [3,29].

In this study we determined spontaneous levels of micronuclei in the blood of the black-headed gull embryos collected from two natural populations. We also examined benzo[a]pyrene for its ability to induce micronuclei in erythrocytes of embryos of black-headed gulls. Some methodological aspects and applicability of the micronucleus test for the monitoring of genotoxic damage in natural populations of black-headed gulls are discussed as well.

2. Materials and methods 2.1. Sampling locations Two colonies of the black-headed gull (L. ridibundus L.) located in two different regions of Lithuania (Fig. 1) have been selected for the study. The distance between the two sites is 148 km. According to the data of the Bird-Ringing Centre of Lithuania, the adults ringed as nestlings have never been recorded breeding in another colony. The majority of the black-headed gulls of Lithuanian populations spend up to 6–7 months in the coastal areas of the North and Baltic (the western part) Seas, which are the main wintering grounds [30]. The first colony is located on an island in the Kriv˙enai water reservoir, central Lithuania. Size of the colony ∼400 breeding pairs in 1999–2000; ∼800–900 breeding pairs in 2002. Geographical co-ordinates of the island 55◦ 05 N23◦ 48 E. Distance to the edge of the highway Kaunas–Klaipeda with traffic intensity >10,000 cars per day [31]—140 m. Distance to Kaunas town (400,000 inhabitants)—22.1 km. Intensively used agricultural land is dominating in the surroundings. The second colony is located on an island in Kretuonas Lake, eastern Lithuania. Size of the colony ∼200 breeding pairs in 1999; ∼300 breeding pairs in 2001–2002. Geographical co-ordinates of the island 55◦ 15 N26◦ 05 E. Distance to the nearest railway of regional importance—1.46 km. Distance to a road of district importance with a traffic intensity <1000 cars per day [31]—5.25 km. Distance to the nearest Švenˇcion˙eliai town (10,000 inhabitants)—8.6 km. Forests and lakes are dominating in the surrounding landscape. Agricultural land covers insignificant plots. Extensive agriculture is dominating in these

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Fig. 1. Map of the black-headed gull sampling sites in Lithuania.

areas. Agriculture pollution was decreasing in the region over the last decade. Studies performed in 1988–1993 by other authors indicated different amounts of benzo[a]pyrene in air, soil and mosses’ samples collected in the Kriv˙enai and Kretuonas surroundings. Concentration of benzo[a]pyrene in air samples from Kriv˙enai and Kretuonas was 1.8 ± 1.0 and 0.3 ± 0.3 ng/m3 [32], in soil samples 0–15 and 0–10 ␮g/kg [33], in mosses >100 and 10–20 ␮g/kg (dry weight) [34], respectively. 2.2. Collection and incubation of eggs The laying dates of the first egg in the observed nests have been registered at the beginning of the breeding season. The eggs in the observed nests were marked with indelible ink according their laying sequence. The clutches were collected in April–May in 1999–2002 at day 9–17 of incubation. The number of eggs collected from each colony usually was in the range of 2–10% of the estimated total number of laid eggs in the colony. Data on the sequence and interval of hatching indicate that black-headed gulls sometimes may start to incubate before laying the last egg in clutch [27]. However, since it is complicated to establish the start of incubation without direct observation of breeding pairs and since laying of eggs occurs at irregular inter-

vals, we made an assumption that the incubation starts from the day of clutch completion. In 2001 (Kretuonas colony) and in 2002 (both colonies) the eggs were incubated by adult birds in the nests until their collection for blood sampling. In all other cases, eggs were incubated in a vertical position for 24 h in a thermostat at 37.5 ◦ C (±0.5 ◦ C) and a relative humidity of 60% (±10%) before blood sampling. All eggs were measured (length and width) with calliper to the nearest 0.1 mm and weighed with a PESOLA spring balance to the nearest 1 g. 2.3. Treatment with benzo[a]pyrene Benzo[a]pyrene (Acros Organics, NJ, USA, CAS 50-32-8) was dissolved in pasteurised rapeseed oil using ultrasound disintegrator. Concentration of the working solution was 1 ␮g benzo[a]pyrene per 1 ␮l solution. The dose of benzo[a]pyrene was selected during a pilot experiment with three doses tested (20, 30 and 50 ␮g per egg; data not shown). It appeared that a dose of 20 ␮g per egg was ineffective, and a dose of 50 ␮g per egg was highly toxic causing mortality of 21 out of 22 treated embryos. Thus, for further experiments dose of 30 ␮g benzo[a]pyrene per egg was selected.

