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Heavy metals in the mallard Anas platyrhynchos from eastern Austria
STOTEN-21534; No of Pages 7 Science of the Total Environment xxx (2016) xxx–xxx
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Heavy metals in the mallard Anas platyrhynchos from eastern Austria Christof Plessl a, Peter Jandrisits a, Regina Krachler a, Bernhard K. Keppler a, Franz Jirsa a,b,⁎ a b
University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Währingerstrasse 42, A-1090 Vienna, Austria University of Johannesburg, Dept. of Zoology, P. O. Box 524, Auckland Park 2006, South Africa
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Mercury levels appear elevated, feathers show highest concentrations. • 3.9% of the specimen show lead levels indicating lead poisoning. • Silver levels were recorded for the first time for this species from Europe. • Chromium and copper levels appear elevated in comparison to other studies.
a r t i c l e
i n f o
Article history: Received 21 October 2016 Received in revised form 1 December 2016 Accepted 2 December 2016 Available online xxxx Editor: F.M. Tack Keywords: Water fowl Trace elements Lead Silver Mercury
a b s t r a c t A total of 77 specimens of the mallard Anas platyrhynchos were sampled from the eastern part of Austria before the ban on lead gun shot for hunting water fowl. Samples of muscle and liver were analyzed for their content of Cr, Cu, Zn, Ag, Cd, Hg and Pb using atomic absorption spectrometry. In addition the Hg content of feather samples from this aquatic bird species was evaluated. Results generally show higher concentrations of the metals in the liver compared to muscle; for mercury the concentrations were feathers N liver N muscle. Elevated, in some cases critical concentrations of Cr, Cu, Cd, Hg and Pb were measured. Levels of Ag were recorded for the first time for this species from Europe, providing basic information for future evaluation of this upcoming pollutant in aquatic environments. © 2016 Published by Elsevier B.V.
1. Introduction Although the use of heavy metals in the past has had a great positive impact on human economy, negative consequences for humans and the environment have been demonstrated (Reisinger et al., 2009). The ⁎ Corresponding author at: University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Währingerstrasse 42, A-1090 Vienna, Austria. E-mail address: [email protected] (F. Jirsa).
global anthropogenic distribution of heavy metals has led to multiplied environmental concentrations in particular for lead, copper, zinc and others in all regions of the world (Nriagu, 1996). The abundance of heavy metals in the anthroposphere and their potential emissions in the future are still on the rise, calling for national and international measurements (Reisinger et al., 2009). Waste streams, mining runoffs and other anthropogenic sources of heavy metals still often end up in wetlands (Levengood, 2003), where all aquatic biota, including waterfowl, may be exposed to this pollution. Mallards can be exposed to heavy
http://dx.doi.org/10.1016/j.scitotenv.2016.12.013 0048-9697/© 2016 Published by Elsevier B.V.
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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metal uptake through their diet. For example freshwater mussels have been identified as a food component of these birds and have been described as metal accumulating organisms (Phillips, 1976; Luschützky, 2005). In addition to this common route of metal uptake, an additional hazard has been observed for waterfowl: uptake of shotgun pellets. These pellets consisting of lead have been used by hunters for ages, and a considerable portion of lead has been distributed into the environment in the form of such pellets. Combined with lost plummets from fisheries, an annual input of 600 t of metallic lead into the Austrian landscape has been estimated (Reisinger et al., 2009), for Germany the value is 9000 t and for Italy 25,000 t per year (Kurz, 2004). Although metallic lead is not readily bioavailable, it has been shown that the low pH and mechanical abrasion in the bird's stomach leads to dissolution and uptake of lead into the organism (Yamamoto et al., 1993). This combined exposure to contamination of food and direct uptake may lead to accumulation of metals in the tissues of these birds. Ducks, as one of the most common waterfowl, therefore have been recognised as biomonitors for contamination by e.g. Henny et al. (2000), Levengood (2003). About 73,000 wild ducks, of which most are mallards Anas platyrhynchos L.1758, are shot every year during the hunting season in Austria, with the majority of app. 60,000 in the easternmost parts of the country (Statistics Austria, 2014). They are mostly sold on regional markets and used for human consumption. Concern about lead poisoning of water birds and other wildlife through exposure to these lead pellets has led to a restriction of lead ammunition in at least 29 countries around the world (Avery, 2009), also leading to a first measure taken in Austria in 2012: lead shotgun pellets for hunting water fowl have been banned since June 1st (Berlakovich, 2011) - which still leaves fishing and hunting on small game as a source for metallic lead in the environment. Regarding surface waters, new legacy has led to a reduction in the use of hazardous metals, and a constant decrease in the heavy metal content of surface waters has been recorded from Europe, including Austria (BMLFUW, 2011). Nevertheless, pollution with elements like cadmium, zinc and to a lesser degree copper and mercury is still a problem in sediments throughout the country, especially in mining regions or around large cities (BMLFUW, 2003). There are no threshold levels legally set for any metals in river or lake sediments, and therefore they are no longer included