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Effects of heavy metal exposure on the condition and health of nestlings of the great tit (Parus major), a small songbird species
Environmental Pollution 126 (2003) 267–274 www.elsevier.com/locate/envpol
Effects of heavy metal exposure on the condition and health of nestlings of the great tit (Parus major), a small songbird species Ellen Janssensa, Tom Dauwea, Rianne Pinxtena, Lieven Bervoetsb, Ronny Blustb, Marcel Eensa,* a Department of Biology, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Wilrijk, Belgium Department of Biology, University of Antwerp (R.U.C.A.), Groenenborgerlaan 171, B-2020, Antwerp, Belgium
b
Received 4 December 2002; accepted 14 May 2003
‘‘Capsule’’: Pollutants reduced nestling body mass and condition and delayed fledging time. Abstract In this study we examined the possible effects of heavy metal exposure on the condition and health of great tit nestlings (Parus major) at four study sites along a pollution gradient near a large non-ferrous smelter in Belgium during three consecutive breeding seasons. Our results showed that nestlings were indeed exposed to large amounts of heavy metals. Excrements contained significantly higher concentrations of several heavy metals (silver, arsenic, cadmium, mercury, lead) near the pollution source than at study sites farther away. When taking into account the number of young in the nest at the time of sampling, nestling body mass and condition were significantly reduced at the most polluted site. Nestlings at the two most polluted sites fledged significantly later than at the least polluted site. We also observed growth abnormalities of the legs near the pollution source. Tarsus length, wing length and haematocrit values did not differ significantly among study sites. # 2003 Published by Elsevier Ltd. Keywords: Heavy metals; Parus major; Nestling condition; Health state; Haematocrit; Fecal concentrations
1. Introduction Heavy metals are widespread contaminants that are highly persistent. Earlier experimental studies have shown that the reproduction and health of birds can be profoundly affected by heavy metals (Edens and Garlich, 1983; Heinz and Fitzgerald, 1993, Burger and Gochfeld, 1994). Evaluation of the impact of heavy metal pollution in the natural environment should be founded on the knowledge of pollution-related health disturbances in the organisms naturally occurring in the ecosystems affected (Nyholm, 1995). However, effects of increased metal exposure on the reproduction in terrestrial free-living birds, or in any other vertebrate, have rarely been demonstrated (Ho¨rnfeldt and Nyholm, 1996).
* Corresponding author. Tel.: +32-3-820-22-84; fax: +32-3-82022-71. E-mail address: [email protected] (M. Eens). 0269-7491/03/$ - see front matter # 2003 Published by Elsevier Ltd. doi:10.1016/S0269-7491(03)00185-4
Using nestling birds as biomonitors is ideal because their body burdens of metals closely reflect the amounts in the food they are fed during their development, making that they can be used to identify local pollution in the foraging area (Burger, 1993; Furness, 1993; Janssens et al., 2002). Their condition or health status may therefore provide important and accurate information regarding the possible effects of heavy metal pollution. Another advantage of nestlings as study objects is that their whole life history can be studied in detail and sampling can be standardized (Furness, 1993). Nevertheless, studies on the effects of heavy metals on the condition and health of wild nestling birds are scarce and results are sometimes contradictory. Nestlings of the European starling (Sturnus vulgaris) exposed to high concentrations of heavy metals, had similar prefledgling weights than control nestlings (Grue et al., 1986). Nestlings of the pied flycatcher (Ficedula hypoleuca) grew equally well along a pollution gradient, while great tit nestlings (Parus major) grew poorer near the pollution source than farther away (Eeva and Lehikoinen, 1996).
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The purpose of our study was to examine the possible effects of exposure to high concentrations of heavy metals on the condition and health of 15-day old great tit nestlings. We determined their tarsus and wing length, body mass and condition along a pollution gradient during three consecutive breeding seasons (1998– 2000). Exposure of the nestlings to heavy metals was studied by fecal concentrations. We also looked for eventually occurring growth abnormalities at the most polluted sites and studied haematocrit values along the pollution gradient. Previous studies along this gradient have already shown that levels of several heavy metals in the feathers of adult great tits from the most polluted site are among the highest reported in literature (Janssens et al., 2001). At the most polluted site, lead and cadmium concentrations in the outermost tail feathers of adult great tits were on average respectively 230.5 and 9.3 ppm, which is respectively 16 and 6 times higher compared to the non-polluted reference site 4 km farther away (Janssens et al., 2001). Levels of lead and cadmium in nestling feathers and eggs of great tits from the polluted site were also extremely high for passerine species (Dauwe et al., 1999, 2000). Since the metal concentrations in tit feathers and eggs reported in these studies are strikingly high, this may pose an enormous strain on the surrounding natural environment and the organisms that live there and it seemed likely that these organisms might experience negative effects of this increased heavy metal exposure on their health and/or reproduction. In agreement with this, our results showed that near the pollution source significantly more great tit females interrupted their laying period than did females farther away. Also, hatching and overall breeding
success was significantly reduced towards the pollution source (Janssens et al., 2003).
2. Methods 2.1. Study sites The present study was performed along a pollution gradient emanating from a non-ferrous smelter in the south of Antwerp, Belgium (see Fig. 1). This metallurgic factory is the most extensively heavy metal emitting point source in Flanders. Lead, cadmium, arsenic, copper and zinc are especially common pollutants in this area and they form an exponentially decreasing pollution gradient away from the factory complex (Janssens et al., 2001). Moderate levels of pollution are documented 1–2 km from the factory, and levels of contaminants approach background levels approximately 4–5 km from the complex (VMM, 1999; Janssens et al., 2001). In 1997, three study sites (UM, F8 and UIA), each with 30–50 great tit nest boxes, were established along a pollution gradient eastwards from the factory. One more intermediate site (F7) with 30 great tit nest boxes was established in 1998. Special attention was paid in selecting study sites so that they would represent a similar habitat type (deciduous park areas). To standardize exposure and nesting conditions among study sites, we installed nest boxes that this species readily occupies. All sites had nest boxes installed at least 6 months prior to the breeding season. The first site (called UM) is located in the immediate vicinity of the pollution source (0–350 m). The second
Fig. 1. Location of our study area (&) in Flanders (Belgium) and the four study sites.