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Before treatment, the eggs were set up in an upright position with the blunt end at the top. The eggshell was opened using a preparatory needle at this end. The experimental solution of 30 ␮l, corresponding to a dose of 30 ␮g benzo[a]pyrene per egg was pipetted directly onto the inner shell membrane of the air cell. The hole was covered by a piece of parafilm (American National Can, Greenwich, CT), to ensure the embryo’s vitality until blood sampling. The shell of the control eggs was left intact. After the administration of benzo[a]pyrene the experimental and control eggs were incubated in a vertical position for 24 h in an incubator before blood sampling.

In every blood sample, 10,000 intact erythrocytes (polychromatic and normochromatic) were examined. The blood samples having ∼70% of squashed or damaged cells as well as blood samples from dead embryos were not examined. Prior to statistical analysis, frequencies of micronucleated erythrocytes (MNE) were transformed through average square root transformation Y = 0.5[(X)0.5 + (X + 1)0.5 ], where X is the number of MNE per 10,000 erythrocytes, and Y the transformed variable. All statistical analyses were performed using InStat V2.02 and SPSS/PC+ statistical packages. Statistical tests were chosen according to the nature of the data analysed.

2.4. Blood sampling and slide preparation After the incubation the shell and the inner shell membrane of the experimental and control eggs were removed at the blunt end before collection of the blood sample. Blood was collected with standard 5-ml single-use syringe from the highest volume vessel of the peripheral circulatory system of chorioallantoic membrane. One blood sample per embryo was collected. The obtained blood was spread out on slides immediately after sampling. The blood smears were air-dried and fixed for 10–15 min in 99.5% methanol (Merck, Darmstadt, Germany, CAS 67-56-1). Fixed blood smears were stained for 10 min in standard 4% Giemsa stain (Merck, Darmstadt, Germany, CAS 51811-82-6). After staining the slides were flushed out and intensively rinsed in demineralised water, then air-dried.

3. Results Eggs of similar dimensions were used for the analysis of micronucleated erythrocytes. Their average weight was 33.9 ± 0.2 g (95% confidence limits: 33.5–34.2 g), length 52.2 ± 0.1 mm (95% confidence limits: 51.9–2.4), and width 36.6 ± 0.1 mm (95% confidence limits: 36.5–36.7 mm). The spontaneous MNE frequency was determined in 119 blood samples of the black-headed gull embryos from the Kriv˙enai colony and in 97 blood samples from the Kretuonas colony (Table 1). Preliminary analysis indicated that the frequency of Table 1 Mean frequencies of micronucleated erythrocytes in embryos of black-headed gulls from two different locations in Lithuania

2.5. Analysis of micronuclei

Location

Year

Incubation time (days)

No. of embryos

MNE (‰) ± S.E.M

The examination of the stained slides was carried out using a Jenaval (Carl Zeiss, Germany) microscope under bright-field illumination (1250× with oil-immersion). The criteria described by Wolf and Luepke [15] were used for classifying the structure as micronucleus:

Kriv˙enai

1999 1999 2000 2002

≤13 ≥14 ≥14 ≤13

49 7 24 39

0.435 0.100 0.100 0.380

± ± ± ±

0.061 0.069a 0.050a 0.049

Kretuonas 1999 1999 2001 2002

≤13 ≥14 ≤13 ≤13

8 7 47 35

0.288 0.057 0.521 0.360

± ± ± ±

0.069 0.020b 0.063 0.056

• three-dimensionality of an object and its similarity to cell nucleus; • similar staining reaction and texture; • the size of the object does not exceed two-third of the size of cell nucleus; • distinct border and round or oval shape.

a P < 0.05 when compared to samplings from the Kriv˙ enai colony done at incubation time ≤13 days, Student’s t-test performed on average square root transformed data. b P < 0.05 when compared to all other samplings from the Kretuonas colony, Student’s t-test performed on average square root transformed data.

