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Immune Status, Carotenoid Coloration, and Wing Feather Growth in Relation to. Organochlorine Pollutants in Great Black-Backed Gulls. Jan Ove Bustnes,1 Kai ...
Arch. Environ. Contam. Toxicol. 53, 96–102 (2007) DOI: 10.1007/s00244-005-0269-3

Immune Status, Carotenoid Coloration, and Wing Feather Growth in Relation to Organochlorine Pollutants in Great Black-Backed Gulls Jan Ove Bustnes,1 Kai Ove Kristiansen,1,2 Morten Helberg1,2 1 2

Norwegian Institute for Nature Research, The Polar Environmental Centre, N-9296 Tromsø, Norway Faculty of Science, Department of Biology, University of Tromsø, N-9037 Tromsø, Norway

Received: 4 January 2006 /Accepted: 25 November 2006

Abstract. Previous ecotoxicological studies have documented relationships between residues of various organochlorines (OCs) and immune status, carotenoid colors, and wing feather growth in different bird species. In this study, the density of white blood cells (WBC), carotenoid colors, and length of the same feathers on each wing were measured in breeding great black-backed gulls (Larus marinus) on the coast of northern Norway, and related to the blood residues of five OCs, including HCB (hexachlorobenzene), b-HCH (b-hexachlorocyclohexane), p,p¢-DDE (p, p¢-dichlorodiphenyldichloroethylene), oxychlordane, and polychlorinated biphenyl (PCB), in addition to SOC. Neither, WBC density nor carotenoid colors were significantly related to blood residues of any of the OCs, suggesting that OC levels may have been too low to significantly affect these outcome parameters. However, in the colony where the OC concentrations were highest, there was a weak but significantly positive relationship between the probability of having different length of feathers on each wing and levels of PCB and SOC, in males. Thus varying length of the wing primaries may reflect adverse impacts of OCs in great black-backed gulls. However, in gulls with moderate levels of OCs, it is probably not a sensitive indicator of progressing ecological impacts of OCs, since such adverse ecological relationships were found in the breeding colonies where there were no relationships between differences in wing feather lengths and OCs.

Organochlorine compounds (OCs), such as polychlorinated biphenyls (PCB) and p,p¢ - dichlorodiphenyldichloroethylene (p,p¢-DDE), are known to cause a number of adverse effects in birds, even in areas distant from their sources. For example, in the glaucous gull (Larus hyperboreus), an arctic marine top predator, a number of effects have been related to OCs at Bear Island, in the Norwegian Arctic (Fig. 1), including poor reproductive performance and reduced survival (Bustnes 2006). Prior to severe ecological impacts one would, however,

Correspondence to: J. O. Bustnes; email: [email protected]

expect measurable biochemical and physiological changes at the individual levels, which may be used as biomarkers of such ecological effects (Peakall 1994). In the glaucous gull, such potential parameters include endocrine disruption (Verreault et al. 2004), immunological effects (Bustnes et al. 2004), and wing feathers asymmetries (Bustnes et al. 2002). Hence, changes in these parameters may function as early warnings for progressing ecological effects, which finally will result in population declines (e.g., Peakall 1994; Handy et al. 2003). The levels and effects of OCs on gull species in the near Arctic areas, such as the coast of northern Norway, have not been studied extensively. The great black-backed gull (Larus marinus) is common all over the northern Atlantic, and previous studies have shown that it may have relatively high levels of OCs (Weseloh et al. 2002; Pusch et al. 2005). Moreover, on the coast of northern Norway poor reproductive performance has been linked to high OC levels (Helberg et al. 2005). The aim of this study was to examine if different parameters were related to various OCs, and may thus be useful as early warning indicators of progressing ecological effects. Firstly, white blood cell counts provide a general and nonspecific indicator of the immune status of individuals (Averbeck 1992; Sheldon and Verhulst 1996), and in herring gulls (Larus argentatus), Caspian terns (Sterna caspia) and glaucous gulls, OC concentrations have been positively related to the level of some white blood cells (Grasman et al. 1996, 2000; Grasman and Fox 2001; Bustnes et al. 2004). In this study, we related blood residues of different OCs to the ratio between white and red blood cells (lymphocyte/erythrocyteratio) on blood smears. Secondly, carotenoids are central components in animal color signals, and thus of importance for the social behavior in many bird species (Møller et al. 2000). They also play important roles in immune functions by stimulating different immunological components (Bendich 1989; Chew 1993; van Poppel et al. 1993; Olson and Owens 1998), and act as antioxidants and protectors of biologically important molecules from the damaging potential of free radicals (Krinsky 1998; Edge et al. 1997). A recent experimental study of American kestrel (Falco sparverius) found that PCB disrupted both coloration and plasma carotenoid levels (Bortolotti et al.

