Trace metal concentrations are higher in cartilage than in bones of scaup and pochard wintering in Poland

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Science of the Total Environment 388 (2007) 90 – 103 www.elsevier.com/locate/scitotenv

Trace metal concentrations are higher in cartilage than in bones of scaup and pochard wintering in Poland Elżbieta Kalisińska a,⁎, Wiesław Salicki a , Katarzyna M. Kavetska a , Marek Ligocki b b

a Department of Zoology, Agricultural University of Szczecin, 20 Doktora Judyma St., 71-466 Szczecin, Poland Department of Poultry and Ornamental Birds, Agricultural University of Szczecin, 20 Doktora Judyma St., 71-466 Szczecin, Poland

Received 8 January 2007; received in revised form 25 July 2007; accepted 27 July 2007 Available online 12 September 2007

Abstract Bones and cartilage of two species of diving ducks: the scaup Aythya marila (n = 24) and the pochard A. ferina (n = 24) were studied. Scaup is protected in Poland where it spends only the winter, while pochard is a game bird, abundant and breeding in Poland. In winter, the two species form large flocks off the southern coast of the Baltic, particularly in the Szczecin Lagoon where they were collected for this study. The bones and cartilage (trachea) were assayed for concentrations (dry weight-based) of three essential metals: iron (Fe), copper (Cu), and zinc (Zn); concentrations of the two toxic metals: lead (Pb) and cadmium (Cd) were assayed as well. These hard tissues of the two species showed the following order of metal concentrations Zn N Fe N Pb N Cu N Cd. In scaup and pochard bones, the respective geometric mean concentrations of Zn, Fe, Pb, Cu, and Cd were 94.4 and 102.0; 20.2 and 24.7; 6.2 and 9.6; 0.19 and 0.26; 0.114 and 0.162 mg/kg. The levels of all the metals in cartilage (Zn 149.1 and 165.8; Fe 58.4 and 116.3; Pb 10.6 and 14.9; Cu 1.41 and 3.31; Cd 0.144 and 0.175 mg/kg, respectively) were higher than in the bones of A. marila and A. ferina. However, statistically significant differences were found in respect to the essential metals only (Zn, Fe, Cu). The inter-species comparisons showed the two species to differ in their cartilage concentrations of Fe, Cu, Zn, and Cd and in their bone concentrations of Pb and Cd. In each case, the pochard exhibited higher concentrations of metals. This study showed distinct differences between trace element accumulation by two heavily mineralised avian body parts: leg bones (tarsometatarsus) and cartilage (trachea). The results are in agreement with data reported by other workers who analysed trace metals in cartilaginous and bone components of the femoral head in homoiotherm vertebrates, including humans. Therefore it is important that intra- and inter-species comparisons of hard biological components be based on corresponding body parts, and that relevant biochemical and ecotoxicological research be pursued. © 2007 Elsevier B.V. All rights reserved. Keywords: Bone; Cartilage; Trace elements; Lead; Cadmium; Iron; Zinc; Copper; Birds; Scaup; Pochard; Biomonitoring

1. Introduction Accelerated development, particularly that of industry, transport, and agriculture, results in increased addition of ⁎ Corresponding author. Tel.: +48 91 4541 521x344; fax: +48 91 4541642. E-mail address: [email protected] (E. Kalisińska). 0048-9697/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2007.07.050

numerous pollutants such as trace metals to the natural environment. They tend to disperse to areas located far away from the sources and to pollute all the ecosystems, including wetlands and water bodies. Numerous metals contribute to profound changes in those ecosystems and adversely affect both humans and animals (Nriagu, 1996; Bowman et al., 2003). During recent decades, particular attention has been paid to rare species, those threatened

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with extinction and with clearly reduced population sizes. Unfortunately, the list of such species becomes ever longer, birds featuring prominently among them (Henny et al., 1995; Austin et al., 2000; BirdLife International, 2004). Palaearctic and Nearctic waterfowl are usually migratory. The breeding and wintering areas of many waterfowl species are separated by hundreds or thousands of kilometres and information about those areas, including contamination, is far from complete (Di Giulio and Scanlon, 1984; Henny et al., 1995; Hothem et al., 1998; Cohen et al., 2000). Diving ducks, the abundance of which has diminished in recent years, include the scaup Aythya marila (breeding in Arctic and subarctic areas of Eurasia and North America) and the pochard Aythya ferina. Breeding grounds of the pochard, occurring only in Eurasia, are located to the south in relation to the breeding sites of the scaup (del Hoyo et al., 1992). In 1990–2000, the abundance of scaup in Europe was reduced by 50%; such a diminishing trend has been reported for North America as well (Austin et al., 2000). Consequently, the scaup threat status in both Europe and globally is recorded, according to the European Union (EU) scale, as EN (endangered) and LC (least concern) respectively (BirdLife International, 2004; IUCN Red List, 2006). Over the same period, the European population of pochard was reduced by 10%, imparting the EU classification-based declining threat status to the species (BirdLife International, 2004). In Poland, the pochard is a game bird, while the scaup is protected. The pochard is one of the most abundant ducks that breed in Poland (20–30 thousand pairs), the population being stable (Tomiałojć and Stawarczyk, 2003). The scaup breeds in Poland only sporadically, the major breeding areas of the species being located in Scandinavia and Iceland (del Hoyo et al., 1992; Tomiałojć and Stawarczyk, 2003). During the breeding season, the scaup feeds on freshwater invertebrates and aquatic plants, the diet of pochard being based mainly on aquatic plants. The winter food of both species consists primarily of mesozoobenthos which account for up to 90% of the diet (del Hoyo et al., 1992; Winfield and Winfield, 1994; Cohen et al., 2000). In European terms, the southern Baltic Sea, and in particular the mouth area of the Odra River, named the Szczecin Lagoon, are among the most important breeding and wintering waterfowl sites. There are an estimated thirty-five thousand scaup and about ten thousand pochard wintering in the Szczecin Lagoon (Durnick et al., 1994). The scaup populations wintering in the southern Baltic breed in Siberia, while the pochard population includes birds bred in Poland and in areas east and northeast of the country (Hagemeijer and Blair, 1997; Werham