in water quality reports. It has been shown that elevated concentrations in sediments can significantly influence aquatic biota due to bioaccumulation or -magnification (e.g. Handy et al., 2005). The former is of major importance for hydrophilic substances like heavy metal cations, mainly taken up via the gills (Hofer et al., 1995), the latter encompasses lipophilic compounds like methylmercury mainly uptaken with food (Hall et al., 1997). To our knowledge there are no data available on heavy metals in mallards from central Europe. We had the opportunity to investigate levels of heavy metals in muscle and liver tissues from mallards collected before the ban of lead shotgun pellets, to ensure a record of the situation and to save data for later comparisons. In addition to Pb it seemed appropriate to analyse Cd and Hg as metals of priority concern within the EU (EU, 2001b). For mercury the analyses of feathers were included, as these are known to be a major pathway for the storage and excretion of Hg in birds (Monteiro and Furness, 2001). Cu, Cr and Zn, which are essential trace metals showing an enhanced anthropogenic distribution in the environment, were also measured in the tissue samples. Moreover we included Ag in the study, as nanoparticles of this metal have started to be widely used in consumer products and therefore will be increasingly included in waste streams, threatening also the aquatic environments (Yu et al., 2013). 2. Materials and methods 2.1. Study site Sampling for this study took place in the northeastern part of Austria (Fig. 1). Within the region lies Austria's capital Vienna (1.7 million
inhabitants), with its industrial impact on the environment. The surrounding region is a typical man-made environment characterized by a population density of 84 inhabitants/km2 (Statistics Austria, 2014) dominated by agricultural activity and managed forests. Regarding water bodies, the region lies within the catchment area of the Danube River, and floodplains, smaller rivers and numerous lakes and ponds are present. Hunting takes place within the whole region, except for congested areas. By far the most abundant water fowl is the mallard A. platyrhynchos, which is native, widespread over Eurasia and well adapted to cultivated landscape. Its population is estimated to be 10,000–20,000 breeding pairs in Austria (Bauer, 2005). 2.2. Animal sampling In total 77 specimens of A. platyrhynchos were received from eight hunting grounds situated in the eastern parts of Austria (Fig. 1). They were shot by hunters during late autumn in 2009 and 2010, using commercially available lead shots. Freshly killed animals were transported to the University of Veterinary Medicine in Vienna, where sex and weight were determined. A dissection followed and tissue samples (app. 2–3 g of liver and pectoral muscle, respectively) and about 0.5 g of feather samples (from the pectoral region) were obtained using stainless steel instruments, avoiding tissue visibly affected by intruded lead bullets. Samples were transferred into acid-prewashed polypropylene (PP) tubes and stored at −21 °C until further analysis. 2.3. Trace element determination Frozen samples were transferred to the lab of the Institute of Inorganic Chemistry at the University of Vienna. Microwave digestion was performed in a MARS XPRESS system (CEM Corporation). 1 g (wet weight) of tissue or 0.2 g of feathers were digested in 9 mL of HNO3 34% (TraceSELECT® Fluka) and 1 mL H2O2 30% (TraceSELECT® Fluka). Afterwards, samples were transferred quantitatively into 15 mL flasks and brought to volume with millipore water. Before analysis, samples were filtered through 0.2 μm PTFE syringe filters (VWR) and, where necessary, diluted. Recovery rates were determined using certified reference material (DORM-3 for Cr and DOLT-3 for all other elements, Natural Research Council Canada). Reference samples comprising 0.2 g (dry weight) of fish protein DORM-3 and dogfish liver DOLT-3 were digested and diluted in the same manner. To determine the detection limits (LOD), analytical blanks were prepared without insertion of a sample. Mercury was detected in the samples immediately after digestion with cold vapor atomic absorption spectrometry (CV-AAS) (FIMS 400 by Perkin Elmer). Zinc was measured with a Perkin Elmer AAnalyst 200 flame atomic absorption spectrometer (F-AAS). For the detection of Cr, Cu, Ag, Cd and Pb a Perkin Elmer PinAAcle 900Z graphite furnace atomic absorption spectrometer (GF-AAS) was used. Recovery rates were within a range from 91.8–104.2%, demonstrating the appropriateness of the method used (Table 1). 2.4. Data processing Data processing and statistics were performed using Microsoft Excel 2010 and SPSS Statistics Version 20. A Kolmogorov-Smirnov-test was performed to check for normal distribution. Differences between hunting grounds and sex were validated using a t-test (if normally distributed) or a Mann-Whitney-U test (if not normally distributed). Comparison between the different tissues was performed with a t-test or a Wilcoxon-test. Results for mercury were checked with a Kruskal-Wallistest, as three kinds of tissues had to be compared. For calculation of the means, randomly chosen values between zero and the LOD were allocated to the samples, if measured concentrations were below the LOD. To compare mercury levels of muscle and liver with feathers, a water content of 72% in tissue (liver & muscle) was assumed following Ristic et al. (2006) to convert wet weight into dry weight. In the results and
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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Fig. 1. Geographical positions of the hunting grounds for mallard sampling in eastern Austria; BE: Bernhardsthal, EB: Ebreichsdorf, FR: Frauenhofen, GR: Groß-Schweinbarth, HA: Haag, LA: Langenschönbichl, LI: Litschau, SA: Saxen.
