Lead concentrations in feathers and blood of common blackbirds (Turdus merula) and in earthworms inhabiting unpolluted and moderately polluted urban areas

Science of the Total Environment 371 (2006) 197 – 205 www.elsevier.com/locate/scitotenv

Lead concentrations in feathers and blood of common blackbirds (Turdus merula) and in earthworms inhabiting unpolluted and moderately polluted urban areas R. Scheifler a,⁎, M. Cœurdassier a , C. Morilhat a , N. Bernard a , B. Faivre b , P. Flicoteaux a , P. Giraudoux a , M. Noël a , P. Piotte c , D. Rieffel a , A. de Vaufleury a , P.-M. Badot a a

Université de Franche-Comté, Department of Environmental Biology, EA 3184 UsC INRA, Place Leclerc, 25030 Besançon Cedex, France b Université de Bourgogne, UMR CNRS 5561 Biogéosciences, Equipe Ecologie Evolutive, 6 Boulevard Gabriel, 21000 Dijon, France c Réseau d'Observation de la Faune Vertébrée de Franche-Comté, Groupe Naturaliste de Franche-Comté, Maison Régionale de l'Environnement, 15 rue de l'industrie, 25000 Besançon, France Received 28 June 2006; received in revised form 8 September 2006; accepted 9 September 2006 Available online 19 October 2006

Abstract Despite the dramatic decrease of atmospheric lead (Pb) concentrations in urban areas of most industrialised countries, we hypothesised that urban common blackbirds (Turdus merula) may still be contaminated by Pb concentrations of toxicological concern due to transfer from soil through the food chain. We sampled blackbirds and earthworms, one of their main preys, in Besançon, a middlesize city of Eastern France (where atmospheric Pb concentrations decreased from 0.5 μg/m3 in 1987 to nearly 0 in 2002) and in a rural reference site. Lead concentrations were determined in the tissues of the different functional groups of earthworms (anecic, epigeous and endogeous) and in blood, washed and unwashed outermost tail feathers and breast feathers of blackbirds. Fresh masses and an index of individual body condition were measured in the two blackbird populations as biomarkers of possible toxic effects. Lead concentrations in earthworms did not differ among functional groups but were significantly higher in urban individuals than in rural ones. Concentrations in outermost tail feathers, breast feathers and blood were significantly higher in urban blackbirds (7.75 ± 4.50, 3.15 ± 1.77 and 0.15 ± 0.09 μg/g, respectively) than in rural individuals. In urban blackbirds, concentrations in washed and unwashed outermost tail feathers allowed estimating the external contamination (probably due to deposition of dusts and/or to excretion of the uropygial gland) at 37% of the total Pb concentration of the unwashed feathers. Remaining 63% should be linked to food chain transfer of persistent Pb from urban soils. Among the 23 sampled blackbirds, 4 of them (3 in the urban site and 1 in the rural site) exhibited blood Pb concentrations higher than the benchmark value (0.20 μg/g) related to subclinical and physiological effects in birds. Variations in body condition index were not correlated to Pb concentrations in blackbird tissues. Present results suggest that Pb may still be of environmental concern for blackbirds in urban areas because of the persistence of Pb in soils and its transfer through the food chain. © 2006 Elsevier B.V. All rights reserved. Keywords: Urban areas; Bird populations; Heavy metals; Ecotoxicological risk

⁎ Corresponding author. Tel.: +33 381 665 740; fax: +33 381 665 797. E-mail address: [email protected] (R. Scheifler). 0048-9697/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2006.09.011