91

et al., 2002). About 750 pairs nest in the Szczecin Lagoon area (Tomiałojć and Stawarczyk, 2003). Owing to its rich avifauna and the abundance of some species, the Odra mouth together with the Szczecin Lagoon are considered the European-scale avian strongholds (Sidło et al., 2004). In 2004, the Szczecin Lagoon was officially designated a Special Protected Area for Birds within the framework of the European Natura 2000 system. As shown by reports of the Helsinki Commission (HELCOM), the executive body of the Helsinki Convention on the Protection of Marine Environment of the Baltic Sea, Poland has for many years been one of the major polluters of the Baltic, the pollutants discharged including trace metals. The pollution load originating in Poland enters the Baltic mainly in the runoff of the two major rivers: the Odra and the Vistula, the areas off their mouths being regarded as particularly heavily polluted with mercury, zinc, copper, lead, and cadmium (Glasby et al., 2004). However, as evidenced by comparative studies conducted by HELCOM, the pollution level in the southern Baltic showed a distinctly decreasing trend over the past 25 years (HELCOM, 1993, 2004). Successful persistence of birds in a habitat depends on a variety of biotic and abiotic factors prevailing there. Heavy metals taken up from the water, bottom sediments and avian food are incorporated into the bird bodies, some of the metals being the cause of lethal or sublethal toxicity (Di Giulio and Scanlon, 1984; Scheuhammer and Norris, 1996; Hernández et al., 1999; Burger and Gochfeld, 2004). Trace metals are analysed in various avian body parts, mainly in the liver and kidney where they are temporarily deposited. In contrast, many metals are deposited permanently (for life), or at least for a long time, in hard tissues (Honda et al., 1986; Kim et al., 1998; Kalisińska et al., 2004; Pain et al., 2005). Highly mineralised materials (bones, cartilage, horn structures) accumulate, at a varying intensity, both the essential (including Fe, Zn, Cu, Mn) and non-essential (Pb, Cd, Hg) trace elements. The skeletal structures account for about 1/5 of the avian body weight. The skeletal pool of trace metals plays an important role in metabolic processes. Some trace elements of skeleton contribute from a few (Cu, Cd, Cr) to more than 50% (Zn, Mn, V, Pb) of the metals accumulated in the whole body, the amount being frequently higher than the pooled amount of a metal accumulated in the liver and kidneys (Agusa et al., 2005; Nam et al., 2005). Avian bones have for years been successfully used to estimate the birds' exposure to toxic effects of lead originating from various anthropogenic sources, as lead shows a high affinity to bones (Scheuhammer and Norris, 1996; Pain et al., 2005;

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Swaileh and Sansur, 2006; Ethier et al., 2007). Cadmium, another highly toxic metal, has been only sporadically assayed in avian bones and very low concentrations have usually been reported (Maedgen et al., 1982; Honda et al., 1986; McFarland et al., 2002), in contrast to the concentrations found in kidneys and livers (Henny et al., 1995; Hothem et al., 1998; Kim et al., 1998; Nam et al., 2005). Cadmium accumulates in bones and is associated with some osteodiseases. Even low levels of environmental exposure to the metal may promote skeletal demineralisation leading to increased bone fragility and risk of fractures, especially in mammals, including man (Bhattacharyya et al., 1988; Silver and Nudds, 1995; Scheuhammer, 1996; D'Haese et al., 1999; Järup, 2002; McFarland et al., 2002). Different pathological conditions have been diagnosed mainly in long-lived animals (including numerous avian species), because cadmium has a long biological half-life (about 20 years). The maximum life span of anseriforms, including those of the genus Aythya, is several years (Clapp et al., 1982; del Hoyo et al., 1992), which places them among homoiotherm vertebrates in danger of longterm exposure to effects of cadmium and other trace metals. Trace metal concentrations in avian cartilage are less well known than in bones, although some attempts to analyse the cartilaginous trachea have been made (Szefer and Falandysz, 1986; Kim et al., 1998; Kalisińska et al., 2005). Scaup and pochard belong to a group of species the populations of which declined in recent years. One of the causes underlying the reduction in the population size may be sought in the excessive exposure of birds to some elements. The breeding areas of the two species are located far away from one another, but the two duck species share wintering and feeding grounds in Europe, e.g., the Szczecin Lagoon. This study aims to determine concentrations in the bone and cartilage, strongly mineralised tissues, of three elements (Fe, Zn, Cu) indispensable for various bodily functions and two (Pb, Cd) that are highly toxic. Deposition of numerous metals in bones and cartilage is relatively slow, but the metals, once deposited, may remain in those components of the body for as long as the entire life span of the bird. For this reason, research on concentrations of some trace metals in hard tissues may prove helpful in assessing the degree of scaup and pochard exposure to the toxic metals over the last several years. There are but a few publications which report on comparisons between heavy metal concentrations in bones and cartilage of wild birds, therefore analysis of trace metals in scaup and pochard bones and cartilage is aimed at addressing at least two questions:

1) are the concentrations of the assayed metals similar in both tissues? and 2) do the two species accumulate similar amounts of the metals over their respective life spans? 2. Materials and methods This study involved 48 anseriform individuals representing the tribe Aythyini (diving ducks): 24 scaups A. marila (21 males and 3 females) and 24 pochards A. ferina (17 males and 7 females). The birds were collected in the Szczecin Lagoon (Fig. 1) where they had perished after becoming entangled in fishing nets while diving to feed. In the late winters of 2000, 2003, and 2004, 6, 0, and 18 scaup, and 14, 10, and 0 pochard respectively were collected, due care being taken to observe Polish regulations with respect to game hunting and environmental conservation. Prior to dissection, the birds were kept at − 20 °C. Assays were performed on the tarsometatarsus bone and the trachea collected from each individual. The biological materials to be analysed were dried at 105 °C to a constant weight. The dried materials were crushed in an agate mortar and 0.5–1.0 g samples were weighed out (to 0.0001 g). The bone and cartilage samples were ashed in glass vessels of a Velp Scientifica, mineralised in a 4:1 mixture of 65% nitric acid (HNO3) and 70% perchloric acid (HClO4) (Suprapur Merck®). Following mineralisation, the samples were diluted and brought to 10 ml with bidistilled water (Kalisińska et al., 2004). Concentrations of lead, cadmium, iron, zinc, and copper were determined using inductively coupled argon plasma atomic emission spectrometry (ICP AES) in a PerkinElmer Optima 2000 DVapparatus in the laboratory of the Department of Poultry and Ornamental Birds, Agricultural University of Szczecin. Detection limits (mg/l) for Pb, Cd, Fe, Zn, and Cu were 1, 0.1, 0.1, 0.2 and 0.4, respectively. To cross-check the analytical procedures applied, a Standard Reference (SRM) 1577b Bovine Liver, manufactured by the National Institute of Standards and Technology, USA was assayed. The reference levels of the elements studied, specified by the SRM 1577b Bovine Liver manufacturer and the corresponding values measured in our laboratory are reported in Table 1. Recoveries of the 5 metals assayed were good, with the exception of Pb (b 0.20 mgPb/kg dry weight) which had an almost 180% recovery compared to the manufacturer-specified concentrations (Table 1). However, Pb concentrations in hard tissue of wild ducks are much higher than 0.5 mg/kg d.w., generally. As concentrations of trace metals frequently deviate from the expected normal distribution, the actual