discussion part, values from other authors, which were available on a dry weight basis, were converted into wet weight, assuming the same water content, to make values comparable. Bioconcentration factor (BCF): The Bioconcentration factors (BCF) were determined using the formula: BCF ðx=yÞ ¼
concentration of trace element in x concentration of the trace element in y
in which the variables x and y stand for matrices that are compared to each other, such as liver, muscle and feathers. To calculate BCF, only values above the LOD were used. 3. Results and discussion Forty female and 37 male mallards with a mean weight of 1214 ± 229 g were sampled. Comparing the sampling regions, for some metal concentrations weak but significant differences occurred, but not coherent regarding the single elements compared in muscle and liver. The sample size differed greatly between hunting grounds; in addition, mallards do not have a steady territory and show opportunistic migration behaviour (Aubrecht and Holzer, 2000). This does not guarantee that sampled specimens fed for a longer period of time in the district they were hunted and thus were exposed to identical environmental
Table 1 Element concentrations in DOLT-3 and DORM-3 (*), certified by NRCC and as detected with GF-AAS(a), F-AAS(b), CV-AAS(c): mean ± SD of six measurements, mean accuracy and detection limits (LOD), all values in mg/kg dw. Element
NRCC (mg/kg) Mean
±SD
Mean (mg/kg)
±SD
Accuracy (%)
LOD (mg/kg)
Cr (a)⁎ Cu (a) Zn (b) Ag (a) Cd (a) Hg (c) Pb (a)
1.89 31.2 86.6 1.20 19.4 3.37 0.32
0.17 1.0 2.4 0.07 0.6 0.14 0.05
1.88 31.43 79.5 1.19 16.68 3.29 0.33
0.11 0.31 6.7 0.09 0.54 0.07 0.01
99.3 100.8 91.8 99.5 101.5 97.8 104.2
0.008 0.015 1.5 0.008 0.005 0.008 0.008
conditions. Therefore a separate presentation of results for each hunting ground seemed inappropriate, and results are presented for the whole sample. Mean level, minimum and maximum levels as well as median levels of the analyzed elements in liver and muscle are given in Table 2. For none of the investigated element concentrations was a significant difference between male and female specimens recorded; this is in accordance with the results of Mansouri and Majnoni (2014), who came to identical results for mallards from Southern Iran. Statistical evaluation (Wilcoxon-test, p b 0.05) showed significantly higher levels in liver compared to muscle for all elements under investigation. Mean BCFs ranged from 3.6 for Hg up to 139 for Cd. This agrees with the literature, confirming the liver as the major storage organ for metals and as a major organ for detoxification as well. These properties are due to a content of up to 30% metallothioneins, a group of proteins rich in cysteine, showing a high affinity towards so-called weak metals like Cd, Cu, Ag and Zn (Kaim and Schwederski, 2005). Especially for Cu- and Cd-regulation the involvement of these proteins has been proven for mallards and other aquatic birds by Nam et al. (2005b). In the following the respective metals are discussed briefly. 3.1. Chromium Chromium was well detectable in all samples. Although Vincent (2001) concluded in his review that the element appears to be a trace nutrient for mammals, its essentiality for plants and animals is still under discussion (Markert et al., 2015). Mean levels in liver (1.08 ± 0.66 μg/g ww) were higher but still comparable with those reported by Mateo and Guitart (2003) from Spain, where mean levels from 0.336 to 1.064 μg/g ww were stated in the liver of A. platyrhynchos from different regions. In contrast, Nam et al. (2005b) reported 0.073 ± 0.025 μg/g ww in the liver of mallards from Japan; Eisler (1986) reported mean Cr levels of 0.2 μg/g ww from the liver of various birds, stating that 1.1 μg/g ww should be considered as evidence for chromium contamination. Our results of 0.110 ± 0.046 μg/g ww in muscle are much higher compared to 0.042 ± 0.022 μg/g ww published by Nam et al. (2005b) from Japan. To our knowledge, chromium levels from muscle of mallards from other studies are missing. Whether the elevated chromium levels in the tissues of mallards from Austria are due
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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Table 2 Heavy metal concentration in tissues of mallards from Eastern Austria (μg/g ww), n = 77, Med: median, BCFli/mu: mean bioconcentration factor liver/muscle. Liver
Cr Cu Zn Ag Cd Hg Pb
Muscle
BCFli/mu
Mean
±SD
Min
Med
Max
Mean
±SD
Min
Med
Max
1.08 37.0 38.2 0.060 0.228 0.170 0.289
0.657 21.4 11.5 0.071 0.184 0.187 0.801
0.361 2.87 14.2 b0.002 0.016 0.020 b0.002
0.916 35.3 37.1 0.037 0.173 0.123 0.088
4.74 99.3 74.9 0.329 0.762 1.55 6.70
0.110 5.28 7.66 0.002 0.002 0.049 0.177
0.046 2.01 2.90 0.002 0.002 0.040 0.996
0.056 1.27 2.67 b0.002 b0.001 0.007 b0.002