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1. Introduction Since the 1970s, most of the industrialised countries have adopted environmental measures to phase out Pb additives in motor fuels (Ancillotti and Fattore, 1998). In France, Pb compounds in gasoline are forbidden since January, 1st 2000 and Pb remains in fuel as trace only. Consequently, Pb atmospheric emissions in Metropolitan France dropped from 4264 t in 1990 to 217 t in 2002 (CITEPA, 2004) and, nowadays, in most French cities, average annual Pb concentrations in the air are around 0.03 μg/m3 (http://www.ecologie.gouv.fr/ article.php3?id_article=1950). The reduction of atmospheric Pb concentrations may incite policy makers to consider that Pb is no longer of environmental concern, at least in industrialised countries. However, like other metallic trace elements (MTE), Pb is non-degradable and, due to past high emissions, urban soils will remain contaminated for a long time. Although background concentrations in unpolluted soils usually do not exceed several tens of μg/g (dry soil), concentrations of several hundreds of μg/g are common in urban areas and values as high as 15,000 μg/g have been reported in urban gardens of Great Britain (Kabata-Pendias, 2000). In birds, Pb residues were commonly assessed in different tissues (e.g. liver, kidney; Weyers et al., 1985; Burger et al., 1997; Dauwe et al., 2005a) but measures of feather and blood concentrations are more advantageous because they constitute a non-destructive indicators of bird exposure. It is generally assumed that concentrations in feathers are representative of longterm exposure, i.e. the few weeks of feather growth (Burger, 1993), while blood concentrations reflect shortterm exposure (immediate dietary intake; Furness, 1993). Many studies used MTE concentrations (including Pb) in various types of feathers for biomonitoring purposes (Dauwe et al., 2003; Dauwe et al., 2004; Battaglia et al., 2005; Dauwe et al., 2005a) but blood concentrations, moreover, can be compared to benchmark values that relate to subclinical, clinical or lethal effects (Franson, 1996; Pain, 1996; Friend and Franson, 1999). In blood, 0.20 μg/g Pb (wet mass) was retained as the lowest limit for subclinical and physiological effects, e.g. delta-aminolevulinic acid dehydratase (ALAD) depression (Franson, 1996). Most of the literature available on Pb concentrations in birds concerns waterfowls (Blus et al., 1999; Tavecchia et al., 2001; Levengood, 2003) and raptors (Pain et al., 1995; Mateo et al., 1999; Clark and Scheuhammer, 2003), both groups being mainly intoxicated by lead shot ingestion or by water, sediments or soils contaminated by hunting activities. Passerines have

also been investigated but mainly in highly-contaminated areas like mining or metallurgic plant vicinities (Eens et al., 1999; Dauwe et al., 2000; Janssens et al., 2002; Dauwe et al., 2005a) and trap and skeet ranges (Vyas et al., 2000). Little data is available on Pb concentrations in birds inhabiting moderately polluted areas like cities (Chandler et al., 2004) although one can hypothesise that persistence of Pb in soils and possible transfer through the food chain may still lead to contamination of bird populations. The risk may be higher for bird species that consume a large (or even predominant) part of soil fauna in their diet (Grue et al., 1984), notably earthworms, because these invertebrates may accumulate high concentrations of Pb in their tissues (Sample et al., 1999; Heikens et al., 2001). Among insectivorous birds, the common blackbird (Turdus merula), a widespread and ubiquitous species of Western Palearctic, feeds on insects and fruits (from late summer to early winter) but its annual diet is dominated by earthworms (Weyers et al., 1985; Cramp, 1988). In this study, we hypothesised that urban blackbirds were more contaminated than rural individuals and that there was still Pb concentrations of toxicological concern in the urban population despite the huge reduction of atmospheric Pb concentrations in French cities. To test these hypotheses, we compared Pb concentrations in blood and feathers from a reference population inhabiting a rural unpolluted area to an urban population inhabiting the middle-size city of Besançon (Eastern France, 176,000 inhabitants in the city and its suburbs). To document the possible trophic transfer, Pb concentrations in earthworms were determined in both rural and urban sites. Then, the Pb concentrations in the blood of the blackbird populations were compared to concentrations reported to be toxic for birds in the literature. Relationships between Pb concentrations in feathers or blood and individual body condition index were also investigated. 2. Materials and methods 2.1. Sample collection The study was conducted in 1999 and 2000 on an urban population living in 2 parks of Besançon, Eastern France, and on a rural population living in Vernierfontaine, a small village where landscape is dominated by grassland with dense hedgerow network, at a 25 km distance, south of Besançon. Atmospheric Pb concentrations in Besançon (http://www.ecologie.gouv.fr/article. php3?id_article=1950 and data graciously provided by the “Association pour la surveillance de la qualité de