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study area (F8) is situated 400–600 m to the east of the pollution source while the third (F7) and fourth area (UIA) are respectively 2500 and 4000 m eastwards from the factory (see Fig. 1).
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nest, range 1–9) were sampled. All samples were analyzed separately but means per nest were used in the calculation of means and in the statistical analysis. 2.3. Metal analysis
2.2. Data sampling Nest boxes were checked once every two days at UM, F8 and UIA during the breeding season of 1998 and daily at the four study sites in 1999 and 2000. Therefore, we were able to determine the date of hatching accurately for each nest. Fifteen days after the first egg hatched all nestlings were ringed with individually numbered metal rings and tarsus length, wing length and body mass were measured. We measured body mass using a pesola spring balance (to the nearest 0.25 g), tarsus length using digital calipers (to the nearest 0.01 mm) and wing length using a ruler (to the nearest 1 mm). Body mass and tarsus length were used to determine body condition (body condition was defined as residuals from the linear regression in order to avoid the effect of body size on body mass, see Ots et al., 1998). Nestlings were also checked for visible growth abnormalities of wings and legs (see Eeva and Lehikoinen, 1996). In 1998, no data were gathered regarding the length of the tarsi and the wings. In 1999 and 2000, data were gathered in all study areas and all parameters were considered. In total, we collected morphological data of 426 young in 54 nests in 1998, 467 young in 57 nests in 1999 and 521 young in 71 nests in 2000. In 1999, in addition to determining the condition of the nestlings, we collected blood to determine haematocrit values and excrements for metal analysis (see below). Blood samples (75 ml) from the brachial vein were taken of half of the young in each nest (nestlings were all 15 days of age and were chosen randomly) to examine the haematocrit levels in nestlings along the gradient. After puncturing the branchial vein with a sterile needle (Terumo, 0.420 mm), approximately 75 ml of blood was transferred into a heparinized microhaematocrit capillary. After the capillary was filled with blood, it was closed at one side with wax and left to stand vertically in a cooled transport box (maximum temperature 7 C). Within 3 h after sampling, the capillary tubes were spun for 4 min at 12,000 rpm. Haematocrit height was measured using digital calipers (to the nearest 0.01 mm) and corrected by dividing it with total blood height (Merila¨ and Svensson, 1995). In total, 206 nestlings in 53 nests were sampled. Means per nest were used in the calculation of means and in the statistical analysis. Nestlings were induced to defecate upon handling and excrements were immediately collected in metal-free plastic containers. Fecal samples were taken from, on average, three 15-day old nestlings per nest. In total, 87 nestlings in 30 nests (on average 2.7 0.3 nestlings per
Since excrements were collected directly into metalfree vials, external contamination was neglectable and the samples were only briefly washed with deionised water. We determined the dry weight of the excrements after they were put in an oven (60 C) for 24 h. Subsequently the excrements were digested in a 1:1 mixture of HNO3 (70%) and H2O2 (30%). We completed the digestion with the microwave procedure described by Blust et al. (1988). After digestion, the samples were diluted by adding 4 ml deionized water and stored at 20 C until analysis. We measured silver, aluminium, arsenic, cadmium, cobalt, chromium, copper, iron, mercury, manganese, nickel, lead and zinc levels in samples with an axial Inductively Coupled Plasma–Mass Spectrometer (ICP– MS, Varian Ultramass 700). The concentrations are expressed in mg g 1 based on dry weights. All samples were analyzed in batches with certified reference material of the Community Bureau of Reference (i.e. mussel sample, CRM 278), blanks and a standard calibration curve. Recovered concentrations of the certified samples were within 10% of the certified values, which is an acceptable margin (Gochfeld and Burger, 1998). All samples were measured on the same day. 2.4. Statistical analysis We used SPSS for Windows (SPSS, 1999) statistical software to perform statistical analysis. A significance level of 0.05 was chosen for all statistical tests. All statistical analyses followed procedures outlined in Sokal and Rolhf (1981). For all statistical analyses and each variable considered, we used mean values per nest. According to Shapiro-Wilk’s W tests all data sets [PCVvalues were log transformed and metal concentrations were log (x+1)-transformed] had normal distributions so parametric tests were used. Differences in body mass, tarsus and wing length, and nestling condition among years and sites were investigated using a two-way ANOVA followed by Tukey HSD tests when appropriate. The differences in body mass, tarsus and wing length and nestling condition were also investigated taking into account the number of young in the nest at the time of sampling. For this we used ANCOVA’s with the independent factors being year and site, the dependent factors were body mass, tarsus and wing length and condition, and the number of young in the nest at the time of sampling was used as a covariate. We applied a one-way ANOVA (+Tukey HSD test) to test for significant differences in PCV-values and metal
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concentrations in the excrements among the study sites. Values given are arithmetic means standard errors (SE).