0.103 4.87 7.93 b0.002 0.001 0.040 b0.002
0.458 10.9 13.8 0.012 0.012 0.288 7.89
to elevated Cr levels in the environment, for which there is no evidence so far, or whether they are specific to A. platyrhynchos, remains a subject for further investigations. 3.2. Copper Copper was well detectable in both, muscle and liver. It is an essential trace element which is part of many proteins associated with electron-transfer in respiration and the metabolism of oxygen (e.g. oxidases and oxygenases) (Kaim and Schwederski, 2005). It has been described to be of higher prevalence in liver compared to muscle, which is confirmed in our results displaying a BCF liver/muscle of 8.0. Copper levels in muscle (mean 5.28 ± 2.01 and median 4.87 μg/g ww) in our study are well comparable to values from two different sites in north-western Poland (mean 5.90 ± 1.79 and 6.23 ± 1.81) reported by Kalisińska et al. (2004) and from southern Poland (median 4.21 μg/g ww) reported by Binkowski et al. (2013), but are much higher compared to mallards from western Iran (mean 2.02 ± 0.52 μg/g ww) (Mansouri and Majnoni, 2014). For Cu levels in the liver, numerous publications are available and our values (mean 37.0 ± 21.4 μg/g ww) are on the higher end of the scale - e.g. mean values of 35.2 μg/g ww were described from Doñana Park, Spain, by Mateo and Guitart (2003), 29.26 ± 20.02 μg/g ww from Poland (Kalisińska et al., 2004) and 35.7 ± 19.3 μg/g ww in mallards from Japan by Nam et al. (2005b). In contrast to these values are those described by Kim and Oh (2012) from the liver of mallards in Korea (11.9 ± 4.0 μg/g ww) and the mean of 3.88 ± 0.38 μg/g ww reported by Mansouri and Majnoni (2014) from Iran. Eisler (2000) suggested that Cu concentrations in birds from areas of high anthropogenic copper use appear to be elevated. This could explain the big differences in copper levels from our study and Poland, compared to Korea and Iran, due to lesser anthropogenic influence in the rural parts compared to European regions. Elevated Cu levels have been reported from Austrian rivers before, amongst others by Jirsa et al. (2008) who found signs of Cu pollution in fish from eastern Austria and in the official report by the Austrian Ministry for Agriculture, Forestry, Environment and Water Management, where 25% of sampling sites showed an increase of Cu concentration in surface water samples between 1998 and 2004 – compared to 1991–1997 (BMLFUW, 2006). 3.3. Zinc As one of the important essential trace elements, Zn was well detectable in both, muscle and liver, displaying a mean BCFliver/muscle of 5.6. Mean levels in muscle from our study (7.66 ± 2.90 μg/g ww) agree well with the results of Kalisińska et al. (2004) for mallards from two sites in north-western Poland (12.25 ± 3.19 and 11.99 ± 1.88 μg/g ww, respectively) and to the results from southern Poland by Binkowski et al. (2013), who reported a median concentration of 7.7 μg/g ww compared to a median of 7.93 μg/g ww from our study. Again these values are higher compared to the results by Mansouri and Majnoni (2014), who report 5.7 ± 0.8 μg/g ww from musculature in mallards from Iran, but the difference is not as pronounced as for copper. Levels in the liver are generally higher compared to muscle, again
10.4 8.0 5.6 25.5 139 3.6 17.3
confirming the importance of the liver for metal transport and storage in the body. Liver concentrations from other publications are in good accordance to our results: we report a mean of 38.2 ± 11.5 μg/g ww, Mateo and Guitart (2003) report a median of 37.6 μg/g ww from the Ebro Delta and 36.5 μg/g ww from Spanish wetlands, Kalisińska et al. (2004) present 39.72 ± 11.46 and 43.32 ± 7.62 from two sites in Poland, and Di Giulio and Scanlon (1984) report 40.3 μg/g ww in mallard livers from Chesapeake Bay. Again, in contrast to these values stand those from Korea reported by Kim and Oh (2012), who found 28.9 ± 13.8 μg/g ww and from Iran reported by Mansouri and Majnoni (2014), who found 18.0 ± 1.8 μg/g ww Zn in their liver samples. The levels in our study appear to be background levels and a sign that Zn pollution is not present; a potential pollution would result in significantly elevated levels both in the liver and muscle, as shown by Gasaway and Buss (1972) during a toxicity test with mallards over 60 days. 3.4. Silver Silver was detectable in only 10% of the muscle samples in very low concentration, resulting in a mean of 0.002 μg/g ww. To our knowledge there are no other reports of Ag levels in the muscle of birds. In contrast, 94.8% of the liver samples showed Ag levels above the LOD. Our mean values of 0.060 ± 0.071 μg/g ww are in perfect accordance with the liver data published by Nam et al. (2005b) for mallards from Japan, who found 0.058 ± 0.076 