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Fig. 1. Temporal trend of average annual atmospheric Pb concentrations (μg/m3) in Besançon from 1987 to 2003. Data from ASQUAB and http://www.ecologie.gouv.fr/article.php3?id_article=1950.

l'air dans l'agglomération bisontine et le Sud FrancheComté”, ASQUAB) decreased from 0.5 μg/m3 in 1987 to 0.01 μg/m3 in 2002 and were not subsequently measured (Fig. 1). The rural site is far from any potential source of pollution such as major roads, mining and smelting sites, and industrialised areas. Five samples of top soil (50 × 50 × 25 cm depth) were randomly selected in both urban and rural sites. Then, earthworms were extracted manually, fasted for 1 week and then stored in pure grade glycerine–alcohol 65%. Each individual was identified at species level and attributed to functional groups (epigeous, anecic and endogeous) according to Bouché (Bouché, 1972). Blackbirds were trapped with mist nets. All captured birds were ringed on the right leg with a numbered aluminium ring (provided by the “Centre de Recherche sur la Biologie des Populations d'Oiseaux”, Museum National d'Histoire Naturelle, Paris, France) and with a unique combination of 3 coloured plastic rings on the left leg. Sex was determined on each captured bird, right and left tarsus lengths were measured to the nearest 0.02 mm using a digital calliper and mass was determined with a precision of 0.1 g with a portable balance. Breast feathers and the terminal part (around 3 to 4 cm) of the right and left outermost tail feathers were taken. A blood sample of 0.2 to 0.5 ml was also taken via venepuncture of the brachial vein using a needle and heparin-coated capillaries. 2.2. Lead concentration determination Earthworms and feathers were dried in an oven (60 °C) to dry mass and weighed to the nearest 0.001 g.

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Blood was only weighed and stored at − 20 °C until analysis. One of the tail feathers of each blackbird was randomly chosen, washed vigorously with 0.25 M sodium hydroxide solution (NaOH) to remove the external contamination, rinsed in ultra-pure water and dried again. Most of works dealing with MTE concentrations in bird feathers have used alternated washings with deionized water and acetone but it has not been shown that such organic solvent had no effect on the stability of the Pb profile in the feather (Burger, 1993). Therefore, NaOH, which is a relatively mild agent compared to organic solvents, was preferred for feather washing, as previously done by Thompson et al. (1998) and Scheifler et al. (2005). The other tail feather was not washed. Earthworms, blood and feathers were then digested in polystyrene crystal tubes with 5 mL HNO3 65% in a drying oven at 60 °C for 48 h. All reagents were of analytical grade and obtained from Carlo Erba (Val de Rueil, France). Total Pb concentrations were measured by furnace atomic absorption spectrophotometry (Perkin–Elmer 3110 furnace HGA 600, Courtaboeuf, France). Validity of analytical methods was checked by means of standard biological reference material (TORT-2, lobster hepatopancreas; National Research Council of Canada–Institute for National Measurement Standard, Ottawa, ON, Canada). 2.3. Body condition index and statistics Non-destructive estimates of individual body condition were used. Individual body condition index corresponded to the residuals obtained from a reduced major axis regression (RMA) between log(body mass) and log(tarsus length). According to Green (2001), RMA residuals are more appropriate as estimates of body condition than ordinary least square linear regression when error measurements in X (i.e. tarsus length) may occur and when Y and X are measured in different units. Then, relationships between body condition indices and Pb concentrations in blackbird tissues were checked by integrating the sex of the individual as explanatory variable (Green, 2001). To compare earthworm data from urban and rural samples, a General Linear Model (Grafen and Hails, 2002) was used with mass or Pb concentrations as measured variables and with site (rural versus urban) and functional group as explanatory variables. General Linear Models were also used to compare blackbird data with mass or Pb concentrations in the various tissues as measured variables and site and sex as explanatory variables. Normality of datasets and of residuals was checked using a Kolmogorov–Smirnov