3. Results 3.1. Metal concentrations in excrements All measured elements were detected in the excrements of great tit nestlings. Moreover, we found significant differences in metal concentrations among sites for seven out of 13 metals (Table 1). Great tit nestlings from UM, the site closest to the pollution source, had significantly higher concentrations of silver, arsenic, cadmium and lead in their excrements than did nestlings at the other three sites (Table 1). Mercury levels were also significantly higher at the two most polluted sites. Silver and lead concentrations in excrements of nestlings near the pollution source were on average 30 times higher than 4 km further east. Arsenic levels were 520 times higher at the most polluted site (Table 1). The level of the essential metal copper was significantly lower at the second most polluted site, while zinc was significantly higher at the least polluted site compared to the other sites along the gradient. Aluminium, cobalt, chromium, iron, manganese and nickel concentrations did not differ significantly among sites (Table 1). 3.2. Nestling condition The 15-day old nestlings along the pollution gradient weighed on average 16.71 0.08 g (data of all years and sites combined). Although nestlings at the most polluted site weighed less compared to nestlings at the less polluted sites, we found no significant differences in nest-
Table 1 Mean concentrations (mg g Metal
UM (N=12)
Ag Al As Cd Co Cr Cu Fe Hg Mn Ni Pb Zn
3.50.5 814259 5212 9.41.6 3.90.7 4.70.9 20934 2464517 112.9 21943 459 28.84.8 26138
1
ling body mass among the four study sites (two-way ANOVA, F3,159=1.9, P=0.13, see Fig. 2 A) or among years (two-way ANOVA, F2,159=0.54, P=0.58). The interaction was also not significant. However, when taking into account the number of nestlings in the nest at the time of sampling (the mean number of nestlings per brood at each site was 7.7 0.3 nestlings at UM, 7.9 0.3 at F8, 8.4 0.4 at F7 and 7.9 0.3 at UIA), the difference in weight among study sites was significant (ANCOVA, F3,144=3.95, P=0.01). Nestlings at UM weighed 3% less than nestlings at F7, and this difference was significant (Tukey HSD test, P=0.007). The condition of the 15-day old nestlings did not differ significantly among sites (Two-Way ANOVA, F3,117=1.17, P=0.3). The difference in condition among sites was however significant when taking into account the number of young per nest (ANCOVA, F3,106=3.03, P=0.03). The average tarsus length of all 15-day old nestlings along the gradient was 17.48 0.04 mm. The great tit nestlings at the most polluted site had slightly smaller tarsi than nestlings at the other three sites, but this difference was not significant (two-way ANOVA, F3,120=1.13, P=0.34, see Fig. 2D). Tarsus length also did not differ significantly among years (two-way ANOVA, f1,120=0.75, P=0.39). The interaction site year was not significant. The difference in tarsus length among sites was also not significant when taking into account the number of young per nest (ANCOVA, F3,106=1.99, P=0.12). The average wing length of all great tit nestlings in the pollution gradient was 46.4 0.28 mm. We found no significant differences in wing length among years or among the different study sites (two-way ANOVA, respectively F1,120=0.95, P=0.33 and F3,120=0.75, P=0.53, see Fig. 2B). The interaction siteyear was not significant. The difference in wing length among sites
dry weightstandard error) of heavy metals in the excrements of great tit nestlings along a pollution gradient F8 (N=9) A A A
A A
A A
0.50.1 17526 8.91.9 4.21.0 2.90.8 2.00.6 6627 2013448 4.11.1 13049 13.65.3 7.21.0 20758
F7 (N=6) B B B
B A
B A
0.30.1 419173 2.61.1 2.70.6 4.82 3.20.8 16733 1863536 1.10.3 22213 5820 1.90.3 25548
UIA (N=3) B B B
A B
B A
0.1 0.07 96 19 0.1 0.1 2.9 1.4 7.8 6.5 2.9 0.2 201 73 1378856 1.4 1.0 523 324 24 19 1.1 0.5 526 159
B B B
A B
B B
F
P
17.79 1.57 7.19 5.03 1.01 2.41 3.33 0.39 4.18 2.39 2.69 8.66 3.02
0.000002 0.2 0.001 0.007 0.4 0.09 0.04 0.8 0.015 0.1 0.07 0.0004 0.05
The results from a One-Way ANOVA are given. Significant differences among sites tested using a Tukey HSD-test are shown by letters. Means followed by the same letter do not differ significantly from each other. Sample sizes (number of nests) are also given.
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was also not significant when taking into account the number of young per nest (ANCOVA, F3,107=1.2, P=0.3).
ferences in fledging age among years (two-way ANOVA, F1,146=3.17, P=0.13).
3.3. Nestling health
4. Discussion
Nestlings had on average a haematocrit value of 41.06 0.4% (data of all sites combined). There was no significant difference in haematocrit values among sites (one-way ANOVA, F3,50=0.72, P=0.5, see Fig. 2C). In 1998 and 2000 we observed respectively one and two nests which contained several young with growth abnormalities at their legs (defectively developed tibiotarsi and tarsometatarsi). Such nests were only observed at the two most polluted sites, UM and F8.
The purpose of this study was to examine possible detrimental effects of heavy metals on the condition and health of 15-day old great tit nestlings along a pollution gradient. The higher concentrations of many heavy metals in the excrements of the nestlings, combined with earlier data on heavy metals in feathers of nestling (Janssens et al., 2002) and adult great tits (Janssens et al., 2001), provide good evidence that great tits near the factory complex were exposed to higher concentrations of certain heavy metals than individuals at the lesser polluted areas. Great tit nestlings from the most polluted site had significantly higher concentrations of silver, arsenic, cadmium, mercury and lead in their excrements. More significant differences in metal concentrations in excrements among sites might have been detected if sample sizes at F7 (N=6) and UIA (N=3) had not been so small. Other studies have found comparable concentrations of heavy metals in excrements of
3.4. Fledging age Fledging age differed significantly among sites (twoway ANOVA, F3,146=5.44, P=0.001, see Fig. 3). Nestlings at the two most polluted sites fledged significantly later than those at the least polluted site (Tukey HSD test, P < 0.05 in both cases). Other differences among sites were not significant. We found no significant dif-
Fig. 2. The average (A) weight; (B) wing length; (C) haematocrit values and (D) tarsus length of great tit nestlings along the gradient. Data from 1998, 1999 and 2000 are combined. Numbers denote the number of nests at a study site.
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Fig. 3. The average fledging age of great tits along the gradient. Data from 1998, 1999 and 2000 are combined. Numbers denote the sample sizes.