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backed gulls at the small island Lamøya. In 2001, about 70 pairs of great black-backed gulls bred on Lamøya. In all three colonies, nests were marked and followed from the start of egg laying in late April and first half of May, until hatching. In May and June, adult breeding birds were caught on their nests using a nest trap (Bustnes et al. 2001a) and individually marked with PVC leg bands and numbered steel bands. Blood (10 ml) was sampled from the wing vein with a syringe. Body condition was indexed using body mass (to the nearest 10 g), controlling for body size (head and bill length [€ 0.1 mm] measured with sliding calipers) (Garca-Berthou 2001). In gulls, males are larger than females (Coulson and Thomas 1983). In all nests where both partners were caught, the largest bird was assumed to be the male. Even when only one bird from each pair was caught, the sex determination of the birds was possible after catching by comparing the color-ringed bird with the other bird in the pair using a telescope. The males were assumed to be larger, and had a different head profile with a more prominent eyebrow and a relatively larger bill, compared to females. When comparing head and bill length between the largest (males) and the smallest birds (females) in pairs, there was no overlap, suggesting that this measurement can be used for sex determination in great black-backed gulls (Helberg et al. 2005), as has been documented for other gulls by Coulson and Thomas (1983; see also Helberg et al. 2005, Butler and Butler 1983, Mawhinney and Diamond 1999).

Immune status

Fig. 1. The study colonies of great black-backed gulls along the coast of northern Norway, including Bear Island

2003a). However, so far no study has to our knowledge related carotenoid coloration to levels of OCs in wild birds. Here we measured carotenoid color expressions in hard and soft tissues in both males and females, and related it to OC concentrations in the blood. Finally, asymmetry in morphological measurements, often referred to as fluctuating asymmetry (FA), may provide an indication of developmental stress (Møller and Swaddle 1997). Bustnes et al. (2002) showed that glaucous gulls with high concentrations of various OCs in the blood were more likely to have % different lengths of the same primary feathers on each wing. In this study, we measured primaries on each wing of individual great black-backed gulls and related this measurement to concentrations of different OCs in the blood.

Materials and Methods Great black-backed gulls were studied in three colonies (Fig. 1) in 2001: Hornøya (70 22¢ N, 31 10¢ E) in eastern Finnmark County; Loppa (70 20¢ N, 21 24¢E) in western Finnmark; and Sleneset (66 35¢ N, 12 60¢E) Nordland County. Details about the study area at Hornøya may be found in Furness and Barrett (1985). In 2001, there were about 200 breeding pairs of great black-backed gulls at this island. The Loppa study area has been described in Helberg et al. (2005). In 2001, there were about 150 pairs of great black-backed gulls breeding at Loppa. At Sleneset, we studied the great black-

Immune status was only studied at Loppa. Blood smears were fixed in methanol and dried in room temperature. The smears were stained with the May-Grnewald-Giemsa method. To determine the white blood cell/red blood cell (erythrocyte) ratio, the average of white blood cell and erythrocyte counts from six randomly chosen areas central on the smear were used. We counted both lymphocytes and heterophils, but only lymphocytes were sufficiently frequent on the smears to be used in further analysis. Hematocrit (the percentage of red blood cells in a blood sample) for each bird was determined by centrifuging a portion of the blood sample in a capillary tube for 195 seconds at 11500 rpm with a Compur Mini Centrifuge. Then, we measured the length of the red blood cells and plasma on the capillary tube with sliding calipers, and calculated the percentage of red blood cells (hematocrit value). Furthermore, we multiplied the hematocrit value by the lymphocyte/erythrocyte ratio on the smears to get an estimate of the level of circulating lymphocytes for each bird. Both lymphocyte densities and hematocrit were quantified twice (repeatability between the two estimates: r2 = 0.54 and r2 = 0.98, respectively; n = 20 in both cases).