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Fig. 1. Geographical location of the scaup and pochard collection site.

distributions were checked with the chi-square test. The outcome of the test in each case dictated the selection of an appropriate significance test: parametric or nonparametric (Mann–Whitney test, M–W). Due to the low number of females in the samples, no between-sexes differences were analysed. In addition, correlation coefficients (Pearson's or Spearman's, depending on the outcome of the chisquare test) were calculated for: a) concentrations of various metals in bones or in the cartilage; b)

concentrations of each metal in bone vs. cartilage. All the calculations were performed (following recommendations of Sokal and Rohlf, 1995; Moran and Solomon, 2002) with Statistica 6.0 software. 3. Results The chi-square test showed as few as 6 distributions out of the 20 analysed to fit the normal distribution (bone and cartilage Zn concentrations in both species;

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bone Cd concentration in scaup; and cartilage Cu concentration in pochard; p N 0.10). The remaining distributions deviated (70%) from normal (p ≤ 0.05). Therefore, non-parametric tests were used in the further statistical treatment of data. Bone and cartilage concentrations (in mg/kg dry weight, d.w.) of the elements studied in scaup and pochard are shown in Tables 2 and 3. Table 2 summarises the 2000–2004 data on scaup and the 2000–2003 data on pochard, while Table 3 reports corresponding data on all the individuals analysed, regardless of the year. 3.1. Essential metals The scaup sampled in 2000 (n = 6) and in 2004 (n = 18) as well as pochard sampled in 2000 (n = 14) and 2003 (n = 10) showed concentrations of the three essential metals to be higher in the cartilage than in the bone (Table 2). The only exception is Zn in the scaup sampled in 2000, with concentrations being similar in both types of tissue (geometric mean: 123.7 and 123.0 mgZn/kg d.w. in bone and cartilage, respectively). Concentrations of Fe, Cu, and Zn in bones of both scaup and pochard sampled in 2000 were higher than those found in the individuals sampled in 2004 (scaup) and 2003 (pochard). The cartilage of individuals of the two species sampled in 2000 showed higher concentrations of Fe and Cu, and lower concentrations of Zn, compared to the corresponding values found in scaup sampled in 2004 and pochard sampled in 2003. All those differences between the 2000 individuals and those sampled in 2004 or 2003 proved significant; the differences in the bone and cartilage concentrations of the two species showed a similar pattern (Table 2). Consequently, the subsequent intra- and inter-specific comparisons with respect to the bones and cartilage are carried out without regard to year of sampling (n = 24 for scaup and n = 24 for pochard). Concentrations of the metals (GM) in bones of the diving duck studied form the following series: Zn N Fe N Cu (scaup: 94.4, 20.2, 0.19; pochard: 102.0, Table 1 Manufacturer's values (CR) and the present authors' data (OR) of heavy metal concentrations in the certified reference materials SRM 1577b (Lyophilised Bovine Liver) Material

Fe

Cu

Zn

Pb

Cd

CR 184 ± 15 160 ± 8 127 ± 16 0.129 ± 0.004 0.50 ± 0.03 OR 177 ± 17 140 ± 2 126 ± 3 0.231 ± 0.029 0.48 ± 0.02 OR/CR (%) 96.2 87.5 99.2 179.1 96.0 All values given as mg/kg in dry weight (arithmetic mean ± standard deviation).

24.7, 0.26 mg/kg d.w., respectively; Table 3). An identical sequence of Zn, Fe, and Cu concentrations was observed in the cartilage of both species (scaup: Zn 149.1, Fe 58.4, Cu 1.41; pochard: Zn 165.8, Fe 116.3, Cu 3.31 mg/kg d.w.). Comparison of concentrations of individual essential metals in the two types of tissue showed the concentrations to be much higher in the cartilage than in bones, both in scaup and in pochard (p ≤ 0.01; Table 3). The ratios of cartilage (C) to bone (B) concentrations of the metals (ZnC/ZnB, FeC/FeB, CuC/CuB) in scaup and pochard were 1.6, 2.9, 7.4 and 1.6, 4.7, 12.7, respectively. Inter-species comparisons revealed no significant differences between scaup and pochard bone concentrations of Zn, Fe, and Cd, significant differences being observed between concentrations of those metals in cartilage (Table 3). Compared with that of scaup, the pochard cartilage was richer in Zn, Fe, and Cu by 11.2, 99.1 and 134.8%, respectively. 3.2. Non-essential metals Concentrations of Pb and Cd showed no unequivocal differences and distinct patterns such as those described for concentrations of the essential metals in scaup and pochard sampled in different years (Table 2). It was in the scaup bone and cartilage only that clearly higher Cd concentrations were typically found in those individuals sampled in 2000, compared to those sampled in 2004. Moreover, the scaup individuals showed higher Pb and Cd concentrations in the cartilage than in the bone, but the differences concerned only those individuals sampled in 2004 and 2003, respectively. Subsequent intra- and inter-specific comparisons are carried out on larger samples, without division between years (Table 3). The scaup and pochard bones exhibited Pb concentrations ranging from 1.8 to 27.6 and from 1.7 to 56.2 mg/kg d.w., respectively, with corresponding GM values of 6.2 and 9.6 mg/kg. The ranges of Pb concentrations in the cartilages of scaup and pochard were much wider (2.5–76.8 and 2.3–101.1 mgPb/kg d.w. respectively), the respective GM being higher as well (10.6 and 14.9 mgPb/kg). Cadmium in the scaup bones and cartilages showed concentrations from 0.062 to 0.233 and from 0.098 to 0.428 mg/kg d.w., respectively (GM: 0.11 and 0.162 mg/kg), the respective ranges of pochard being 0.065–0.353 and 0.112–0.597 mg/kg d.w. (GM: 0.144 and 0.175 mg/kg d.w.). In neither of the diving duck species did Pb and Cd bone and cartilage concentrations show significant differences; however, the concentrations tended to be higher in cartilage (Table 3).