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Table 1 Pb concentrations (μg/g, mean ± standard deviation) in anecic, epigeous and endogeous earthworms from rural and urban sites

Rural site Urban site

Epigeous

Anecic

Endogeous

1.17 ± 0.54 1.70 ± 1.25⁎

1.01 ± 0.86 2.63 ± 0.25⁎

0.77 ± 0.53 1.80 ± 0.90⁎

Asterisks indicate significant difference between rural and urban samples (ANOVA, p b 0.05).

test for goodness of fit (Sokal and Rohlf, 1997). Statistics and graphical display were performed using R 2.3.0. (R Development Core Team, 2006). The percentage of external contamination on the total Pb concentration in feathers was calculated using the following equation: %½Pbext ¼ ðð½PbunwR −½PbwR Þ=½PbunwR Þx100 With %[Pb]ext, the percentage of external contamination, [Pb]unwR, the average Pb concentration in the unwashed rectrices and [Pb]wR, the average Pb concentration in the washed rectrices. Whether this percentage of external contamination differed between the rural and the urban sites was tested by the Wilcoxon–Mann– Whitney test (Siegel and Castellan, 1988). 3. Results Biomass of earthworms in the rural site (0.541 ± 0.419 g per soil sample, i.e. 0.075 m3) was not significantly different from that of the urban sites (0.197 ± 0.160 g per sample, ANOVA, p = 0.39). At the rural site, the earthworm community was mainly composed of anecic, followed by endogeous and epigeous species with 48.4, 29.0 and 22.6% in biomass, respectively. In the urban site, endogeous were dominant (51.8% in biomass), followed by epigeous (30.9%) and anecic (17.3%). Pb concentrations in earthworms

Table 2 Pb concentrations (mean ± standard deviation) in breast, unwashed and washed outermost tail feathers (in μg/g dry mass) and blood (in μg/g wet mass) of rural and urban common blackbirds (Turdus merula)

Rural Urban

Breast feathers

Outermost tail feathers Unwashed

Washed

1.37 ± 1.07 3.15 ± 1.77⁎

1.82 ± 1.14 7.75 ± 4.50⁎

0.73 ± 0.65 4.89 ± 3.18⁎

Blood

0.05 ± 0.10 0.15 ± 0.09⁎

Asterisks indicate significant difference between rural and urban samples (ANOVA, p b 0.05).

15 [Pb] in unwashed feathers (µg/g)

200

10

5

Urban blackbirds Rural blackbirds

0 0

2

4

6

8

10

[Pb] in washed feathers (µg/g) Fig. 2. Relationship between Pb concentrations in unwashed and washed outermost tail feathers of urban and rural blackbirds.