great tit nestlings hatched in polluted areas (Dauwe et al., 2000; Nyholm et al., 1995; Eeva and Lehikoinen, 1996). In 1998, Dauwe et al. (2000) studied metal concentrations in excrements of great and blue tit nestlings at the most and least polluted site in the pollution gradient. The highest metal concentrations were found at the most polluted site (UM), although concentrations found in this study were slightly different from those found in our study. Possibly, this was due to the fact that Dauwe et al. (2000) only sampled one nestling per brood and intraclutch variation in metal levels in excrements tends to be high. Excrements of great tit nestlings in Finland had significantly higher concentrations of copper, nickel and lead towards the pollution source (Eeva and Lehikoinen, 1996). Metal concentrations found in excrements of Polish great tit nestlings were of the same magnitude than concentrations found at our study sites (Nyholm et al., 1995). Chemical analyses of excrements for heavy metals provided direct evidence of recent exposure at a local site. Although adult birds might be more exposed to heavy metals than nestlings (Sawicka-Kapusta et al., 1986), growing young are believed to be the most sensitive to the detrimental effects of pollutants (Scheuhammer, 1987). Nestling growth and fledgling weight may be more sensitive indicators of environmental conditions than the number of fledged young, suggesting that these may be better measures of overall reproductive performance, because of their influence on post-fledging survival (Furness, 1993). Despite the obvious exposure to high concentrations of heavy metals and the previously reported effects on the breeding performance (reduced hatching and breeding success) of great tits (Janssens et al., 2003), we could only detect a few effects of this high exposure on the quality of the great tit nestlings. Nestling tarsus and wing length did not differ significantly among study sites, although nestlings at the most polluted site had on average smaller tarsi and shorter wings than those at the other three sites. A reason why we may
have failed to detect differences might be that the weakest and most unhealthy nestlings may already have died by the time morphological measurements were taken. Overall breeding success was significantly lower towards the factory complex (Janssens et al., 2003). This was not only caused by the lowered hatching success towards the pollution source, but also by an increase in nestling mortality. At UM and F8 respectively 87 and 89% of the hatchlings fledged while at F7 and UIA this was respectively 98 and 91% (Janssens et al., 2003). Nestlings of the European starling hatched in highway roadside verges and therefore exposed to high concentrations of heavy metals, had similar prefledgling weights than control nestlings (Grue et al., 1986). Hatchling weight in curlews (Numenius arquata) was not influenced by the distance to the pollution source (Currie and Valkema, 1998). Eeva and Lehikoinen (1996) reported that nestling great tits grew poorer near the pollution source than farther away. However, they found a considerable amount of variation in growth in the background areas. This is partly consistent with our data. Although nestlings at the most polluted site weighed less compared to nestlings at the less polluted sites, we found no significant differences in nestling body mass along the pollution gradient. However, when taking into account the number of nestlings in the nest at the time of sampling, the difference in weight among study sites became significant. Nestling condition also differed significantly among sites when taking into account the number of nestlings in the nest. This difference in body mass and condition on day 15 may have important effects on the survival and recruitment of great tits along our pollution gradient. In great tits, body mass on day 15 after hatching can affect local recruitment rates (Tinbergen et al., 1987; Gebhardt-Henrich and van Noordwijk, 1991) and future fertility (Haywood and Perrins, 1992). Fledgling body size measurements may also affect survival and the variation in fledgling size seems to be one of the most important components of the variation in fitness (Gebhardt-Henrich and Richner, 1998). Fledging age differed significantly among sites in our study. Nestlings at the more polluted sites fledged on average 0.5–1 day older than nestlings at the lesser polluted sites. Previous studies have stated that nestling development is the most important determinant of fledging age (Michaud and Leonard, 2000). Once a critical wing length has been reached, sibling interactions and parental behavior may influence the time of fledging. Although we could not detect any significant differences, tarsus and wing length were smaller towards the pollution source. Lead has been shown to affect neurobehavioral development in nestlings. Burger and Gochfeld (1997) showed that there were significant leadinduced differences in righting response, locomotion, thermoregulation, begging, and feeding behavior in nestling herring gulls (Larus argentatus). Lead-injected
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herring gull chicks were less able to compete for food with their siblings, with a resultant significant difference by weight at 16 days of age. The differences in nestling weight at 15 days among sites might have been sufficiently large to induce differences in fledgling age. Another reason why great tit nestlings fledged significantly later near the pollution source might not only be due to poorer growth as a result of neurobehavioral effects of heavy metals, but also as a result of reduced food availability near the pollution source. During our three-year-study, we observed three nests at the most polluted sites that contained young with badly developed legs. Growth abnormalities can occur as a result of the impairing effect of dietary heavy metals on the calcium-metabolism. Similar results have been shown in pied flycatchers (Nyholm, 1995; Eeva and Lehikoinen, 1996), but not in great tits (Eeva and Lehikoinen, 1996). We did not find any differences in haematocrit values among sites. Haematocrit values represent the relative amount of red blood cells in the total blood volume and reflect the extent and efficiency of oxygen uptake and transfer to tissues (Ots et al., 1998). Assessment of haematological variables can provide important information on health and physiological status. Factors such as nutrition, stress, seasonal cycles and toxic chemicals have been shown to alter haematological values. Low haematocrit values are indicative of bacterial infections or may reflect nutritional deficiencies (Ots et al., 1998). Until now, several studies on the effects of heavy metals on haematocrit values reported depressed values as a result of increased metal exposure (Grue et al., 1986; Nyholm, 1998). However, other studies did not find any effects of increased metal exposure on haematocrit values (Fair and Myers, 2002). Perhaps in this study, the elevated iron concentrations at the most polluted sites reversed the detrimental effects of certain heavy metals on haematocrit values and other haematological parameters (Crowe and Morgan, 1997). In conclusion, our results showed that exposure to elevated concentrations of heavy metals resulted in reductions in body mass and condition in nestling great tits. Since nestling body mass can affect local recruitment rates (Tinbergen et al., 1987; Gebhardt-Henrich and van Noordwijk, 1991), future fertility (Haywood and Perrins, 1992) and survival (Gebhardt-Henrich and Richner, 1998), this might have an important effect on the great tit population close to the pollution source. Further research on recruitment rates and survival of great tits along our pollution gradient is therefore necessary.
Acknowledgements We thank Els Van Duyse and Joke Beernaert for help with the fieldwork and Valentine Mubiana for help with
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the metal analysis. This study was supported by an I.W.T.-grant to Ellen Janssens. Marcel Eens was a Research Associate of the F.W.O, while Lieven Bervoets is supported by a FWO-Postdoctoral Fellowship. This study was also supported by FWO project G.0397.00 and by the Research Council of the University of Antwerp (NOI-BOF UA-2000 and GOA-2001 project).