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Fig. 2. Dependence of spontaneous frequency of micronucleated erythrocytes in blood of embryos (n = 216) of the black-headed gull (L. ridibundus L.) in the Kretuonas and Kriv˙enai colonies on the eggs’ incubation length. Two eggs were analyzed after 9 days, 61—after 10 days, 51—after 11 days, 46—after 12 days, 18—after 13 days, 4—after 14 days, 15—after 15 days, 7—after 16 days, and 12—after 17 days of incubation. Vertical bars represent the standard deviation.

MNE per 10,000 erythrocytes did not follow a normal distribution (data not shown). However, average square root-transformed data were roughly normally distributed (P > 0.05, Kolmogorov–Smirnov test). Thus, average square root transformation was used for all further statistical analyses. A marked drop in the spontaneous MNE frequencies was observed after the 13 day of the eggs’ incubation (Fig. 2). The frequency of MNE was 0.424 ± 0.028‰ in the embryos incubated for ≤13 days versus 0.092 ± 0.035‰ in the embryos incubated for ≥14 days (P < 0.0001, Mann–Whitney U-test). Due to this reason, for further analysis all embryos were divided into two groups according to the length of incubation: incubated for ≤13 and for ≥14 days. There were no statistically significant differences in the frequency of MNE between the eggs incubated by adult birds in the nests until their collection for blood sampling and eggs incubated in a laboratory for the last 24 h in an incubator. Indeed, the mean frequency of MNE was 0.310±0.095‰ in 10 embryos incubated for 10 days in the nests plus 24 h in an incubator, and 0.503 ± 0.078‰ in 40 embryos incubated for 11 days in the nests (P = 0.1389, Mann–Whitney U-test). Similarly, in 18 embryos incubated for 11 days in the nests plus 24 h in an incubator the mean frequency of MNE was 0.400 ± 0.065‰, while in 27 embryos

incubated for 12 days in the nests it was 0.412 ± 0.073 (P = 0.8518, Mann–Whitney U-test). Thus, for further analysis the data of both incubation types were pooled together. Results of analysis of variance of mean square root transformed data (Table 2) indicated that the eggs’ incubation time was the only variable significantly (P = 0.0001) affecting the spontaneous frequency of MNE in embryos of black-headed gulls. Such variables as location of colony and year of sampling were not

Table 2 Results of analysis of variance of mean square root transformed spontaneous frequency of micronucleated erythrocytes in embryos of black-headed gulls from the Kriv˙enai and Kretuonas colonies Source of variability Main effects Location Year Incubation timeb Residual

Sum of squares 0.5183 4.0440 9.7037 135.2125

Total (corrected) 175.6262 a

d.f.

Mean square

1 0.5183 3 1.3480 1 9.7037

F-ratioa

P-value

0.80 2.09 15.07

0.3696 0.1021 0.0001

210 0.6439 215

All F-ratios are based on the residual mean square error. Incubation time is an indicator variable where 1: incubation time ≤13 days, 2: incubation time ≥14 days. b

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Table 3 Results of analysis of variance of mean square root transformed frequency of micronucleated erythrocytes in control and benzo[a]pyrenetreateda embryos of black-headed gulls Source of variability

Sum of squares

d.f.

Mean square

F-ratiob

P-value

Main effects Dose (A) Incubation timec (B) Interaction A × B Residual

1.5995 35.1483 2.5233 179.7857

1 1 1 293

1.5995 35.1483 2.5233 0.6136

2.61 57.28 4.11

0.1064 <0.0001 0.0426

Total (corrected)

224.8184

296

30 ␮g benzo[a]pyrene per egg was pipetted directly onto the inner shell membrane of the air cell of the egg. All F-ratios are based on the residual mean square error. c Incubation time is an indicator variable where 1: incubation time ≤13 days, 2: incubation time ≥14 days. a

b

statistically significant (P = 0.3696 and P = 0.1021, respectively). Eighteen embryos from the Kretuonas colony and 60 embryos from the Kriv˙enai colony were treated with benzo[a]pyrene for the last 24 h of incubation time. Analysis of variance indicated (Table 3) that eggs’ incubation time was the most significant variable affecting the frequency of MNE in both treated and control embryos (P < 0.0001), while influence of dose of benzo[a]pyrene was not significant (P = 0.1064).