did not differ significantly among functional groups (ANOVA, p = 0.82; Table 1). However, concentrations of urban earthworms were significantly higher than those of rural earthworms, functional group being taken into account (ANOVA, p = 0.0004; Table 1). Assuming that only anecic and epigeous earthworms are accessible to blackbirds (endogeous ones living mainly under soil surface, Bouché, 1972; Gobat et al., 1998), we calculated, taking into account the respective proportions and Pb concentrations of anecic and epigeous earthworms, that urban blackbirds were exposed to 2.0fold higher Pb concentrations via worm ingestion than rural population. Eleven and 12 blackbirds were captured in the urban and the rural site, respectively. The sex-ratio was unbalanced with only 3 females at each site. Average body mass of urban blackbirds (84.1 ± 5.2 g) did not differ significantly from that of rural birds (88.0 ± 6.8 g, ANOVA, p = 0.14). Average Pb concentrations in breast feathers of urban blackbirds were 2.3-fold higher than those of rural birds (ANOVA, p = 0.02; Table 2). Pb concentrations in washed and in unwashed outermost tail feathers were respectively 4.3 and 6.7-fold higher in urban blackbirds than in the rural population (ANOVA, p = 0.003 and p = 0.002, respectively; Table 2). On average, concentrations in washed feathers were 37 (for urban birds) to 60% (for rural birds) lower than the concentrations in the unwashed feathers. This percentage of external contamination did not differ significantly between the

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rural and the urban sites (Wilcoxon–Mann–Whitney test, p = 0.06). Concentrations in unwashed and washed feathers were highly (r 2 = 0.86) and significantly correlated for the urban site but not for the rural one (Fig. 2). Lead residues in blood were significantly higher in urban blackbirds than in rural specimens (ANOVA, p = 0.041; Table 2), while no difference was detected between males and females (ANOVA, p = 0.74). The ratio of Pb blood concentrations of urban birds to rural ones was in the same range as the ratio of Pb residue in urban earthworms to rural earthworms (3.0 and 2.0, respectively). Except the correlation between Pb concentrations in washed and unwashed feathers in the urban site, there was no relationship between Pb concentrations in outermost tail and breast feathers or between feathers and blood, whatever the site. Pb concentrations in feathers or blood were not correlated with variations in body condition indices estimated from RMA residuals. 4. Discussion This study showed, as hypothesised, that Pb concentrations in earthworms sampled in Besançon, a French middle-size city, were significantly higher than those found in individuals sampled in a remote rural place, far from any important source of pollution. Though, present atmospheric Pb concentrations are nearly 0 in French cities (including Besançon) due to the progressive banning of Pb compounds in gasoline. Therefore, the higher Pb concentrations found in urban earthworms suggest that Pb in urbanized soils is still bioavailable to worms as it was shown for Lumbricus terrestris L. in urban soils from Montréal (Canada) area (Kennette et al., 2002). Present results show that Pb concentrations in feathers and blood of urban blackbirds are higher than those found in similar tissues in rural individuals, suggesting, as hypothesised by Kennette et al. (2002), that Pb contained in worms, which constitute one of the main diet items of blackbirds, is available to birds in urban environments. 4.1. Lead concentrations in feathers The average concentration (3.15 ± 1.77 μg/g) in breast feathers of urban blackbirds is higher than that found in breast feathers of mourning doves (Zenaida macroura) sampled in two agricultural fields contaminated with Pb shots in South Carolina, United States (Burger et al., 1997). This comparison must however be handled with care because breast feathers were not washed in our