Color measurements Color was also studied at Loppa only. Analysis of the carotenoidbased tissue color at the ring, gape, and bill was taken using a portable spectrometer (Avantes). Measurements were taken with a 2.5-mm reflection probe (FCR-7UV200) connected to a Halogen Light Source (HL-2000) and a spectrometer (AVS-USB2000) by fiber optic cables. The light from the light source was coupled via a standard SMA905 connector into a fiber bundle consisting of 6 fibers and carried to the probe end. The surface reflected light back into a 7th fiber. This fiber transferred the data to the output SMA905 connector, which was coupled to the spectrometer. The data from the spectrometer was passed into a computer with Spectrawin 4.2 software. The measurements were relative and referred to a standard white reference tile (WS-2). The color measurements hue, saturation and darkness were

J. O. Bustnes et al.

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Table 1. Concentrations of different OCs in blood (ng/g, wet weight) and length of the third primaries on the wings of female and male great black-backed gulls in three colonies at the coast of northern Norway Females Colony Variables Homoya HCB b-HCH Oxychlordane p,p¢-DDE PCB SOC Web length right (mm) Web length left (mm) Loppa HCB b-HCH Oxychlordane p,p¢-DDE PCB SOC Web length right (mm) Web length left (mm) Sleneset HCB b-HCH Oxychlordane p,p¢-DDE PCB SOC Web length right (mm) Web length left (mm)