Table 2 Trace metal concentrations (mg/kg d.w.) in bone, B, and cartilage, C, of scaup and pochard sampled in different years, and significance of differences between years (AM, arithmetic mean; SD, standard deviation; GM, geometric mean; M-W, Mann-Whitney test; NS, non-significant) Species

AM ± SD min-max GM AM ± SD min-max GM

Copper

Zinc

Lead

Cadmium

B

C

B

C

B

C

B

C

B

C

34.8 ± 9.8 20.1-46.0 33.5A 18.4 ± 7.4 10.9-33.1 17.1A

176.7 ± 26.2 135.1-208.1 174.9B 43.1 ± 16.9 19.1-88.4 40.5B

0.40 ± 0.08 0.32-0.53 0.39A 0.17 ± 0.09 0.07-0.40 0.15A

5.41 ± 1.79 4.02-8.93 5.21B 0.96 ± 0.33 0.40-1.69 0.91B

124.2 ± 11.5 107.1-137.6 123.7 88.6 ± 20.7 61.3-122.8 86.3A

123.3 ± 9.5 111.0-136.0 123.0 159.7 ± 15.7 129.5-196.8 159.0B

9.5 ± 8.9 4.7-27.6 7.5 8.1 ± 7.4 1.8-27.3 5.8A

10.2 ± 17.2 3.1-45.3 5.0 20.3 ± 20.9 2.5-76.8 13.5B

0.197 ± 0.026 0.167-0.233 0.195 0.100 ± 0.032 0.062-0.185 0.095A

0.251 ± 0.097 0.185-0.428 0.238 0.123 ± 0.013 0.098-0.147 0.122B

NS 19.3 ± 16.3 7.7-56.2 14.7 6.6 ± 4.3 1.7-13.2 5.3

p ≤ 0.05 27.7 ± 30.3 4.1-101.1 16.4 26.1 ± 30.0 2.3-83.7 13.0

p ≤ 0.001 0.260 ± 0.056 0.167-0.353 0.255A 0.164 ± 0.061 0.112-0.354 0.157A

p ≤ 0.001 0.164 ± 0.061 0.112-0.354 0.157B 0.223 ± 0.135 0.150-0.597 0.203B

p ≤ 0.001

NS

p ≤ 0.0001

p ≤ 0.05

Aythya marila: comparison between 2000 and 2004 M-W test Pochard Aythya ferina 2000 n = 14 2003 n = 10

AM ± SD min-max GM AM ± SD min-max GM

M-W test

p ≤ 0.01 34.0 ± 12.9 18.0-70.6 32.2A 20.4 ± 15.0 9.4-53.3 17.1A

p ≤ 0.001 153.7 ± 52.9 70.6-282.0 146.0B 89.0 ± 30.4 52.6-137.4 84.5B

p ≤ 0.001 0.34 ± 0.12 0.18-0.60 0.33A 0.20 ± 0.10 0.11-0.46 0.19A

p ≤ 0.001 5.52 ± 1.89 2.88-8.67 5.21B 1.83 ± 0.60 1.18-3.26 1.75B

p ≤ 0.01 110.2 ± 15.1 85.4-134.0 109.2A 94.1 ± 17.6 75.7-119.6 92.7A

p ≤ 0.001 155.1 ± 20.0 124.5-198.3 154.0B 185.5 ± 25.5 156.7-229.1 184.0B

p ≤ 0.001

p ≤ 0.01

Aythya ferina: comparison between 2000 and 2003 p ≤ 0.001 p ≤ 0.0001 p ≤ 0.05 p ≤ 0.01

Comparison between metal concentration in bone and cartilage (M-W test; p ≤ 0.05): existing differences were signed by different letters (A and B).

Table 3 Trace metal concentrations (mg/kg d.w.) in bone, B, and cartilage, C, of scaup and pochard, and significance of differences between species (AM, arithmetic mean; SD, standard deviation; GM, geometric mean; M-W, Mann-Whitney test; NS, non-significant) Species

Scaup Aythya marila n = 24 Pochard Aythya ferina n = 24

M-W test

Iron

AM ± SD min-max GM AM ± SD min-max GM

Copper

Zinc

Lead

Cadmium

B

C

B

C

B

C

B

C

B

C

22.5 ± 10.7 10.9-46.0 20.2A 28.3 ± 15.1 9.4-70.6 24.7A

76.5 ± 62.1 19.1-208.1 58.4B 126.8 ± 54.8 52.6-282.0 116.3B

0.23 ± 0.13 0.07-0.53 0.19A 0.29 ± 0.13 0.11-0.60 0.26A

2.07 ± 2.16 0.40-8.93 1.41B 3.98 ± 2.37 1.18-8.67 3.31B

97.5 ± 24.4 61.3-137.6 94.4A 103.5 ± 17.8 75.7-134.0 102.0A

150.6 ± 21.5 111.0-196.8 149.1B 167.8 ± 26.7 124.5-229.1 165.8B

8.5 ± 7.6 1.8-27.6 6.2A 14.0 ± 14.1 1.7-56.2 9.6A

17.8 ± 20.2 2.5-76.8 10.6A 27.1 ± 29.5 2.3-101.1 14.9A

0.124 ± 0.053 0.062-0.233 0.114A 0.189 ± 0.097 0.065-0.353 0.162A

0.155 ± 0.073 0.098-0.428 0.144A 0.189 ± 0.101 0.112-0.597 0.175A

NS

p ≤ 0.01

Comparison between Aythya marila and Aythya ferina NS p ≤ 0.001 NS p ≤ 0.05

p ≤ 0.05

NS

p ≤ 0.05

p ≤ 0.01 95

Comparison between metal concentrations in bone and cartilage (M-W test; p ≤ 0.01): significant differences denoted by different letters (A and B).

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Scaup Aythya marila 2000 n=6 2004 n = 18

Iron

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Compared to scaup, pochard bones showed higher concentrations of Pb and Cd, while the cartilage showed higher concentrations of Cd. Pochard bone and cartilage Pb concentrations were respectively more than 50% and 40% higher than those in scaup. Cadmium concentrations in pochard bone and cartilage were by 42 and 21% respectively, higher than those in scaup. 3.3. Metal vs. metal relationships Table 4 summarises Spearman's rank correlation coefficients (rS) for the relationships examined. There were numerous (exclusively positive) correlations between different elements in bones of the scaup and pochard (Fe with Cu, Zn, Cd; Cu with Zn and Cd) but not in the cartilage metal concentrations (Table 4). Additionally, correlation coefficients were calculated for relationships between bones and cartilage with respect to individual metals. Significant (p ≤ 0.05), positive correlations in the scaup were seen in the concentrations of Fe, Pb, and Cd (the respective rS values were 0.56, 0.67, 0.45). The zinc concentration in the bone is negatively correlated with that in the cartilage (rS = − 0.60). In the pochard, there were significant positive correlations (0.53, 0.64, and 0.83, respectively) between Fe, Cu and Pb. Bone and cartilage Cd concentrations were negatively correlated (rS = − 0.44).