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study while it was the case (washing with deionized water and acetone) in the study of Burger et al. (1997). Comparison of our data with concentrations in other types of feathers was not made because several works underlined that concentrations of MTE (including Pb) may dramatically vary among the different types of feathers (Burger, 1993; Dauwe et al., 2003). These differences are generally attributed to molting pattern (Furness et al., 1986), pigmentation (Dmowski et al. 1984 in Dauwe et al., 2003) and external contamination (deposition from the environment and from excretion of the uropygial gland during preening; Dauwe et al., 2003). Lead concentrations found in the washed outermost tail feathers of urban blackbirds are low compared to the concentrations found in similar feathers of other adult passerines inhabiting polluted areas. On the university campus of Antwerpen, Belgium, Pb concentrations in outermost tail feathers of blue and great tits were 64.0 ± 15.2 and 16.3 ± 1.8 μg/g, respectively (Eens et al., 1999). Concentrations ranging from 43 to 82 μg/g were found in a mixture of rectrices and remiges of barn swallows (Hirundo rustica) collected near a major highway in Maryland, United States (Grue et al., 1984). Lead concentrations as high as 230.5 ± 31.2 μg/g were found in outermost tail feathers of adult great tits (Parus major) inhabiting the vicinity of a metallurgic factory near the city of Antwerpen, Belgium (Janssens et al., 2001). In the same study, Pb concentrations decreased dramatically along the pollution gradient to reach 8.1 ± 1.3 μg/g at a site considered as an “unpolluted” control one because it was located 20 km away from the factory, remote from industrialisation and urbanization (Janssens et al., 2001). Even in our polluted site, the Pb concentrations measured in blackbirds were lower than those found in these “reference” tits (Janssens et al., 2001). This may be due to the high urbanization and the dense road network of the area of Antwerpen. This highly anthropogenic area of Flanders obliged Eens et al. (1999) to use the site of Tenerife, Spain, as a reference “unpolluted” site because “unpolluted sites are probably difficult to find in the Flanders”. In Tenerife, Pb concentration in outermost tail feathers of blue tits was indeed much lower (3.7 ± 1.5 μg/g). This underlines the need for studies on background concentrations in bird tissues, which could be done in very remote and unpolluted areas or by using, under appropriate washing methods, feathers of preserved birds in Museums. 4.2. Importance of the external contamination One way to limit the influence of the external contamination is to wash the feathers, which allows the

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removal of –at least gross– external contamination (Burger, 1993). In our study, both washed and unwashed outermost tail feathers were analysed, allowing estimating the external contamination in rural and urban blackbirds at 60 and 37%, respectively, of the total Pb concentration of the unwashed feathers. Because the atmospheric Pb concentration in Besançon nowadays is nearly 0, this relatively high external contamination is probably related to Pb-contaminated soil particles and dusts that adsorbed on plumage when bird were foraging on the ground. In a strongly polluted area in Germany, Weyers et al. (1988) found that 87% of Pb in blackbird first primaries was exogenous. The remaining 13% were attributed to “constitutive” Pb and to food chain transfer. In our moderately polluted urban site and according to our calculations, a larger part of the Pb concentration found in the outermost tail feathers (63%) could be attributed to “constitutive” Pb and food chain transfer, which is in agreement with the higher Pb concentrations found in urban earthworms than in rural individuals. A last –and generally least considered– exposure source is the excretion by the uropygial gland during preening behaviour. Indeed, Dauwe et al. (2002) suggested that Pb concentration in outermost tail feathers of zebra finches (Taeniopygia guttata) exposed to Pb in drinking water was due to internal deposition via blood in growing feathers but also to an external contamination due to excretion of the uropygial gland (i.e. preening). Calculations from their data suggest that the contribution of this source to the total concentration of Pb in the feathers could be 39%. Because this uropygial contamination could not be removed by washing in ultra-pure water, these authors suggest that, in natural environments, Pb in feathers partially originates from this source. It is unknown whether stronger washing –that was used in our study for instance– may remove this contamination and we cannot exclude that a part of the concentration measured in the washed outermost feathers of urban blackbirds may come from this source. More generally, it is somewhat surprising to notice that few data are available in the literature about the relative importance of external contamination on the concentrations of metals in feathers. This paucity of data is also underlined Dauwe et al. (2003) who add that such work was done in detail for mercury (Hg) only. This issue is however of importance to better understand the factors that affect deposition in or on feathers (soil and atmospheric contamination, bioavailability of MTE in variously-contaminated sites, diet, behaviour and physiology of the birds…) and, thus, to improve interpretations of feather MTE concentrations (Burger, 1993; Dauwe et al., 2002).