N

Mean

Median

Males

SD

Min

Max

N

Mean

Median

SD

Min

Max

62 62 62 62 62 62 62 62

3.47 0.16 1.64 11.11 59.13 74.77 286.59 286.78

2.96 0.14 1.41 10.71 50.44 66.19 286.95 286.30

2.18 0.12 1.24 5.66 42.50 49.42 7.02 6.55

0.62 0.03 0.22 2.31 10.54 13.62 271.90 272.90

16.26 0.84 8.15 28.81 257.35 308.58 302.00 300.00

51 51 51 51 51 51 51 51

4.35 0.24 2.63 18.29 110.25 134.78 299.47 300.67

3.72 0.16 2.13 15.46 92.58 113.85 300.80 301.70

2.54 0.22 2.44 10.85 85.95 98.92 7.27 8.01

1.75 0.06 0.60 5.13 24.57 33.92 275.00 276.10

17.14 1.36 16.84 55.98 508.09 594.48 314.50 314.80

23 23 23 23 23 23 23 23

6.37 0.32 4.24 32.56 103.90 145.87 289.83 289.65

4.14 0.20 1.79 20.42 78.51 112.22 286.50 287.50

7.59 0.33 7.24 36.52 84.64 130.07 8.04 7.63

0.69 0.07 0.41 3.08 8.66 12.59 279.00 279.00

35.20 1.52 35.11 158.07 349.60 538.49 306.00 307.00

30 30 30 30 30 30 30 30

4.34 0.22 3.76 26.10 99.65 132.77 307.23 306.95

4.03 0.19 2.54 24.47 91.57 112.52 305.50 306.75

2.13 0.15 4.01 13.44 57.47 70.33 8.73 9.04

0.85 0.05 0.74 4.03 16.42 21.95 294.00 294.00

9.52 0.74 19.1 58.67 233.01 305.31 329.00 332.00

14 14 14 14 14 14 14 14

4.28 0.29 3.77 41.27 163.66 212.03 285.64 285.21

4.36 0.26 2.93 39.13 107.89 176.22 286.00 285.00

1.94 0.15 2.35 29.85 113.21 124.91 6.28 6.93

1.47 0.09 0.84 4.96 54.01 60.96 276.00 276.00

6.76 0.66 7.91 119.59 397.49 442.74 294.00 294.00

10 10 10 10 10 10 10 10

3.93 0.30 3.30 38.27 178.40 222.98 299.00 299.10

3.70 0.30 3.51 34.82 131.53 185.71 295.00 295.50

1.27 0.11 1.56 23.11 111.34 127.87 12.84 12.85

2.37 0.16 0.87 8.56 86.66 110.83 283.00 283.00

5.94 0.51 5.73 79.83 431.94 505.60 324.00 324.00

recorded. Hue represents the wavelength of the color, while saturation and darkness can, respectively, be understood as the density of pigmentation and the amount of black pigmentation. Hue is given in inverse scale, that is, lower values of hue means redder coloration. To avoid misunderstandings, we have, therefore, inverted the scale so higher values of hue mean redder coloration. The colors were measured at 12 standard points; two points at each eye-ring, four points in the gape, and four points at the bill (two at the top of the bill and one at each bill spot). One of the points was measured twice and used for estimating the repeatability (hue: r2 = 0.94, p < 0.0001; saturation: r2 = 0.93, p < 0.0001; darkness: r2 = 0.94 p < 0.0001; n = 50). The mean value of hue, saturation, and darkness for these points were used in statistical analyses. We will use two different mean values in the following: ‘‘soft tissues’’ is the mean value of soft tissues (eye ring and gape) and ‘‘bill’’ is the mean value of points taken at the bill. We focus on hue and saturation in further analyses because they represent, respectively, the type and amount of carotenoids pigmentation. Consequently, they are a qualitative and quantitative measure of carotenoids and, therefore, are the most relevant parameters. We use the term ‘‘color intensity’’ to refer to both parameters.

exceeded the maximal measuring range of calipers (200 mm). Since this is a long-term study, it was imperative not to harm the birds, and we did not remove any wing feathers. To measure the Primaries, we separated the feathers at the base, and to reduce stress we chose to measure only one feather on each wing. In this study, repeatability of the feather measurement was tested at Loppa by measuring the feathers twice (r2 = 0.96, p < 0.0001; n = 21). In glaucous gull, we have also shown high repeatability when the feathers are measured twice by the same person (Bustnes et al. 2002), but true repeatability may only be measured by recapturing the birds, which turned out to be impossible for great black-backed gulls, However, since we only used the deviation in wing feather length correlated to the blood concentrations of OCs, and made no attempts to compare wing feather asymmetries between the different populations, we argue that repeatability of the measurement is not critical. If measuring error was larger than potential differences between feathers on each wing, finding any relationships with OCs, for which we had no information at the time of feather measurement, would be very unlikely.

Chemical analyses Wing feather length To get a measure of wing symmetry, we used the deviation between the web lengths (the distance between the start of the growth zone of the web and the feather tip) of the third primary on each wing (Bustnes et al. 2002). The web length was used because a distinct color pattern (white vs. grey) could be identified at the lower base of the primary shaft, which was suitable for measuring (nearest mm) with a ruler. A ruler was used because the length of the web (> 273 mm; Table 1

The OC analyses were carried out at the Environmental Toxicology Laboratory at the Norwegian School of Veterinary Science/National Veterinary Institute. All details regarding analyses of OCs can be found in Andersen et al. (2001) and Bustnes et al. (2004). We used wet weight of the different OCs in the blood as a measure of an individualÕs OC burden. The following PCB congeners were determined (IUPAC numbers; 99, 118, 138, 153, 170, and 180). In the analyses, we used the sum of these congeners, denoted PCB. The other OCs analysed were hexachlorobenzene (HCB), b-hexachlorcyclohexane (b-HCH),

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oxychlordane, and p,p¢-dichlorodiphenyldichloroethylene (p,p¢-DDE). We also used the sum of all measured OCs, denoted: SOC. Other studies suggest a relatively high short- and long-term stability of the blood levels of OCs in incubating gulls and that it is a reliable, relative (compared to other individuals) measurement for body burden of an individual (Henriksen et al. 1998; Bustnes et al. 2001b). This has also been shown in great black-backed gulls (Bustnes et al. 2005).

Statistical analyses Statistical analyses were performed with the SAS System software (SAS 1999). The OC values were Log10-transformed to approximate normal distributions. After transformation, we tested the concentrations of the different OCs for normality for each colony, using the ShapiroWilk test in SAS; all distributions were normal (0.087 p < 0.96; totally 18 tests) apart from weak deviations for oxychlordane at Loppa (p = 0.03) and HCB at Hornøya (p = 0.03). Since F-statistics are robust to a small deviation from normality (Kleinbaum et al. 1998), we applied parametric statistics on the whole data set. In studies with multiple comparisons, one might adjust p values. However, we did not conduct such adjustments in accordance with Rothman (1990) and Nakagawa (2004). All statistical tests were, however, two-tailed and p values < 0.05 were considered statistically significant. We used the GENMOD Procedure, Type 1 statistics. Immune status and colors were analyzed by multiple regressions while feather asymmetry was analyzed by logistic regression with a binary response variable (equal primary length on the two wings [0] or unequal primary length [1]). In the analysis, the best statistical models were chosen by backward selections, with white blood cell density, color property, or wing feather asymmetry as the response variables, and concentration of OCs in the blood and a set of potential confounding variables, such as gender and body condition, as independent variables. We started with a full model, removing independent variables one at a time if they were not significant (p > 0.05).