Table 4 Spearman's rank correlation coefficients calculated for metal vs. metal relationships in bone and cartilage of two duck species (genus Aythya) Correlation

Iron with Cu Zn Pb Cd Copper with Zn Pb Cd Zinc with Pb Cd Lead with Cd

Scaup A. marila (n = 24)

Pochard A. ferina (n = 24)

bone

cartilage

bone

cartilage

+0.87xxxx +0.86xxxx NS +0.88xxxx

+0.79xxxx -0.78xxxx NS +0.41x

+0.83xxxx +0.60xx NS +0.66xxx

+0.77xx NS NS NS

+0.89xxxx NS +0.84xxxx

-0.53xx NS +0.53xx

+0.75xxxx NS +0.78xxxx

-0.43x NS -0.56xx

NS +0.81xxxx

+0.43x NS

NS +0.73xxxx

NS NS

NS

Significance level: xp b 0.05; NS, non-significant.

NS p b 0.01;

xx

NS p b 0.001;

xxx

NS xxxx

p b 0.0001;

4. Discussion Bones are not structurally uniform: in addition to the spongy and compact bone tissue, they contain cartilaginous tissue (covering joint surfaces), long bones containing also marrow (Stevens and Lowe, 1992). The trachea is a typically cartilaginous structure. Hard bone and cartilage structures play a significant part in the mineral metabolism of homoiotherm vertebrates, including humans, and are sites of a relatively slow deposition and release of trace metals (Sánchez et al., 1987; Brandão-Neto et al., 1995; King et al., 2000). This is probably the reason for their being relatively seldom used in ecotoxicological studies, in contrast to kidneys and liver, which are very important in detoxification processes, and, in a relatively short time, accumulate high concentrations of trace metals (Wapnir, 1998; Myklebust and Pedersen, 1999; King et al., 2000; Barjaktarovic et al., 2002; Stout et al., 2002). However, hard tissues may prove very useful in the assessment of cumulative, multiannual exposure of homoiotherm vertebrates to various trace metals. At present, an exception is provided by bones assayed mainly for their Pb concentrations, Pb showing a high affinity to the bones (Pain et al., 2005; Swaileh and Sansur, 2006; Ethier et al., 2007). Despite numerous similarities between birds and mammals, their mineral metabolisms are substantially different. The differences are a result of at least two factors: oviparity of birds and their periodic moulting (Myklebust and Pedersen, 1999; Harr, 2002). The skeletal structure, accounting for about 20% of the bird weight, contains high amounts of trace metals accumulated in the body: for instance, from 50 to 90% of Pb, Zn, Mn, V, and Sr, while other elements (e.g., Cu and Cd) are deposited in contents not exceeding 5% (Ojanen et al., 1975; Di Giulio and Scanlon, 1984; Cosson et al., 1988; Agusa et al., 2005; Nam et al., 2005). Both groups of metals affect avian mineral metabolism, but concentrations of those metals are seldom determined in hard tissues of wild birds, including those whose population has a declining trend. In a situation involving calcium deficiency, protein-deficient diet, and acidified environment, accumulation of toxic metals (Pb and Cd) by bones may be greatly intensified, which is particularly the case for females during the breeding season (Silver and Nudds, 1995; Scheuhammer, 1996; Dauwe et al., 2006). Concentrations of trace metals in bird bones are described rarely (Hutton, 1981; Szefer and Falandysz, 1986; van Eeden and Schoonbee, 1992; Agusa et al., 2005; Nam et al., 2005; Swaileh and Sansur, 2006;

Table 5 Summary of 5 trace metal concentrations in bone and trachea (mg/kg dry weight, except source 6: wet weight) in birds from different habitats (ad, adult; dif, different; M, male; F, female; ecological category and time of material collection: mig, migrant; win, wintering; bre, breeding; sed, sedentary; C, control group; P, birds from polluted area; ND, not detected) Species

Anas platyrhynchos Anas strepera Aythya ferina Netta rufina Aythya ferina Aythya marila Aythya marila other species Egretta alba modesta Bubulcus ibis Larus atricilla Larus crassirostris Larus argentatus Haemantopus ostralegus Catharacta skua Phalacrocorax carbo Fulica cristata Fulica cristata Fulica atra Porphyrio porphyrio oceanic seabirds ⁎ Tyto alba guttata Taeniopygia guttata Passer domesticus Columba livia trachea Aythya marila Aythya marila Aythya ferina oceanic seabirds ⁎

n

Sex

Age

Ecol. category

Location

64 13 82 23 157 11 19 40 P 6P 11 P 9P 24 24 6 10

F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F M

dif dif dif dif dif ad ad dif dif dif dif dif dif dif

win win win win win sed/win sed/win sed/bre sed/bre sed/bre sed/bre bre/win win win

USA, Chesapeake Bay

5 9 25 5 15 25 11 4 6P 18 P 58 P 11 P 22 43

M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M F+M

ad ad ad ad dif dif dif ad ad dif dif dif dif dif

bre/mig bre bre bre bre mig bre bre bre/sed bre/sed bre/sed bre/sed mig sed

30 C 30 C 10 P 7C 41 P

F M F+M F+M

ad ad ad dif

sed sed

Palestine Great Britain, London GB, Chelsea Great Britain, Chelsea

1 4 24 24 22

F M F+M F+M F+M

dif

win

Poland, Gulf of Gdańsk

dif dif dif

win bre/win mig

Poland, Szczecin Lagoon Poland, Szczecin Lagoon North Pacific

Fe

Zn

Cu

Pb

0.006 0.014 3.04 2.94 3.22 3.32 0.18 0.14 1.1 ± 0.3 1.2 ± 0.2

7 1.9 4.8 24.2 22.1 2.461 0.247 2.23 2.19 4.47 4.05 9.7 6.1 1.7 ± 0.4 8.4 ± 1.9

Poland, Szczecin Poland, Słońsk Spain, Donana

16.5 16.1

Poland, Szczecin Lagoon Poland, Szczecin Lagoon Poland, Gulf of Gdańsk

18.7 16.1 210 ± 60 230 ± 30

157 150 176 145 118 96.12 85.64 154.9 211.1 193.1 191.3 91.4 82.9 160 ± 20 190 ± 40