μg/g ww in their samples. Other data for silver from birds are scarce, e.g. Fredricks et al. (2009) reported Ag in only one sample of 70 above the LOD of 0.050 μg/g ww in white winged doves from Texas, but Custer and Hohman (1994) reported that 10% of liver samples from canvasback ducks Aythya valisineria had detectable Ag levels between their LOD of 0.56 and 3.1 μg/g ww, which are significantly higher levels compared to the studies mentioned before. Silver is not known to have any biological function and has not been in the focus of research for a long time. The enhanced anthropogenic use as an antibacterial agent not only in medical products but increasingly in household products, especially in the form of nanoparticles, has led to rising concern for this metal in the environment (Yu et al., 2013). Although the mechanism of Ag-nanoparticle toxicity in the body of vertebrates including humans is still under discussion, it is suggested that the effects induced by these particles are mediated via Ag+, which is released from the particle surface (Hadrup and Lam, 2014). Studies on Ag toxicity in aquatic birds are still missing, but the adverse effects of Ag-nanoparticles have been demonstrated for aquatic invertebrates and fish, e.g. Choi et al. (2010) and Girilal et al. (2015). Therefore we strongly suggest including Ag analyses in environmental samples to monitor the trend of its distribution in the aquatic environment in the future. 3.5. Cadmium Cadmium was above the LOD of 0.001 μg/g ww in 36 (46.8%) of the muscle samples; only one sample contained N0.010 μg/g ww, specifically 0.012 μg/g ww. In contrast to that, Cd was well detectable in all liver samples, reaching up to 0.762 μg/g ww. The mean BCFliver/muscle of 139
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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was the highest for all metals under investigation. Compared to other studies the Cd levels in muscle from our study are rather low: Kalisińska et al. (2004) found similar mean levels of 0.005 ± 0.009 μg/ g ww in mallards from one site in Poland, but the other site in their publication shows elevated levels of 0.027 ± 0.029 μg/g ww. Even more elevated levels are reported by Mansouri and Majnoni (2014) with 0.14 ± 0.02 μg/g ww from Iran, and Binkowski et al. (2013) give a median of 0.27 μg/g ww from southern Poland. Cd concentrations in the liver in our study (mean 0.228 ± 0.184 μg/g ww) are in good accordance with the results of Mateo and Guitart (2003), who found a mean of 0.182 μg/g ww and 0.15 μg/g ww in mallards from two regions in Spain, respectively, by Kalisińska et al. (2004) who reported 0.282 ± 0.161 μg/g ww from the less polluted site in Poland, as well as by Kim and Oh (2012) who found 0.22 ± 0.28 μg/g ww in mallard livers from Korea. Similar concentrations were reported by Nam et al. (2005a), noting 0.253 ± 0.109 μg/g ww from Japan. More elevated levels were reported by Mansouri and Majnoni (2014) (0.51 ± 0.06 μg/g ww) from Iran, Binkowski et al. (2013) who found a median concentration of 0.969 μg/g ww in liver from Poland, and by Kalisińska et al. (2004) (0.869 ± 0.682 μg/g ww) from the more polluted site within their study from Poland. Regarding human consumption, note that 6 of the sampled birds had liver concentrations N 0.5 μg/g ww, which is the maximum allowance for poultry liver set by the European Commission (EU, 2001a). Cd is one of the metals of major concern in the EU and worldwide. Although its use as a material itself has a short history of about 80 years, even before that Cd was released into the environment through the processes involved with Pb and Zn production. Cd toxicity is well reported and this metal has no known biological function – with one exception, where Lane and Morel (2000) showed that Cd can replace Zn in functioning enzymes in marine diatoms. Its toxicity for most other biota is believed to have exactly the same reason: it replaces Zn in enzymes, but blocks their function by doing so. Cd is a common contaminant in phosphate fertilizers (Tarras-Wahlberg et al., 2002), but levels of Cd in the different phosphorites differ greatly from region to region. Agricultural activities in catchment areas therefore doubtlessly represent a source of elevated Cd levels in inland waters; additionally, atmospheric deposition of Cd has been reported to be of great importance for pollution with this element (Alloway, 1994). Rising Cd concentrations in eider ducks from Canada over the last 20 years, reported by Mallory et al. (2014), certainly demonstrate still increasing levels of this toxic element in the environment.
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Fig. 2. Total mercury concentrations in different compartments of mallards Anas platyrhynchos.