4.3. Lead concentrations in blood and their toxicological significance We could not find in the literature Pb concentrations in blood of the presently studied species, T. merula. Therefore, the present results were compared to those obtained from a species belonging to the same genus, the American robin T. migratorius, for which an abundant literature is available (Beyer et al., 2004; Roux and Marra, 2005; U.S. Fish and Wildlife Service 2005). In the Little North Fork Coeur d'Alene River reference area, Pb residues in blood, 0.050 ± 0.047 μg/g (wet mass, U.S. Fish and Wildlife Service 2005), were similar to the concentrations found in the blood of the rural blackbird population in the present study. Beyer et al. (2004) reported Pb blood concentrations in populations of T. migratorius inhabiting reference sites of 0.129 ± 0.046 μg/g (assuming that blood water content was 85%). It is questionable whether or not 0.129 μg/g can be considered as a reference value because such a level was related to 50% of ALAD inhibition in 3 passerines, including the American robin (U.S. Fish and Wildlife Service 2005). In non-polluted rural environments, Pb concentrations in the blood of American robin populations were 0.079 ± 0.009 μg/g (Roux and Marra, 2005). These results, related to present data, suggest that values ranging from 0.05 to 0.08 μg/g could be considered as reference Pb blood concentrations for at least these two species of Turdus. For urban populations, our results were in the range of Pb blood concentrations assessed in American robins inhabiting the moderately-contaminated area of Government Gulch, i.e. 0.163 μg/g (U.S. Fish and Wildlife Service 2005), but are lower than those of several Pb polluted sites: Tri-State Mining District (0.255 ± 0.008 μg/g; Beyer et al., 2004), urban environments (0.264 ± 0.035 μg/g, Roux and Marra, 2005; Magnet Gulch, 0.715 ± 0.383 μg/g, U.S. Fish and Wildlife Service 2005) and Deadwood Gulch (0.656 ± 0.522 μg/g, U.S. Fish and Wildlife Service 2005). Compared to the benchmark value for blood Pb residues related to subclinical signs in birds, i.e. 0.20 μg/ g (Franson, 1996; Pain, 1996; U.S. Fish and Wildlife Service 2005), 3 specimens of the urban populations presented higher blood concentrations (1 male with 0.30 μg/g and 2 females with 0.21 and 0.29 μg/g). Moreover, 1 male of the rural population exhibited the highest blood concentration measured in this study, 0.31 μg/g. All these Pb residue values were below the lowest blood Pb residues related to clinical and toxic effects, i.e. 1.0 μg/g in falconiformes (Franson, 1996). In the American robin, Pb blood concentrations higher

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than 0.20 μg/g measured in populations inhabiting metal-contaminated sites were shown to correspond with ALAD inhibition of 67% and more (U.S. Fish and Wildlife Service 2005). Johnson et al. (1999) reported that ALAD inhibitions higher than 50% corresponded to levels sufficient to cause injury in birds and high inhibitions (N 75%) may induce physiological impairments such as anemia, reduced hematocrit and haemoglobin or cerebral damage (Dieter and Finley, 1979; Hoffman et al., 2000). So, it cannot be excluded that a significant part of the urban populations of blackbirds suffered the exposure of Pb. Further investigations are needed to document possible individual and population responses induced by Pb in these urban blackbirds. Several laboratory studies have demonstrated that pollutants adversely affect the body condition of birds. Most of field studies were interested in relationships between organic chemicals such as organochlorines and body condition of waterbirds or raptors assessed by either destructive (e.g. measurement of lipids) or nondestructive methods (White et al., 1981; Bustnes et al., 2002; Kenntner et al., 2003). Biological effects of metal pollution on birds were poorly investigated in natural circumstances (Dauwe et al., 2005b). Significant relationships were reported between some indices of body condition and metal level in the liver of diving duck carcasses (Takekawa et al., 2002). The approach we used allowed relating a general indicator of individual health, body condition index, to contaminant exposure, both being assessed in a non-destructive way. However, the use of residuals obtained from log(body mass) = f(body size indicator) as estimates of body condition should be handled with care and cautions about the assumptions of the method (both biological and statistical ones) are to be taken (see Green, 2001 for details). We did not found any correlation between Pb concentrations in blackbirds and body condition index. This result is consistent with Pb residues in blood that could only be related to subclinical signs in the most contaminated birds. About other passerines, no differences of both great and blue tit (P. major and Parus caerulens, respectively) body condition indices were found between populations inhabiting along a pollution gradient of metals (Dauwe et al., 2006). 5. Conclusions Although atmospheric Pb emissions were reduced dramatically in most of the European and North-American cities, we demonstrate here that urban birds may remain exposed to Pb concentrations of ecotoxicological concern. In agreement with literature data, present results suggest