Korea USA, Texas USA, Galveston Bay Japan Great Britain, Isle of May Great Britain, Dyfed Great Britain, Foula Japan South Africa (pollut. area) South Africa, Natalspruit Spain, Donana

50.2 ± 5.5

87.3 ± 6.4

0.79 ± 0.13

126 ± 17 146.8 ± 3.7 158.9 ± 4.9 220.1 ± 8.9

1.17 ± 0.14

North Pacific The Netherlands Laboratory experiment

122.9 ± 28

297.7 ± 93.8 159.0 139.3 167

45 (8-896) 904 914

600 510 ± 50 40.7 93.6

59.9 57.2 150.4 ± 8.6 157.7 ± 5.8 197.7 ± 5.1 200 190 ± 10 159.4 167.2 46

1.09 ± 0.04 10.3 ± 4.5 5.5 ± 1.2 3.34 2.71 0.64 1.8 (0.6-5.5) 0.16 0.16 0.8 ± 0.1

3.6 4.4 ± 0.4 0.92 2.51 2.59

Cd

0.001 0.004

0.117 0.091 0.08 ± 0.02 0.03 ± 0.01

0.25 ± 0.10 7.93 ± 3.84 6.93 ± 0.92 1.50 ± 1.26 37.7 ± 4.7 14.2 ± 1.42 4.52 ± 1.21 0.54 ± 0.01 38.7 ± 32.8 30.1 ± 4.1 2.36 1.26 0.148 1.54 (b 1.1-12) 0.64 0.87 14.3 ± 1.3 5.7 ± 1.0 669 ± 45

0.015 ± 0.002 0.09 ± 0.04 0.21 ± 0.02 0.11 ± 0.11 ND 1.22 0.24 0.11 ± 0.06

19.9 ± 2.1 13.1 21.1 0.184

0.08 0.10 ± 0.02 0.122 0.198 1.92

4.8 ± 0.57

0.157 b0.03 (b 0.03-0.28)

0.03

Source

1 1 1 1 1 2 2 3 3 3 3 4 4 5 5 6 7 7 8 9 9 9 10 11 12 3 3 13 14 14 15 15 16 17 17

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bone ordo Anseriformes Aythya valisineria Aythya marila Aythya affinis Aythya collaris Anas platyrhynchos Anas platyrhynchos

5 5 4 4 13

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Source: 1 Di Giulio and Scanlon (1984); 2 Kalisińska et al. (2004); 3 Taggart et al. (2006); 4 this study; 5 Szefer and Falandysz (1986); 6 Honda et al. (1986); 7 Hulse et al. (1980); 8 Agusa et al. (2005); 9 Hutton (1981); 10 Nam et al. (2005); 11 van Eeden and Schoonbee (1992); 12 van Eeden (2003); 13 Kim et al. (1998); 14 Esselink et al. (1995); 15 Dauwe et al. (2006); 16 Swaileh and Sansur (2006); 17 Hutton and Goodman (1980). ⁎ assays performed on pooled samples of materials collected from three individuals of Procellaria aequinoctialis (n = 3), Diomedea melanophrys (n = 9), and D. chrysostoma (n = 10).

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Taggart et al., 2006). It should be mentioned that assays reported involved various skeletal parts (usually the tarsometatarsus or tibiotarsus bones of the leg and the humerus or femur bones of the wing). According to Szefer and Falandysz (1986) and Ethier et al. (2007), skeletal parts may differ in their concentrations of an element, which should be taken into account in comparative studies. The bird's age is another important variable in such studies, but, unfortunately, it is not always possible to measure. The assays involving the cartilage (trachea) are sporadic only (Szefer and Falandysz, 1986; Kim et al., 1998; Kalisińska et al., 2005). For comparative purposes, Table 5 summarises data on concentrations of Fe, Cu, Zn, Pb, and Cd in bones and the trachea mainly of various waterfowl and waterbirds. Unfortunately, it is not in each case that identical bones were involved. 4.1. Essential metals Iron, zinc, and copper occur in bones and cartilages of birds and mammals in various concentrations depending on the stage in ontogenetic development, physiological status, and the amount of metals taken up from the habitat (Sánchez et al., 1987; Brandão-Neto et al., 1995; Wapnir, 1998; King et al., 2000; Tuormaa, 2000; van Eeden, 2003). In addition, biological antagonism between Zn and Fe and between Zn and Cu has to be taken into account (Kang et al., 1977). Moreover, synergistic and/or antagonistic behaviours of those metals with respect to the non-essential ones (Cd and Pb) have to be considered (Tuormaa, 2000; D'Haese et al., 1999). 4.2. Iron The bones of adult birds contain 17% of the total body Fe and, in this respect, after muscles, are the second iron-richest component, at least in Egretta alba modesta (Honda et al., 1986). There are large differences in bird bone Fe concentrations, which range from 16 to N 900 mg/kg d.w. (Table 5). The highest waterfowl Fe concentrations (more than 200 mg/kg d.w.) are those reported by Szefer and Falandysz (1986) for leg bones and the trachea of scaup from the Gulf of Gdańsk (Fig. 1). Bones of Fulica cristata (Gruiformes), collected from South African areas polluted with heavy metals (van Eeden and Schoonbee, 1992) showed Fe concentrations lower by about 50% than those reported by Szefer and Falandysz (1986) for scaup; on the other hand, scaup and pochard (from the western coast

of Poland) examined in this study as well as Anas platyrhynchos from the north-west of Poland showed the lowest Fe concentrations (b 20 mg/kg d.w.) among the species listed in Table 5. In ducks of the genus Aythya from the Polish coast, Fe concentrations were 2–5 times higher in cartilage than in bone (Table 5). 4.3. Copper The skeleton of adult E. alba modesta and Larus crassirostris (Charadriiformes) contains about 5% of the total body Cu content. The highest Cu contents are typical of muscles and feathers (up to 53 and 34%, respectively) (Honda et al., 1986; Agusa et al., 2005). Still other contents were reported by Nam et al. (2005) for Phalacrocorax carbo (Pelecaniformes), with 70, N11, c.a. 7, and 2% of the total Cu content being deposited in muscles, liver, feather, and bones, respectively. At the same time, Nam et al. (2005) stressed the high Cu contents in the cormorant's muscles to be rather unusual for birds. It should be then assumed that Cu contents and concentrations in various avian body parts are, on the one hand, species- and biology-dependent, and, on the other, reflect the habitat quality, as concluded also by Taggart et al. (2006). Mean bone Cu concentrations of the waterfowl from non-polluted and polluted areas (Donana National and Natural Parks in Spain and South Africa) were found to vary from 0.006 to 1.8 and from 2.7 to 10.3 mg/kg d.w., respectively (Table 5). Compared to those birds, the bones of scaup and pochard examined in this study as well as bones of A. platyrhynchos from the north-west of Poland showed the lowest Cu concentrations, lower than 0.2 mg/kg d.w. Scaup from the Gulf of Gdańsk, studied by Szefer and Falandysz (1986) showed Cu concentrations in bones and cartilage to be 5 and 4 times higher, respectively, than those revealed in this study. Oceanic seabirds and diving ducks from Poland showed cartilage Cu concentrations to be 4 to 14 times those in the bones (Table 5). 4.4. Zinc Zinc plays an important part in metabolic processes and the metal is also involved in ossification and acts on cartilage growth, especially in young organisms, including birds (Brandão-Neto et al., 1995; Lai and Yamaguchi, 2005). However, an excess of Zn leads to poisoning, described also in waterfowl, mainly from examination of the liver or pancreas of the birds affected (Zdziarski et al., 1994; Hernández et al., 1999).