Results The concentrations of OCs in blood of males and females from the different colonies are shown in Table 1. The highest concentrations were found at Sleneset, while the lowest were found at Hornøya.

colors except for the bill, than birds in poor condition. We, therefore, controlled for body condition in all analyses. There were near significant, positive relationships between the saturation of soft tissue and PCB (F 1,45 = 3.15, p = 0.083), and also for saturation in bill color and PCB (F 1,45 = 2.88, p = 0.097). No relationships between the other OCs, including SOC, and color measurements were found (0.11 < p < 0.97). Hence, birds in good condition were more strongly colored, but high PCB also tended to be related to more saturated colors.

Wing feather length and OCs Wing feather lengths in different colonies can be found in Table 1. Body condition did not affect the probability of having different lengths of the wing feathers in any of the colonies (0.14 < p < 0.81). At Loppa (n = 53) and Hornøya (n = 113), there was no effect of sex on the probability of having different feather lengths (p = 0.59 and p = 0.44, respectively), and no interactions between blood residues of any of the five OCs and sex (0.12 < p < 0.85). There were no effects of any OCs on the probability of having different lengths of the wing feathers either at Loppa (0.51 < p < 0.91) or at Hornøya (0.16 < p < 0.90). At Sleneset, however, there were significant interactions between sex and PCB (p = 0.027) and SOC (p = 0.031), and near significant interaction for HCB (p = 0.072), oxychlordane (p = 0.071), and p,p¢-DDE (p = 0.051), but not for b-HCH (p = 0.45). A separate analysis of the sexes showed that males (n = 10) with high blood residues of PCB had a higher probability of having different lengths of the wing feathers than birds with low levels (v2 1,8 = 6.69 p = 0.0097, estimate = 11.35 € 6.52 SE). The same was also found for ROC (v2 1, 8 = 6.15 p = 0.0131; estimate = 10.35 € 5.74 SE), while it was near significant for HCB (p = 0.078), oxychlordane (p = 0.095), and; p,p¢-DDE (p = 0.082), but not for b-HCH (p = 0.57). In females at Sleneset (n = 14), there were no significant relationships between the probability of having different lengths of the wing feathers and any of the OCs (0.36 < p < 0.98).

Discussion White blood cell density and OCs The density of white blood cells (lymphocyte/erythrocyte ratio) was not related to sex (p = 0.86) or body condition (p = 0.52). Furthermore, there were no significant relationships between any of the OCs and density of white blood cells (0.19 < p < 0.50, n = 50).

Carotenoid colours and OCs When controlling for sex, body condition had a significant or near significant effect on the reddishness of the soft tissue (F 1, 46 = 3.86, p = 0.056) and the saturation of both soft tissues (F 1,46 = 6.48, p = 0.014) and the bill (F 1,46 = 4.47, p = 0.04), but not to the reddishness of the bill (p = 0.49). This suggests that birds in good condition in general have brighter

White blood cell density White blood cell counts are general, non-specific indicators of health status, and high counts may indicate either increased infection or high immunocompetence (Siva-Jothy 1995; Sheldon and Verhulst 1996; Norris and Evans 2000). In herring gull, glaucous gulls, and Caspian terns, positive relationships have been found between p,p¢-DDE and PCB and levels of some types of white blood cell levels (Grasman et al. 1996, 2000; Grasman and Fox 2001; Bustnes et al. 2004). However, in these species OC concentrations were higher than in the great black-backed gulls investigated at Loppa in 2001; i.e., glaucous gulls may have 3–6 times higher PCB and 2–6 times higher p,p¢-DDE levels in the blood (Bustnes et al. 2003). In general, herring gulls and Caspian terns in the Great Lakes of North- America have even higher OC concentrations

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than arctic glaucous gulls and sub-arctic great black-backed gulls (Grasman et al. 2000; Grasman and Fox 2001). It is, thus, possible that the OC concentrations at Loppa were too low to affect the proliferation of white blood cells, as measured by the relatively crude method of WBC counts on smears. Consequently, at OC concentrations found in great black-backed gulls in our study area, WBC counts do not seem to be a good indicator of progressing ecological effects of OCs since several OCs have been shown to be related to reproductive performance at Loppa (Helberg et al. 2005).