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Nyholm, N.E.I., Sawicka-Kapusta, K., Swiergosz, R., Laczewska, B., 1995. Effects of environmental pollution on breeding populations of birds in Southern Poland. Water, Air and Soil Pollution 85, 829– 834. Nyholm, N.E.I., 1998. Influence of heavy metal exposure during different phases of the ontogeny on the development of Pied Flycatchers, Ficedula hypoleuca, in natural populations. Archives of Environmental Contamination and Toxicology 35, 632–637. Ots, I., Muruma¨gi, A., Horak, P., 1998. Haematological health state indices of reproducing Great Tits: methodology and sources of natural variation. Functional Ecology 12 (4), 700–707. Sawicka-Kapusta, K., Kozlowski, J., Sokolowska, T., 1986. Heavy metals in tits from polluted forests in Southern Poland. Environmental Pollution 42, 297–310. Scheuhammer, A.M., 1987. The chronic toxicity of aluminium, cadmium, mercury and lead in birds: a review. Environmental Pollution 46, 263–295. Sokal, R.R., Rohlf, F.J., 1981. Biometry. W.H. Freeman and Cie, New York, NY, USA. SPSS, 1999. SPSS Reference Guide. Chicago, Illinois, USA. Tinbergen, J.M., van Balen, J.H., Drent, P.J., Cave´, A.J., Mertens, J.A.L., 1987. den Boer-Hazewinkel, Population dynamics and costbenefit analysis. Netherlands Journal of Zoology 37, 180–213. VMM, 1999. Studie van de luchtverontreiniging in de omgeving van Union Minie`re vestiging Hoboken, jaarrapport 1998. Vlaamse Milieumaatschappij, Erembodegem, Belgie¨.
Effects of heavy metal exposure on the condition and health of nestlings of the great tit (Parus major), a small songbird species Ellen Janssensa, Tom Dauwea, Rianne Pinxtena, Lieven Bervoetsb, Ronny Blustb, Marcel Eensa,* a Department of Biology, University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Wilrijk, Belgium Department of Biology, University of Antwerp (R.U.C.A.), Groenenborgerlaan 171, B-2020, Antwerp, Belgium
b
Received 4 December 2002; accepted 14 May 2003
‘‘Capsule’’: Pollutants reduced nestling body mass and condition and delayed fledging time. Abstract In this study we examined the possible effects of heavy metal exposure on the condition and health of great tit nestlings (Parus major) at four study sites along a pollution gradient near a large non-ferrous smelter in Belgium during three consecutive breeding seasons. Our results showed that nestlings were indeed exposed to large amounts of heavy metals. Excrements contained significantly higher concentrations of several heavy metals (silver, arsenic, cadmium, mercury, lead) near the pollution source than at study sites farther away. When taking into account the number of young in the nest at the time of sampling, nestling body mass and condition were significantly reduced at the most polluted site. Nestlings at the two most polluted sites fledged significantly later than at the least polluted site. We also observed growth abnormalities of the legs near the pollution source. Tarsus length, wing length and haematocrit values did not differ significantly among study sites. # 2003 Published by Elsevier Ltd. Keywords: Heavy metals; Parus major; Nestling condition; Health state; Haematocrit; Fecal concentrations
1. Introduction Heavy metals are widespread contaminants that are highly persistent. Earlier experimental studies have shown that the reproduction and health of birds can be profoundly affected by heavy metals (Edens and Garlich, 1983; Heinz and Fitzgerald, 1993, Burger and Gochfeld, 1994). Evaluation of the impact of heavy metal pollution in the natural environment should be founded on the knowledge of pollution-related health disturbances in the organisms naturally occurring in the ecosystems affected (Nyholm, 1995). However, effects of increased metal exposure on the reproduction in terrestrial free-living birds, or in any other vertebrate, have rarely been demonstrated (Ho¨rnfeldt and Nyholm, 1996).
* Corresponding author. Tel.: +32-3-820-22-84; fax: +32-3-82022-71. E-mail address: [email protected] (M. Eens). 0269-7491/03/$ - see front matter # 2003 Published by Elsevier Ltd. doi:10.1016/S0269-7491(03)00185-4
Using nestling birds as biomonitors is ideal because their body burdens of metals closely reflect the amounts in the food they are fed during their development, making that they can be used to identify local pollution in the foraging area (Burger, 1993; Furness, 1993; Janssens et al., 2002). Their condition or health status may therefore provide important and accurate information regarding the possible effects of heavy metal pollution. Another advantage of nestlings as study objects is that their whole life history can be studied in detail and sampling can be standardized (Furness, 1993). Nevertheless, studies on the effects of heavy metals on the condition and health of wild nestling birds are scarce and results are sometimes contradictory. Nestlings of the European starling (Sturnus vulgaris) exposed to high concentrations of heavy metals, had similar prefledgling weights than control nestlings (Grue et al., 1986). Nestlings of the pied flycatcher (Ficedula hypoleuca) grew equally well along a pollution gradient, while great tit nestlings (Parus major) grew poorer near the pollution source than farther away (Eeva and Lehikoinen, 1996).
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The purpose of our study was to examine the possible effects of exposure to high concentrations of heavy metals on the condition and health of 15-day old great tit nestlings. We determined their tarsus and wing length, body mass and condition along a pollution gradient during three consecutive breeding seasons (1998– 2000). Exposure of the nestlings to heavy metals was studied by fecal concentrations. We also looked for eventually occurring growth abnormalities at the most polluted sites and studied haematocrit values along the pollution gradient. Previous studies along this gradient have already shown that levels of several heavy metals in the feathers of adult great tits from the most polluted site are among the highest reported in literature (Janssens et al., 2001). At the most polluted site, lead and cadmium concentrations in the outermost tail feathers of adult great tits were on average respectively 230.5 and 9.3 ppm, which is respectively 16 and 6 times higher compared to the non-polluted reference site 4 km farther away (Janssens et al., 2001). Levels of lead and cadmium in nestling feathers and eggs of great tits from the polluted site were also extremely high for passerine species (Dauwe et al., 1999, 2000). Since the metal concentrations in tit feathers and eggs reported in these studies are strikingly high, this may pose an enormous strain on the surrounding natural environment and the organisms that live there and it seemed likely that these organisms might experience negative effects of this increased heavy metal exposure on their health and/or reproduction. In agreement with this, our results showed that near the pollution source significantly more great tit females interrupted their laying period than did females farther away. Also, hatching and overall breeding
success was significantly reduced towards the pollution source (Janssens et al., 2003).