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Studies on Zn distribution in adult seabirds and waterfowl showed their bones to contain from 30 to 50% of the total amount of the metal in the body (Honda et al., 1986; Kim et al., 1998; Agusa et al., 2005; Nam et al., 2005). The values mentioned are the highest among Zn data reported from various biological materials examined in birds. Among the avian species listed in Table 5, the highest bone Zn concentrations (in excess of 200 mg/kg d.w.) were found in F. cristata, Anas strepera, and Catharacta skua (Charadriiformes). The zinc liver concentrations in those species were 420, 139, and 139 mg/kg d.w., respectively, and in two of them were distinctly lower than in bones. In the opinion of Agusa et al. (2005), the Zn level in the bones of, e.g., the gull L. crassirostris, was relatively high compared to that in other tissues and organs, because some metals, including Zn, are likely to substitute calcium ions in the hydroxyapatites, the major bone components. A comparison between the A. marila data collected from the Gulf of Gdańsk and the Szczecin Lagoon, shows bones and cartilages of the individuals examined by Szefer and Falandysz (1986) to be zinc-enriched by more than 130% and 20% respectively than the birds examined in this study (Table 5). Cartilage Zn concentrations in the diving ducks from the Polish coast and in oceanic seabirds are 1.2–1.6 times those in the bones (Table 5). 4.5. Non-essential metals Of the two toxic metals assayed in the bones, there is clearly much more information on Pb than on Cd, as Pb is deposited in much higher concentrations than Cd. 4.6. Lead Lead poisoning in birds, most often due to shot pellets and less seldom to anglers' sinkers, is well documented (Di Giulio and Scanlon, 1984; Scheuhammer, 1991; Scheuhammer and Norris, 1996; Fisher et al., 2006; Ethier et al., 2007). Poisoning with metallic Pb also poses a threat to birds of prey which consume animals shot by hunters (Pain et al., 2005). In addition, those birds inhabiting urbanised areas and living in the vicinity of motorways absorb Pb from car fumes which contain lead tetraethyl (Hulse et al., 1980; Grue et al., 1986). This still remains a big problem in those parts of the world where lead-containing fuel is still in use (Swaileh and Sansur, 2006). In some environments, bird plumbism is caused by industrial pollution or leaded paint chips (Sileo and Fefer, 1987; van Eeden, 2003). Scheuhammer (1987) suggested that Pb levels over 5 mg/kg d.w. in adult wild birds might indicate an

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increased environmental exposure to this element. A considerable bird exposure to Pb is evidenced by concentrations exceeding 10 mg/kg bone d.w., according to the opinion of some authors, or 20 mg/kg, as maintained by others (Scheuhammer, 1989; Wickson et al., 1992; Guitart et al., 1994). Of the 48 ducks examined in this study, bones of 6 scaups and 11 pochards, i.e., 25% and 46% respectively, showed Pb concentrations higher than 10 mg/kg. Szefer and Falandysz (1986) found differences in bone Pb concentration between male and female scaup (Table 5). The male mean Pb concentration is 35% higher than the mean calculated for A. marila from the Szczecin Lagoon (8.4 and 6.1 mgPb/kg d.w., respectively; Table 5). In the opinion of Di Giulio and Scanlon (1984), freshwater herbivores of the order Anseriformes accumulate more Pb in their bones than birds classified as brackish-water omnivores and seaducks. Those observations were confirmed in this study: a clearly higher bone Pb concentration was typical of pochard, a game species ingesting preferably plants and breeding abundantly in Poland, than in the omnivorous scaup. Both species overwinter in the brackish-water Szczecin Lagoon, where they feed mainly on macrozoobenthos. In Poland, as in other countries of central and eastern Europe, Pb shot pellets are commonly in use. Of the waterfowl and waterbirds listed in Table 5, the highest bone Pb concentrations, exceeding 30 mg/kg, were reported from F. cristata, and Larus argentatus. Comparison of trachea and bone Pb concentrations in birds (Table 5) shows concentrations in cartilage of diving ducks and seabirds to be higher than in bones (by N 2 and 1.2 times respectively). No significant differences in Pb concentrations between the two tissues were found in scaup and pochard from the Szczecin Lagoon (due to the very wide range of concentrations), but the concentrations were highly correlated. Some interesting data were reported by Kwapuliński et al. (1995) and Zoeger et al. (2006) who studied metal distribution in femoral heads of adult humans. Kwapuliński et al. (1995) found Pb contents in ash to produce the following series in femur head: joint surface cartilage N cortical bone N spongious bone. Zoeger et al. (2006) described a highly specific accumulation of Pb in the tidemark (the transition zone between the calcified and non-calcified articular cartilage). Lead fluorescence intensities in the tidemark were 13 times higher when compared to the subchondral bone. Those studies on the human femur point to clearly higher Pb levels in the cartilaginous part of the bone than in deeper-lying and more strongly mineralised parts, which would seem to correspond with our results on Pb in the cartilaginous trachea and bones of diving ducks.