Carotenoid colors Carotenoids are important for immune functions (Bendich 1989; Chew 1993; Olson and Owens 1998) and detoxification (Edge et al. 1991 Krinsky 1998). Because of these functions, and because the birds must acquire carotenoids through the diet, they are important in animal signaling; i.e., they are considered honest signals. That is, such signals cannot be faked by inferior individuals (Zahavi 1975; Møller et al. 2000). There is now increasing evidence from wild birds that carotenoid signals reflect individual quality (Massaro et al. 2003), including in the great black-backed gull (Kristiansen et al. 2006). Any disruption of such signals is, therefore, potentially serious with regard to social behavior. There are several studies that have linked colors in birds to pollution, both OCs (Bortolotti et al. 2003a, b; McCarty and Secord 2000), heavy metals (Eeva et al. 1998), and radioactivity (Camplani et al. 1999; Møller and Mousseau 2001). Bortolotti et al. (2003a) also showed experimentally in American kestrels that PCB disrupted both color expressions and plasma carotenoid levels, and suggested that PCB confounded carotenoids through endocrine disruption. However, in this study no significant associations were found between levels of OCs and color intensity. The only near significant result was a positive trend for color saturation and PCB in males. This may be of interest since Bortolotti et al. (2003a) found positive associations between PCB and plasma carotenoids in juvenile American kestrels. Hence, under some circumstances, PCB may enhance the carotenoid colors. However, this study suggests that within the range of OC levels found in the great black-backed gulls, other factors, such as body condition, are more important in affecting carotenoid color expressions (Kristiansen et al. 2006). Thus, carotenoid colors are not a sensitive biomarker for progressing ecological effects of OCs in great black-backed gulls.

(2002) showed that blood residues of OCs were related to the probability of showing asymmetry in primaries in arctic breeding glaucous gulls, indicating that OCs were a considerable stressor. In this study, there was no general relationship between the probability of having unequal lengths of wing feather and OCs, but at Sleneset, the colony with the highest OC levels, males with high PCB were significantly more likely to have different lengths of the primaries. On its own, this finding may be of little importance since it was the only significant result, and the sample size from this colony was small. However, data that will be presented in a separate paper show that birds from this colony are especially vulnerable to die during winter and that body condition and OCs influenced their survival probability (J. O. Bustnes et al. unpublished data). This suggests that they suffered from a multitude of stresses, which seem to affect their ability to grow symmetric feathers. Different lengths of wing feathers may, thus, be an indicator of current ecological effects of OCs in great blackbacked gulls. However, it is clearly not a sensitive measure since we found that even in the colonies where there was no relationship between the probability of having different wing feather lengths and OCs, there were several ecological effects linked to OCs (Helberg et al. 2005; J. O. Bustnes et al. unpublished data). One of the problems with measures of wing feathers, as used in this study, is that the feathers are grown prior to the year of investigation. However, based on high correlations of OCs in individual glaucous gulls between years (Bustnes et al. 2001b), Bustnes et al. (2002) concluded that this was a minor problem. Moreover, in great black-backed gulls, the levels of OCs may change between years on the population level, but the relative relationship between individuals seems rather constant (Bustnes et al. 2005). In conclusion, within the range of OCs found in great blackbacked gulls, differences in wing feather lengths do not seem to be a sensitive indicator of progressing ecological effects. However, we would recommend a more thorough test of the method, for example, by using dead birds, where were a very accurate measure of several wing feathers can be acquired.

Acknowledgments. We are grateful to John Andre Henden, Øystein Varpe, and Espen Dahl for valuable help during field work, and Anuschka Polder and her team at the Norwegian National Veterinary Institute for conducting the OC analyses. We also thank two anonymous reviewers for comments that greatly improved an earlier draft of the manuscript. The study was funded by the Norwegian Research Council (Project number 141443/S30).

References Length of wing feathers In birds, the optimal phenotype is perfect wing symmetry and there is a high aerodynamic cost of asymmetry (Thomas 1993). Many studies of fluctuating asymmetry have focused on morphological traits that individuals grow once, such as bones or fins. In seabirds, however, feathers are molted and grown annually, and it has been shown that various stresses may lead to asymmetry in wing feathers (Swaddle and Witter 1994; Witter and Lee 1995; Carrascal et al. 1998). In passerines asymmetry in primaries may increase with proximity to heavy metal pollution sources (Eeva et al. 2000). Bustnes et al.

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