0.847 μg/g. These values are much higher compared to the levels published by Zolfaghari et al. (2009) in mallards from Iran (0.30 ± 0.14 μg/g) but they reflect well the levels Doi et al. (1984) reported from a mercury polluted area in Japan (0.9–1.9 μg/g). Feathers have proven to be a suitable tool for monitoring dietary exposure to MeHg in birds, as Hg is deposited during feather growth and bound strongly to the feather matrix (Scheuhammer, 1987). Even if Hg levels found in our study do not sound too alarming regarding human consumption of mallards, it should be borne in mind that chronic Hg poisoning, even in very low concentrations (e.g. 0.040 μg/g ww in feathers), has been shown to reduce reproductive success in the aquatic bird species common loon Gavia immer by Evers et al. (2008), who conducted a long term investigation. In contrast, Heinz et al. (2010) did not find a high sensitivity of mallards towards MeHg in their food, and the relatively high concentrations in food (up to 4 μg/g ww) and in their eggs did not significantly reduce reproductive success and short term survival under experimental conditions. Until long term data become available for mallards, the elevated Hg concentrations in the birds of our investigation should at least be considered an additional stressor for the development of mallard populations. 3.7. Lead
3.6. Mercury Mercury was well detectable in all samples of muscle, liver and feathers; a statistical comparison of the three matrices is given in Fig. 2. Although Hg has been under investigation in many biota, data for muscle and liver levels from mallards are scarce: Rothschild and Duffy (2005) reported a mean of 0.089 μg/g ww (n = 3) from western Alaska, which is comparable to the levels we found (0.049 ± 0.040 μg/g ww). Four of the mallards in our study had levels N0.1 μg/g ww, which corresponds to the closely related species Anas clypeata that was reported to have 0.132 μg/g ww from the Gulf of California (Ruelas-Inzunza et al., 2009). Interspecific comparisons require caution because especially the Hg content in all biota depends highly on the composition of their diet; especially piscivorous birds have been shown to have elevated levels in all tissues compared to omnivorous or herbivorous species (Becker et al., 2002; Zolfaghari et al., 2009), as Hg has the property of biomagnification in its methylated form (MeHg), which presents the major chemical species in aquatic food chains (Driscoll et al., 2007). The mercury content in livers was significantly higher compared to muscle, and 10 samples contained N 0.3 μg/g ww, a level critically seen for human consumption of fish by the US environmental protection agency (EPA, 2016). Levels in feathers were higher compared to muscle and liver; we measured between 0.070 μg/g dw and 4.38 μg/g dw, resulting in a mean value of 1.031 ± 0.948 μg/g dw, with a median of
Lead levels in the muscle were below the LOD of 0.002 μg/g ww in 75.3% of the samples and two samples contained exceptionally high concentrations of 3.84 and 7.89 μg/g ww, respectively, clearly pointing to lead poisoning. Our mean value of 0.177 ± 0.996 μg/g ww corresponds well to an earlier investigation on lead levels in mallards from Austria in which a mean of 0.145 ± 0.077 μg/g ww was reported (Ferstl, 1993) – in her work 8 of the 30 samples were below the LOD. In liver, lead concentrations were above the LOD of 0.002 μg/g ww in 96.1% of the samples, displaying moderate values up to 1 μg/g in 94.8% of the samples; four samples were above 1 μg/g, of which one specimen had a liver concentration of 6.7 μg/g Pb, which can be classified as clinical poisoning (Franson, 2011 and references therein). Our mean level of 0.289 ± 0.801 μg/g ww is lower than the concentrations measured by Ferstl (1993), who reported 1.537 ± 1.585 μg/g ww Pb. Guitart et al. (1994) found a median concentration of 0.822 μg/g ww (0.059– 21.60 μg/g ww) in mallards from the Ebro delta in Spain, with 25% of the birds having N 1.5 μg/g Pb in the liver, which these authors considered as lead-intoxication. Taking various levels seen as a sign for lead poisoning in consideration, at least 3.9% of the specimen in our study must be classified as poisoned. Comparing the numerous publications on lead in birds, it has to be pointed out that all mallards sampled for our study were able to fly, as they were shot during flight; any specimen unable to fly due to any reason, including potential lead poisoning, was
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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not included. Accordingly, our results may well be an underestimation of lead poisoning cases. Future investigations will have to show if the ban on lead shotgun pellets for hunting water fowl has been a sufficient measure to reduce lead poisoning cases in aquatic bird species. 4. Conclusions Mean heavy metal concentrations in Austrian mallards are generally low, but there are definite signs for pollution, as some specimens displayed elevated concentrations of Cr, Cu, Cd, Hg and Pb. Some of these concentrations must be seen as critical for human consumption of the birds, but more importantly also as signs for persisting metal pollution in Austrian rivers. For Pb, three specimens showed elevated concentrations in muscle as well as in the liver, pointing to an individual poisoning with lead. Mercury concentrations reflect the trend of rising environmental concentrations. Nonetheless, higher levels in feathers compared to muscle and liver also confirm feathers as an important pathway for excretion of this element and point to the biological advantages of birds in contrast to e.g. fish, where this excretion pathway does not exist and mercury is accumulated to a much higher degree in muscle. The concentrations of silver are a first record for this bird species from Europe and should lead