2. Methods 2.1. Study sites The present study was performed along a pollution gradient emanating from a non-ferrous smelter in the south of Antwerp, Belgium (see Fig. 1). This metallurgic factory is the most extensively heavy metal emitting point source in Flanders. Lead, cadmium, arsenic, copper and zinc are especially common pollutants in this area and they form an exponentially decreasing pollution gradient away from the factory complex (Janssens et al., 2001). Moderate levels of pollution are documented 1–2 km from the factory, and levels of contaminants approach background levels approximately 4–5 km from the complex (VMM, 1999; Janssens et al., 2001). In 1997, three study sites (UM, F8 and UIA), each with 30–50 great tit nest boxes, were established along a pollution gradient eastwards from the factory. One more intermediate site (F7) with 30 great tit nest boxes was established in 1998. Special attention was paid in selecting study sites so that they would represent a similar habitat type (deciduous park areas). To standardize exposure and nesting conditions among study sites, we installed nest boxes that this species readily occupies. All sites had nest boxes installed at least 6 months prior to the breeding season. The first site (called UM) is located in the immediate vicinity of the pollution source (0–350 m). The second
Fig. 1. Location of our study area (&) in Flanders (Belgium) and the four study sites.
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study area (F8) is situated 400–600 m to the east of the pollution source while the third (F7) and fourth area (UIA) are respectively 2500 and 4000 m eastwards from the factory (see Fig. 1).
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nest, range 1–9) were sampled. All samples were analyzed separately but means per nest were used in the calculation of means and in the statistical analysis. 2.3. Metal analysis
2.2. Data sampling Nest boxes were checked once every two days at UM, F8 and UIA during the breeding season of 1998 and daily at the four study sites in 1999 and 2000. Therefore, we were able to determine the date of hatching accurately for each nest. Fifteen days after the first egg hatched all nestlings were ringed with individually numbered metal rings and tarsus length, wing length and body mass were measured. We measured body mass using a pesola spring balance (to the nearest 0.25 g), tarsus length using digital calipers (to the nearest 0.01 mm) and wing length using a ruler (to the nearest 1 mm). Body mass and tarsus length were used to determine body condition (body condition was defined as residuals from the linear regression in order to avoid the effect of body size on body mass, see Ots et al., 1998). Nestlings were also checked for visible growth abnormalities of wings and legs (see Eeva and Lehikoinen, 1996). In 1998, no data were gathered regarding the length of the tarsi and the wings. In 1999 and 2000, data were gathered in all study areas and all parameters were considered. In total, we collected morphological data of 426 young in 54 nests in 1998, 467 young in 57 nests in 1999 and 521 young in 71 nests in 2000. In 1999, in addition to determining the condition of the nestlings, we collected blood to determine haematocrit values and excrements for metal analysis (see below). Blood samples (75 ml) from the brachial vein were taken of half of the young in each nest (nestlings were all 15 days of age and were chosen randomly) to examine the haematocrit levels in nestlings along the gradient. After puncturing the branchial vein with a sterile needle (Terumo, 0.420 mm), approximately 75 ml of blood was transferred into a heparinized microhaematocrit capillary. After the capillary was filled with blood, it was closed at one side with wax and left to stand vertically in a cooled transport box (maximum temperature 7 C). Within 3 h after sampling, the capillary tubes were spun for 4 min at 12,000 rpm. Haematocrit height was measured using digital calipers (to the nearest 0.01 mm) and corrected by dividing it with total blood height (Merila¨ and Svensson, 1995). In total, 206 nestlings in 53 nests were sampled. Means per nest were used in the calculation of means and in the statistical analysis. Nestlings were induced to defecate upon handling and excrements were immediately collected in metal-free plastic containers. Fecal samples were taken from, on average, three 15-day old nestlings per nest. In total, 87 nestlings in 30 nests (on average 2.7 0.3 nestlings per
Since excrements were collected directly into metalfree vials, external contamination was neglectable and the samples were only briefly washed with deionised water. We determined the dry weight of the excrements after they were put in an oven (60 C) for 24 h. Subsequently the excrements were digested in a 1:1 mixture of HNO3 (70%) and H2O2 (30%). We completed the digestion with the microwave procedure described by Blust et al. (1988). After digestion, the samples were diluted by adding 4 ml deionized water and stored at 20 C until analysis. We measured silver, aluminium, arsenic, cadmium, cobalt, chromium, copper, iron, mercury, manganese, nickel, lead and zinc levels in samples with an axial Inductively Coupled Plasma–Mass Spectrometer (ICP– MS, Varian Ultramass 700). The concentrations are expressed in mg g 1 based on dry weights. All samples were analyzed in batches with certified reference material of the Community Bureau of Reference (i.e. mussel sample, CRM 278), blanks and a standard calibration curve. Recovered concentrations of the certified samples were within 10% of the certified values, which is an acceptable margin (Gochfeld and Burger, 1998). All samples were measured on the same day. 2.4. Statistical analysis We used SPSS for Windows (SPSS, 1999) statistical software to perform statistical analysis. A significance level of 0.05 was chosen for all statistical tests. All statistical analyses followed procedures outlined in Sokal and Rolhf (1981). For all statistical analyses and each variable considered, we used mean values per nest. According to Shapiro-Wilk’s W tests all data sets [PCVvalues were log transformed and metal concentrations were log (x+1)-transformed] had normal distributions so parametric tests were used. Differences in body mass, tarsus and wing length, and nestling condition among years and sites were investigated using a two-way ANOVA followed by Tukey HSD tests when appropriate. The differences in body mass, tarsus and wing length and nestling condition were also investigated taking into account the number of young in the nest at the time of sampling. For this we used ANCOVA’s with the independent factors being year and site, the dependent factors were body mass, tarsus and wing length and condition, and the number of young in the nest at the time of sampling was used as a covariate. We applied a one-way ANOVA (+Tukey HSD test) to test for significant differences in PCV-values and metal
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concentrations in the excrements among the study sites. Values given are arithmetic means standard errors (SE).