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4.7. Cadmium A negative influence of Cd on bones of homoiotherms, particularly humans, has been well documented; the element is usually deposited in small amounts (Mayack et al., 1981; Barjaktarovic et al., 2002; Järup, 2002; Åkesson et al., 2006). Cadmium is called a nephrotoxin because the highest concentrations and amounts (up to 40–48% of the total amount of Cd in the bird body, Kim et al., 1998) are frequently found in kidneys. The liver is usually the second cadmium-richest organ, for which reason those two soft organs are most frequently assayed in ecotoxicological studies (Hutton, 1981; Honda et al., 1986; Saeki et al., 2000; Agusa et al., 2005). If the mammalian hepatic Cd : renal Cd concentration ratios were less than 1, a prolonged low Cd exposure is implied, while a ratio higher than 1 suggests damage to detoxification mechanisms in the body and poisoning (Mayack et al., 1981; Scheuhammer, 1987; Barjaktarovic et al., 2002). Then, bone Cd contents are usually elevated. The human itai-itai disease observed in Japan over 50 years ago entails a mixed pattern of bone diseases, as well as kidney damage. According to Järup (2002), Cd content in the skeleton of the affected individuals was found to be several times higher than in non-exposed individuals (for men, 2.7 mg/ kg compared with 0.3 mg/kg; for women, 2.8 mg/kg compared with 0.6 mg/kg). Cadmium content in the avian skeleton is usually low and does not exceed 3% of the body burden (Kim et al., 1998; Saeki et al., 2000; Agusa et al., 2005), although some exceptions have been reported. For example, Honda et al. (1986) showed bones of nestling and adult E. alba modesta contain much more Cd, 22.2% and 12.1% respectively. Cadmium concentrations in bones of most of the waterfowl listed in Table 5 are low and did not exceed 0.3 mg/kg d.w. Exceptional cases are furnished by F. cristata, Haematopus ostralegus, and oceanic seabirds which showed concentrations exceeding 1 mg/kg d.w., too. In the extreme cases, e.g., in F. cristata from South Africa, when the body carries an excessive load of heavy metals, the highest Cd concentration (above 4 mg/kg d.w.) may occur in the bones rather than in kidneys and liver. The F. cristata Cd bone concentration was more than 2 and 3–4 times higher than that of the kidneys and liver, respectively (van Eeden, 2003). It is worth mentioning that Agusa et al. (2005) revealed a positive correlation in Cd content between bones and liver (rS = 0.686, p ≤ 0.001) and between bones and kidneys (rS = 0.358, p ≤ 0.01) in gulls. The increasing concentrations of Cd in the liver and kidneys were

accompanied by a gradual, albeit slow, increase in bone Cd concentrations. The adult gulls showed Cd concentrations in the kidneys, liver, and bones to amount to 4.80, 40.70, 0.075 mg/kg d.w., respectively. Our study revealed significant correlations between cartilage and bone Cd concentrations in scaup and pochard. Cadmium concentrations in the trachea of diving ducks from the Polish coast and of oceanic seabirds were higher by 22–69%, compared to those in bones (Table 5). The data seem to correspond with the results reported by Kwapuliński et al. (1995) who, when studying the human femur capitulum, found the cartilage joint surface to contain 70% more Cd than in the cortical and spongiform parts. Bone Cd concentrations in scaup and pochard from the Szczecin Lagoon were low (b 0.20 mgCd/kg d.w.), but the comparison with scaup data of Szefer and Falandysz (1986) shows the bone Cd concentration in the species is at present about 20 times that recorded 20 years ago (Table 5). This type of temporal–spatial information on Cd in avian bones is scant, so perhaps it would be worthwhile to examine museum specimens. Some authors are of the opinion that concentrations of Cd in some birds might have arisen from exposure on migration (Hothem et al., 1998), Cd originating from polluted sediments and benthos, particularly molluscs. The study of De Leeuw (1999) on the tufted duck (Aythya fuligula) and scaup feeding on zebra mussels (Dreissena polymorpha) showed the daily mussel consumption (wet weight-based) was about two to three times the body mass of the birds. Zebra mussels are abundant in the Szczecin Lagoon and may be an important source of trace metals: Zn, Cu, Pb, Cd, and Hg (Protasowicki, 1991; Wiesner et al., 2001). Cadmium biomagnification in the aquatic food chain was pointed out by Croteau et al. (2005), and Luoma and Rainbow (2005). At the same time, birds – at least some of them – are capable of freeing themselves of various trace elements (including Hg and Cd) during moulting, and the females also during egg-laying (McFarland et al., 2002). It is not known, however, if those mechanisms are sufficient to protect the birds from adverse effects of trace elements on reproduction, development, and behaviours – including locomotion, feeding, and cognitive abilities – that in turn affect survival in nature (Burger and Gochfeld, 2004). 5. Conclusions Bones and cartilages of two diving ducks scaup A. marila and pochard A. ferina showed the following order

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of trace metal concentrations: Zn N Fe N Pb N Cu N Cd. Concentrations of all the metals were higher in the cartilage than in the bone, in both species (the differences were not significant for Pb and Cd). Our results point to distinct differences in the level of trace elements in two types of strongly mineralised tissues. The data were found to correspond to results reported by other authors who studied trace metals in cartilaginous and bone parts of the femoral head of mammals, including humans. For those reasons, it is important that intra- and inter-species comparisons of hard biological materials be based on corresponding body parts. Further relevant biochemical and ecotoxicological studies of metal concentrations in bone and cartilage are necessary. So far, bioindicative research on bird skeletons has not treated cartilaginous and bone elements separately due to problems with separating cartilage from bone joint surfaces and due to fragmentary knowledge on differences in trace metal accumulation levels. The trachea is an easily sampled cartilaginous structure. We suggest that it be used more frequently in ecotoxicological research, in addition to assays made on bones, especially in studies on multiannual exposure of birds to trace metals. Noteworthy are large differences between two diving ducks from Szczecin Lagoon in terms of their cartilage (Fe, Cu, and Cd) and bone (Pb and Cd) levels. In each type of the material assayed, higher concentrations were shown in pochard, a game species in Poland. It is abundant here during the breeding season and in winter, for which reason it may be regarded as more exposed to trace metals (particularly Pb), compared to the exposure of scaup which only overwinters on the southern Baltic coast. Compared to waterfowl from other areas of the world, particularly those regarded as strongly heavy metal-polluted, bones of scaup and pochard from the Szczecin Lagoon show very low levels of Zn, Fe, Cu, Cd, but not Pb. Although the use of leaded fuels in Poland was discontinued a few years ago, lead-containing shot pellets are still commonly used by hunters. To protect the environment and the birds, it is mandatory that such pellets are replaced by other types of shot, manufactured from non-toxic materials. Scaup and pochard are longlived, homoiotherm animals and show a tendency for accumulation in their bodies of substantial amounts of certain trace metals. However, inter-specific ecological differences, including food preferences, have to be borne in mind. A correct interpretation of data calls for knowledge of physiological levels of the elements and/or of the levels reflecting the geochemical background concentrations of the metals in addition to the knowledge of threshold sublethal and lethal concentrations for bone and cartilage.

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