At the same time, interaction between the dose and the incubation time was significant (P = 0.0426), indicating that there may be differences in response to benzo[a]pyrene at different incubation times. Indeed, the frequency of MNE did not increase in the embryos incubated for 13 days and exposed to 30 ␮g per egg of benzo[a]pyrene (Fig. 3; t = −0.3359, d.f. = 220, P = 0.7370, Student’s t-test; statistical analysis performed using average square-root transformation). On the contrary, in the group of embryos incubated for 14 days and exposed to 30 ␮g per egg of benzo[a]pyrene a statistically significant (t = 2.6453, d.f. = 73, P = 0.01) increase in MNE frequency was observed.

4. Discussion

Fig. 3. Effects of exposure of embryos of the black-headed gull (L. ridibundus L.) to benzo[a]pyrene for the last 24 h of incubation time on the frequency of micronucleated erythrocytes . Open columns—MNE frequency in embryos from eggs incubated for ≤13 days (55 control and 41 treated with benzo[a]pyrene), filled columns—MNE frequency in embryos from eggs incubated for ≥14 days (38 control and 37 treated with benzo[a]pyrene). Vertical bars represent the standard deviation.

Agricultural and urban areas are typical feeding areas of the black-headed gull during breeding, migration and wintering [35]. Therefore it is an appropriate object in the monitoring of dispersed pollution of genotoxic substances. Colonial breeding of this species enables a relatively simple collection of a sufficient number of samples for statistical analysis. As shown in our study, the micronucleus test can be successfully applied for the embryos of the black-headed gull. It allows avoiding complicated blood sampling from adult birds or nestlings that may cause a significant negative impact on reproduction of population studied. We found that the spontaneous frequency of micronucleated erythrocytes in embryos of black-headed gulls varied from 0.057 ± 0.020 to 0.521 ± 0.063‰

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and was greatly dependent on the length of the eggs’ incubation time (Table 1). The mean MNE frequency at the late stages of incubation (0.092 ± 0.035‰) was quite similar to the frequencies reported in erythrocytes of adult birds by other authors, especially Galliformes [36]. The mean MNE frequency at early stages of incubation (0.424 ± 0.028‰) was quite similar to the cumulated historic control frequency obtained at the same stage of incubation from hen’s embryos [15]. The dependence of the frequency of MNE on the age of the test species is quite well documented [36,37]. Often it is explained by the elimination of MNE from the reticuloendothelial system [38]. It is supposed that variation in efficiency of this elimination determines the different background numbers of MNE in different species [36]. Thus, it is likely that the statistically significant decrease in the spontaneous MNE frequency in the embryos of the black-headed gull after the 13 day of incubation is determined by the start of the spleen function, although precise developmental biology of this species is not known. On the other hand, the incubation period of eggs of the black-headed gull (22–24 days) is similar to that in the domestic hen (21–22 days). It has been demonstrated that on the 11 day of incubation a completely developed spleen is still absent in embryos of the domestic hen [23]. Alternatively, different MNE frequencies at different incubation periods may also be explained by the peculiarities of erythropoiesis in avian embryos. In the chick embryo, the major erythropoietic site up to the 18–20 day of incubation remains the yolk sac, while bone-marrow erythropoiesis starts at the 10–12 day and becomes the major site of erythropoiesis at the time of hatching [23]. Since the yolk sac is also one of the metabolically most important organs [39], there may be differences in the frequency of MNE in erythrocytes produced by yolk sac and bone marrow. No statistically significant difference in spontaneous MNE frequencies was found in the embryos from two colonies of the black-headed gulls, although they differed in anthropogenic load to the breeding and foraging habitats. It may appear that environmental contaminants present in these locations were not able to induce gross chromosomal damage that is usually detected by micronucleus assay [24]. Indeed, from the characteristics of the surrounding areas and some direct measurements [32–34] it may be concluded that the main difference between two colonies is in the