3. Results 3.1. Metal concentrations in excrements All measured elements were detected in the excrements of great tit nestlings. Moreover, we found significant differences in metal concentrations among sites for seven out of 13 metals (Table 1). Great tit nestlings from UM, the site closest to the pollution source, had significantly higher concentrations of silver, arsenic, cadmium and lead in their excrements than did nestlings at the other three sites (Table 1). Mercury levels were also significantly higher at the two most polluted sites. Silver and lead concentrations in excrements of nestlings near the pollution source were on average 30 times higher than 4 km further east. Arsenic levels were 520 times higher at the most polluted site (Table 1). The level of the essential metal copper was significantly lower at the second most polluted site, while zinc was significantly higher at the least polluted site compared to the other sites along the gradient. Aluminium, cobalt, chromium, iron, manganese and nickel concentrations did not differ significantly among sites (Table 1). 3.2. Nestling condition The 15-day old nestlings along the pollution gradient weighed on average 16.71 0.08 g (data of all years and sites combined). Although nestlings at the most polluted site weighed less compared to nestlings at the less polluted sites, we found no significant differences in nest-
Table 1 Mean concentrations (mg g Metal
UM (N=12)
Ag Al As Cd Co Cr Cu Fe Hg Mn Ni Pb Zn
3.50.5 814259 5212 9.41.6 3.90.7 4.70.9 20934 2464517 112.9 21943 459 28.84.8 26138
1
ling body mass among the four study sites (two-way ANOVA, F3,159=1.9, P=0.13, see Fig. 2 A) or among years (two-way ANOVA, F2,159=0.54, P=0.58). The interaction was also not significant. However, when taking into account the number of nestlings in the nest at the time of sampling (the mean number of nestlings per brood at each site was 7.7 0.3 nestlings at UM, 7.9 0.3 at F8, 8.4 0.4 at F7 and 7.9 0.3 at UIA), the difference in weight among study sites was significant (ANCOVA, F3,144=3.95, P=0.01). Nestlings at UM weighed 3% less than nestlings at F7, and this difference was significant (Tukey HSD test, P=0.007). The condition of the 15-day old nestlings did not differ significantly among sites (Two-Way ANOVA, F3,117=1.17, P=0.3). The difference in condition among sites was however significant when taking into account the number of young per nest (ANCOVA, F3,106=3.03, P=0.03). The average tarsus length of all 15-day old nestlings along the gradient was 17.48 0.04 mm. The great tit nestlings at the most polluted site had slightly smaller tarsi than nestlings at the other three sites, but this difference was not significant (two-way ANOVA, F3,120=1.13, P=0.34, see Fig. 2D). Tarsus length also did not differ significantly among years (two-way ANOVA, f1,120=0.75, P=0.39). The interaction site year was not significant. The difference in tarsus length among sites was also not significant when taking into account the number of young per nest (ANCOVA, F3,106=1.99, P=0.12). The average wing length of all great tit nestlings in the pollution gradient was 46.4 0.28 mm. We found no significant differences in wing length among years or among the different study sites (two-way ANOVA, respectively F1,120=0.95, P=0.33 and F3,120=0.75, P=0.53, see Fig. 2B). The interaction siteyear was not significant. The difference in wing length among sites
dry weightstandard error) of heavy metals in the excrements of great tit nestlings along a pollution gradient F8 (N=9) A A A
A A
A A
0.50.1 17526 8.91.9 4.21.0 2.90.8 2.00.6 6627 2013448 4.11.1 13049 13.65.3 7.21.0 20758
F7 (N=6) B B B
B A
B A
0.30.1 419173 2.61.1 2.70.6 4.82 3.20.8 16733 1863536 1.10.3 22213 5820 1.90.3 25548
UIA (N=3) B B B
A B
B A
0.1 0.07 96 19 0.1 0.1 2.9 1.4 7.8 6.5 2.9 0.2 201 73 1378856 1.4 1.0 523 324 24 19 1.1 0.5 526 159
B B B
A B
B B
F
P
17.79 1.57 7.19 5.03 1.01 2.41 3.33 0.39 4.18 2.39 2.69 8.66 3.02
0.000002 0.2 0.001 0.007 0.4 0.09 0.04 0.8 0.015 0.1 0.07 0.0004 0.05
The results from a One-Way ANOVA are given. Significant differences among sites tested using a Tukey HSD-test are shown by letters. Means followed by the same letter do not differ significantly from each other. Sample sizes (number of nests) are also given.
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was also not significant when taking into account the number of young per nest (ANCOVA, F3,107=1.2, P=0.3).
ferences in fledging age among years (two-way ANOVA, F1,146=3.17, P=0.13).
3.3. Nestling health
4. Discussion
Nestlings had on average a haematocrit value of 41.06 0.4% (data of all sites combined). There was no significant difference in haematocrit values among sites (one-way ANOVA, F3,50=0.72, P=0.5, see Fig. 2C). In 1998 and 2000 we observed respectively one and two nests which contained several young with growth abnormalities at their legs (defectively developed tibiotarsi and tarsometatarsi). Such nests were only observed at the two most polluted sites, UM and F8.
The purpose of this study was to examine possible detrimental effects of heavy metals on the condition and health of 15-day old great tit nestlings along a pollution gradient. The higher concentrations of many heavy metals in the excrements of the nestlings, combined with earlier data on heavy metals in feathers of nestling (Janssens et al., 2002) and adult great tits (Janssens et al., 2001), provide good evidence that great tits near the factory complex were exposed to higher concentrations of certain heavy metals than individuals at the lesser polluted areas. Great tit nestlings from the most polluted site had significantly higher concentrations of silver, arsenic, cadmium, mercury and lead in their excrements. More significant differences in metal concentrations in excrements among sites might have been detected if sample sizes at F7 (N=6) and UIA (N=3) had not been so small. Other studies have found comparable concentrations of heavy metals in excrements of
3.4. Fledging age Fledging age differed significantly among sites (twoway ANOVA, F3,146=5.44, P=0.001, see Fig. 3). Nestlings at the two most polluted sites fledged significantly later than those at the least polluted site (Tukey HSD test, P < 0.05 in both cases). Other differences among sites were not significant. We found no significant dif-
Fig. 2. The average (A) weight; (B) wing length; (C) haematocrit values and (D) tarsus length of great tit nestlings along the gradient. Data from 1998, 1999 and 2000 are combined. Numbers denote the number of nests at a study site.