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that food transfer from soil invertebrates may be an important route of Pb exposure for blackbirds. Persistent chemicals may present a risk for wildlife, which should be characterized more precisely in terms of both individual and population effects during long-term survey programs. Acknowledgements Data on atmospheric Pb concentrations in Besançon were graciously provided by the « Association pour la surveillance de la qualité de l'air dans l'agglomération bisontine et le Sud Franche-Comté » (ASQUAB). Jean François is gratefully acknowledged for field assistance. The members of the Office National de la Chasse et de la Faune Sauvage of the Doubs department (particularly E. Renaud and R. Barbier) are acknowledged for their interest and their sound advice. We are grateful to two anonymous referees for their help in improving the manuscript. References Ancillotti F, Fattore V. Oxygenate fuels: market expansion and catalytic aspect of synthesis. Fuel Process Technol 1998;57:163–94. Battaglia A, Ghidini S, Campanini G, Spaggiari R. Heavy metal contamination in little owl (Athene noctua) and common buzzard (Buteo buteo) from northern Italy. Ecotoxicol Environ Saf 2005;60:61–6. Beyer WN, Dalgarn J, Duddin S, French JB, Mateo R, Miesner J, et al. Zinc and lead poisoning in wild birds in the Tri-State Mining District (Oklahoma, Kansas, and Missouri). Arch Environ Contam Toxicol 2004;48:108–17. Blus LJ, Henny CJ, Hoffman DJ, Sileo L, Audet DJ. Persistence of high lead concentrations and associated effects in Tundra Swans captured near a mining and smelting complex in Northern Idaho. Ecotoxicology 1999;8:125–32. Bouché M. Lombriciens de France. Ecologie et Systématique. Paris: Institut National de la Recherche Agronomique; 1972. 672 pp. Burger J. Metals in avian feathers: bioindicators of environmental pollution. Rev Environ Toxicol 1993;5:203–311. Burger J, Kennamer RA, Lehr Brisbin I, Gochfeld M. Metal levels in Mourning Doves from South Carolina: potential hazards to Doves and hunters. Environ Res 1997;75:173–86. Bustnes JO, Erikstad KE, Skaare JU, Bakken V, Mehlum F. Ecological effects of organochlorine pollutants in the arctic: a study of the glaucous gull. Ecol Appl 2002;13:504–15. Chandler RB, Strong AM, Kaufman CC. Elevated lead levels in urban house sparrows: a threat to sharp-thinned hawks and merlins? J Raptor Res 2004;38:62–8. CITEPA. Inventaire des émissions de polluants atmosphériques en France- Séries sectorielles et analyses étendues. CITEPA / CORALIE / format SECTEN; 2004. Clark AJ, Scheuhammer AM. Lead poisoning in upland-foraging birds of prey in Canada. Ecotoxicology 2003;12:23–30. Cramp S, editor. The birds of the Western Palearctic. Tyrant Flycatchers to ThrushesNew York, USA: Oxford University Press; 1988. Dauwe T, Bervoets L, Blust R, Pinxten R, Eens M. Can excrement and feathers of nestling songbirds be used as biomonitors for heavy metal pollution? Arch Environ Contam Toxicol 2000;39:541–6.

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