to rising awareness for this metal because the consequences of rising Ag levels in animals due to increasing use in consumer products are widely unknown. Acknowledgements We cordially thank Anja Joachim from the University of Veterinary Medicine, Vienna, for providing lab space for dissection and space in deep-freeze chambers. Michael J. Mühlegger is thanked for the dissection of the birds. In addition the cooperation with the confederations of hunters from Lower Austria and Upper Austria, mediated by Alois Gansterer, is highly acknowledged. References Alloway, B.J.E., 1994. Heavy Metals in Soils: Springer. Aubrecht, G., Holzer, G., 2000. Stockenten: Biologie, Ökologie, Verhalten. Österr. Agrarverlag, Leopoldsdorf. Avery, D.W., 2009. R.T. Regulation of lead-based ammunition around the world. In: Watson, R.T.F.M., Pokras, M., Hunt, W.G. (Eds.), Ingestion of Spent Lead Ammunition: Implications for Wildlife and Humans. The Peregine Fund, Boise, Idaho. Bauer, H.-G., 2005. Kompendium der Vögel Mitteleuropas. Vol 1. Wiesbaden, AulaVerlag. Becker, P.H., Gonzalez-Solis, J., Behrends, B., Croxall, J., 2002. Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age. Mar. Ecol. Prog. Ser. 243, 261–269. Berlakovich, N., 2011. Verwendung von Bleischrotmunition bei der Jagd auf Wasservögel. Bundesministerium für Land- und Forstwirtschaft, Jahrgang 2011. Republic of Austria. Binkowski, L.J., Stawarz, R.M., Zakrzewski, M., 2013. Concentrations of cadmium, copper and zinc in tissues of mallard and coot from southern Poland. J. Environ. Sci. Health B 48, 410–415. BMLFUW, 2003. Wassergüte in Österreich - Jahresbericht 2003. Bundesministerium für Land- und Forstwirtschaft, Wien, p. 178. BMLFUW, 2006. Wassergüte in Österreich - Jahresbericht 2006. Bundesministerium für Land- und Forstwirtschaft, Umwelt und Wasserwirtschaft & Bundesumweltamt, Wien, p. 186. BMLFUW, 2011. Wassergüte in Österreich - Jahresbericht 2011. Bundesministerium für Land- und Forstwirtschaft, Umwelt und Wasserwirtschaft & Umweltbundesamt, Wien, p. 94. Choi, J.E., Kim, S., Ahn, J.H., Youn, P., Kang, J.S., Park, K., et al., 2010. Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish. Aquat. Toxicol. 100, 151–159. Custer, T.W., Hohman, W.L., 1994. Trace elements in canvasbacks (Aythya valisineria) wintering in Louisiana, USA, 1987–1988. Environ. Pollut. 84, 253–259. Di Giulio, R.T., Scanlon, P.F., 1984. Heavy metals in tissues of waterfowl from the Chesapeake Bay, USA. Ecological and Biological. Environmental Pollution Series A 35, pp. 29–48. Doi, R., Ohno, H., Harada, M., 1984. Mercury in feathers of wild birds from the mercurypolluted area along the shore of the Shiranui Sea, Japan. Sci. Total Environ. 40, 155–167. Driscoll, C.T., Han, Y.J., Chen, C.Y., Evers, D.C., Lambert, K.F., Holsen, T.M., et al., 2007. Mercury contamination in forest and freshwater ecosystems in the northeastern United States. Bioscience 57, 17–28.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Heavy metals in the mallard Anas platyrhynchos from eastern Austria Christof Plessl a, Peter Jandrisits a, Regina Krachler a, Bernhard K. Keppler a, Franz Jirsa a,b,⁎ a b
University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Währingerstrasse 42, A-1090 Vienna, Austria University of Johannesburg, Dept. of Zoology, P. O. Box 524, Auckland Park 2006, South Africa
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Mercury levels appear elevated, feathers show highest concentrations. • 3.9% of the specimen show lead levels indicating lead poisoning. • Silver levels were recorded for the first time for this species from Europe. • Chromium and copper levels appear elevated in comparison to other studies.
a r t i c l e
i n f o
Article history: Received 21 October 2016 Received in revised form 1 December 2016 Accepted 2 December 2016 Available online xxxx Editor: F.M. Tack Keywords: Water fowl Trace elements Lead Silver Mercury
a b s t r a c t A total of 77 specimens of the mallard Anas platyrhynchos were sampled from the eastern part of Austria before the ban on lead gun shot for hunting water fowl. Samples of muscle and liver were analyzed for their content of Cr, Cu, Zn, Ag, Cd, Hg and Pb using atomic absorption spectrometry. In addition the Hg content of feather samples from this aquatic bird species was evaluated. Results generally show higher concentrations of the metals in the liver compared to muscle; for mercury the concentrations were feathers N liver N muscle. Elevated, in some cases critical concentrations of Cr, Cu, Cd, Hg and Pb were measured. Levels of Ag were recorded for the first time for this species from Europe, providing basic information for future evaluation of this upcoming pollutant in aquatic environments. © 2016 Published by Elsevier B.V.
1. Introduction Although the use of heavy metals in the past has had a great positive impact on human economy, negative consequences for humans and the environment have been demonstrated (Reisinger et al., 2009). The ⁎ Corresponding author at: University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Währingerstrasse 42, A-1090 Vienna, Austria. E-mail address: [email protected] (F. Jirsa).
global anthropogenic distribution of heavy metals has led to multiplied environmental concentrations in particular for lead, copper, zinc and others in all regions of the world (Nriagu, 1996). The abundance of heavy metals in the anthroposphere and their potential emissions in the future are still on the rise, calling for national and international measurements (Reisinger et al., 2009). Waste streams, mining runoffs and other anthropogenic sources of heavy metals still often end up in wetlands (Levengood, 2003), where all aquatic biota, including waterfowl, may be exposed to this pollution. Mallards can be exposed to heavy
http://dx.doi.org/10.1016/j.scitotenv.2016.12.013 0048-9697/© 2016 Published by Elsevier B.V.