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Fig. 3. The average fledging age of great tits along the gradient. Data from 1998, 1999 and 2000 are combined. Numbers denote the sample sizes.
great tit nestlings hatched in polluted areas (Dauwe et al., 2000; Nyholm et al., 1995; Eeva and Lehikoinen, 1996). In 1998, Dauwe et al. (2000) studied metal concentrations in excrements of great and blue tit nestlings at the most and least polluted site in the pollution gradient. The highest metal concentrations were found at the most polluted site (UM), although concentrations found in this study were slightly different from those found in our study. Possibly, this was due to the fact that Dauwe et al. (2000) only sampled one nestling per brood and intraclutch variation in metal levels in excrements tends to be high. Excrements of great tit nestlings in Finland had significantly higher concentrations of copper, nickel and lead towards the pollution source (Eeva and Lehikoinen, 1996). Metal concentrations found in excrements of Polish great tit nestlings were of the same magnitude than concentrations found at our study sites (Nyholm et al., 1995). Chemical analyses of excrements for heavy metals provided direct evidence of recent exposure at a local site. Although adult birds might be more exposed to heavy metals than nestlings (Sawicka-Kapusta et al., 1986), growing young are believed to be the most sensitive to the detrimental effects of pollutants (Scheuhammer, 1987). Nestling growth and fledgling weight may be more sensitive indicators of environmental conditions than the number of fledged young, suggesting that these may be better measures of overall reproductive performance, because of their influence on post-fledging survival (Furness, 1993). Despite the obvious exposure to high concentrations of heavy metals and the previously reported effects on the breeding performance (reduced hatching and breeding success) of great tits (Janssens et al., 2003), we could only detect a few effects of this high exposure on the quality of the great tit nestlings. Nestling tarsus and wing length did not differ significantly among study sites, although nestlings at the most polluted site had on average smaller tarsi and shorter wings than those at the other three sites. A reason why we may
have failed to detect differences might be that the weakest and most unhealthy nestlings may already have died by the time morphological measurements were taken. Overall breeding success was significantly lower towards the factory complex (Janssens et al., 2003). This was not only caused by the lowered hatching success towards the pollution source, but also by an increase in nestling mortality. At UM and F8 respectively 87 and 89% of the hatchlings fledged while at F7 and UIA this was respectively 98 and 91% (Janssens et al., 2003). Nestlings of the European starling hatched in highway roadside verges and therefore exposed to high concentrations of heavy metals, had similar prefledgling weights than control nestlings (Grue et al., 1986). Hatchling weight in curlews (Numenius arquata) was not influenced by the distance to the pollution source (Currie and Valkema, 1998). Eeva and Lehikoinen (1996) reported that nestling great tits grew poorer near the pollution source than farther away. However, they found a considerable amount of variation in growth in the background areas. This is partly consistent with our data. Although nestlings at the most polluted site weighed less compared to nestlings at the less polluted sites, we found no significant differences in nestling body mass along the pollution gradient. However, when taking into account the number of nestlings in the nest at the time of sampling, the difference in weight among study sites became significant. Nestling condition also differed significantly among sites when taking into account the number of nestlings in the nest. This difference in body mass and condition on day 15 may have important effects on the survival and recruitment of great tits along our pollution gradient. In great tits, body mass on day 15 after hatching can affect local recruitment rates (Tinbergen et al., 1987; Gebhardt-Henrich and van Noordwijk, 1991) and future fertility (Haywood and Perrins, 1992). Fledgling body size measurements may also affect survival and the variation in fledgling size seems to be one of the most important components of the variation in fitness (Gebhardt-Henrich and Richner, 1998). Fledging age differed significantly among sites in our study. Nestlings at the more polluted sites fledged on average 0.5–1 day older than nestlings at the lesser polluted sites. Previous studies have stated that nestling development is the most important determinant of fledging age (Michaud and Leonard, 2000). Once a critical wing length has been reached, sibling interactions and parental behavior may influence the time of fledging. Although we could not detect any significant differences, tarsus and wing length were smaller towards the pollution source. Lead has been shown to affect neurobehavioral development in nestlings. Burger and Gochfeld (1997) showed that there were significant leadinduced differences in righting response, locomotion, thermoregulation, begging, and feeding behavior in nestling herring gulls (Larus argentatus). Lead-injected
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herring gull chicks were less able to compete for food with their siblings, with a resultant significant difference by weight at 16 days of age. The differences in nestling weight at 15 days among sites might have been sufficiently large to induce differences in fledgling age. Another reason why great tit nestlings fledged significantly later near the pollution source might not only be due to poorer growth as a result of neurobehavioral effects of heavy metals, but also as a result of reduced food availability near the pollution source. During our three-year-study, we observed three nests at the most polluted sites that contained young with badly developed legs. Growth abnormalities can occur as a result of the impairing effect of dietary heavy metals on the calcium-metabolism. Similar results have been shown in pied flycatchers (Nyholm, 1995; Eeva and Lehikoinen, 1996), but not in great tits (Eeva and Lehikoinen, 1996). We did not find any differences in haematocrit values among sites. Haematocrit values represent the relative amount of red blood cells in the total blood volume and reflect the extent and efficiency of oxygen uptake and transfer to tissues (Ots et al., 1998). Assessment of haematological variables can provide important information on health and physiological status. Factors such as nutrition, stress, seasonal cycles and toxic chemicals have been shown to alter haematological values. Low haematocrit values are indicative of bacterial infections or may reflect nutritional deficiencies (Ots et al., 1998). Until now, several studies on the effects of heavy metals on haematocrit values reported depressed values as a result of increased metal exposure (Grue et al., 1986; Nyholm, 1998). However, other studies did not find any effects of increased metal exposure on haematocrit values (Fair and Myers, 2002). Perhaps in this study, the elevated iron concentrations at the most polluted sites reversed the detrimental effects of certain heavy metals on haematocrit values and other haematological parameters (Crowe and Morgan, 1997). In conclusion, our results showed that exposure to elevated concentrations of heavy metals resulted in reductions in body mass and condition in nestling great tits. Since nestling body mass can affect local recruitment rates (Tinbergen et al., 1987; Gebhardt-Henrich and van Noordwijk, 1991), future fertility (Haywood and Perrins, 1992) and survival (Gebhardt-Henrich and Richner, 1998), this might have an important effect on the great tit population close to the pollution source. Further research on recruitment rates and survival of great tits along our pollution gradient is therefore necessary.
Acknowledgements We thank Els Van Duyse and Joke Beernaert for help with the fieldwork and Valentine Mubiana for help with
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the metal analysis. This study was supported by an I.W.T.-grant to Ellen Janssens. Marcel Eens was a Research Associate of the F.W.O, while Lieven Bervoets is supported by a FWO-Postdoctoral Fellowship. This study was also supported by FWO project G.0397.00 and by the Research Council of the University of Antwerp (NOI-BOF UA-2000 and GOA-2001 project).
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