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concentration of PAHs, particularly, benzo[a]pyrene. However, benzo[a]pyrene at a concentration of 30 ␮g per egg was unable to induce micronuclei at early stages of incubation (≥13 days) but induced micronuclei at late stages (Fig. 3). Unfortunately, for the analysis of spontaneous micronuclei at late stage of incubation only 7 embryos from the Kretuonas colony and 31 embryos from the Kriv˙enai colony were available (Table 1). Thus, sensivity of future analyses of the intercolony differences may be improved by increasing sample size of embryos at a single defined stage of incubation. Similarly as in our study, no differences in the frequency of sister chromatid exchanges were found in the embryos of Herring gull collected from colonies on the Great Lakes Basin and from a control colony, despite different levels of contaminants present in the eggs [3]. Thus, analysis methods more sensitive than cytogenetic assays may be needed in order to detect genotoxic effects of contaminants present in the eggs. Indeed, a significantly higher rate of heritable minisatellite mutations in the Herring gull nestlings from industrially polluted areas than in the nestlings from areas with low inputs from anthropogenic sources were determined in one recent study [4,40]. Since the black-headed gull is a migrating species, it may accumulate some genotoxic contaminants in wintering areas and then excrete them into the eggs. Thus, knowledge of local conditions may be not sufficient for real estimation of amounts of xenobiotics present in the body and the eggs of this species. However, several recent studies indicate that even migrating species like common terns and double-crested cormorants are good indicators of local contamination. Concentrations of pollutants in eggs of these species tend to reflect their uptake by female foraging close to the colony in few days prior to egg laying [20,41]. We found a statistically significant increase of MNE numbers in the embryos incubated for 14 days and treated with benzo[a]pyrene for the last 24 h of incubation time. No such increase was detected in embryos incubated for ≤13 days (Fig. 3). This difference might be caused by the higher developmental stage of the embryonic liver and respective increase in metabolic activity of enzymes that are responsible for the activation of promutagens. Indeed, higher activities of xenobiotic-metabolizing enzymes were found at later embryonic developmental stages of such wild bird species as eider duck (Somateria mollissima)

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[42], tree swallow (Tachycineta bicolor) [43], and herring gull (Larus argentatus) [44]. In hen’s eggs, however, the activity of such enzymes remains quite uniform during the whole developmental period [42]. In the experiments with domestic hen’s embryos treated with different genotoxic compounds, the highest MNE frequencies were found when the application of the test substance took place 72 h before blood sampling. The 24-h exposure reveals an extremely weak response [17]. However, in an earlier study of these authors a 24-h exposure to genotoxins was sufficient to induce a significant increase in MNE frequency [15]. Based on the latter findings, we used 24 h exposure to benzo[a]pyrene. Such duration of exposure was enough to induce MNE in embryos incubated for ≥14 days, but not for ≤13 days. As mentioned above, this difference may be explained in terms of different metabolic activity at these periods of incubation. However, this difference may also be explained by high background level of MNE in embryos at early incubation time that simply masks weak effects caused by short exposure time or low dose of the genotoxic compound. Some authors suggest that species with the highest values of MNE are the most suitable for testing of genotoxic effects in the micronucleus test [37]. This might not be true for the black-headed gull in the light of present findings. At least in the case of this species, embryos at the late periods of incubation (14 days) showing lower spontaneous frequency of MNE seems to be more sensitive to genotoxic action of xenobiotics and, thus, more suitable for genetic monitoring purposes. However, experiments on living avian embryos during the second half of the incubation period are considered as animal experiments in some countries, like the UK [45]. In such case the collection of blood samples from the adult birds before or well after the reproductive period is the only non-destructive method in the monitoring of genotoxic damage in wild colonial bird populations. Alternatively, the possibility to use other genetic endpoints such as measured in the Comet assay [7,8] at the first half of incubation period may be evaluated. References [1] N.M. Belfiore, S.L. Anderson, Effects of contaminants on genetic patterns in aquatic organisms: a review, Mutat. Res. 489 (2001) 97–122.

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