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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metal uptake through their diet. For example freshwater mussels have been identified as a food component of these birds and have been described as metal accumulating organisms (Phillips, 1976; Luschützky, 2005). In addition to this common route of metal uptake, an additional hazard has been observed for waterfowl: uptake of shotgun pellets. These pellets consisting of lead have been used by hunters for ages, and a considerable portion of lead has been distributed into the environment in the form of such pellets. Combined with lost plummets from fisheries, an annual input of 600 t of metallic lead into the Austrian landscape has been estimated (Reisinger et al., 2009), for Germany the value is 9000 t and for Italy 25,000 t per year (Kurz, 2004). Although metallic lead is not readily bioavailable, it has been shown that the low pH and mechanical abrasion in the bird's stomach leads to dissolution and uptake of lead into the organism (Yamamoto et al., 1993). This combined exposure to contamination of food and direct uptake may lead to accumulation of metals in the tissues of these birds. Ducks, as one of the most common waterfowl, therefore have been recognised as biomonitors for contamination by e.g. Henny et al. (2000), Levengood (2003). About 73,000 wild ducks, of which most are mallards Anas platyrhynchos L.1758, are shot every year during the hunting season in Austria, with the majority of app. 60,000 in the easternmost parts of the country (Statistics Austria, 2014). They are mostly sold on regional markets and used for human consumption. Concern about lead poisoning of water birds and other wildlife through exposure to these lead pellets has led to a restriction of lead ammunition in at least 29 countries around the world (Avery, 2009), also leading to a first measure taken in Austria in 2012: lead shotgun pellets for hunting water fowl have been banned since June 1st (Berlakovich, 2011) - which still leaves fishing and hunting on small game as a source for metallic lead in the environment. Regarding surface waters, new legacy has led to a reduction in the use of hazardous metals, and a constant decrease in the heavy metal content of surface waters has been recorded from Europe, including Austria (BMLFUW, 2011). Nevertheless, pollution with elements like cadmium, zinc and to a lesser degree copper and mercury is still a problem in sediments throughout the country, especially in mining regions or around large cities (BMLFUW, 2003). There are no threshold levels legally set for any metals in river or lake sediments, and therefore they are no longer included in water quality reports. It has been shown that elevated concentrations in sediments can significantly influence aquatic biota due to bioaccumulation or -magnification (e.g. Handy et al., 2005). The former is of major importance for hydrophilic substances like heavy metal cations, mainly taken up via the gills (Hofer et al., 1995), the latter encompasses lipophilic compounds like methylmercury mainly uptaken with food (Hall et al., 1997). To our knowledge there are no data available on heavy metals in mallards from central Europe. We had the opportunity to investigate levels of heavy metals in muscle and liver tissues from mallards collected before the ban of lead shotgun pellets, to ensure a record of the situation and to save data for later comparisons. In addition to Pb it seemed appropriate to analyse Cd and Hg as metals of priority concern within the EU (EU, 2001b). For mercury the analyses of feathers were included, as these are known to be a major pathway for the storage and excretion of Hg in birds (Monteiro and Furness, 2001). Cu, Cr and Zn, which are essential trace metals showing an enhanced anthropogenic distribution in the environment, were also measured in the tissue samples. Moreover we included Ag in the study, as nanoparticles of this metal have started to be widely used in consumer products and therefore will be increasingly included in waste streams, threatening also the aquatic environments (Yu et al., 2013). 2. Materials and methods 2.1. Study site Sampling for this study took place in the northeastern part of Austria (Fig. 1). Within the region lies Austria's capital Vienna (1.7 million
inhabitants), with its industrial impact on the environment. The surrounding region is a typical man-made environment characterized by a population density of 84 inhabitants/km2 (Statistics Austria, 2014) dominated by agricultural activity and managed forests. Regarding water bodies, the region lies within the catchment area of the Danube River, and floodplains, smaller rivers and numerous lakes and ponds are present. Hunting takes place within the whole region, except for congested areas. By far the most abundant water fowl is the mallard A. platyrhynchos, which is native, widespread over Eurasia and well adapted to cultivated landscape. Its population is estimated to be 10,000–20,000 breeding pairs in Austria (Bauer, 2005). 2.2. Animal sampling In total 77 specimens of A. platyrhynchos were received from eight hunting grounds situated in the eastern parts of Austria (Fig. 1). They were shot by hunters during late autumn in 2009 and 2010, using commercially available lead shots. Freshly killed animals were transported to the University of Veterinary Medicine in Vienna, where sex and weight were determined. A dissection followed and tissue samples (app. 2–3 g of liver and pectoral muscle, respectively) and about 0.5 g of feather samples (from the pectoral region) were obtained using stainless steel instruments, avoiding tissue visibly affected by intruded lead bullets. Samples were transferred into acid-prewashed polypropylene (PP) tubes and stored at −21 °C until further analysis. 2.3. Trace element determination Frozen samples were transferred to the lab of the Institute of Inorganic Chemistry at the University of Vienna. Microwave digestion was performed in a MARS XPRESS system (CEM Corporation). 1 g (wet weight) of tissue or 0.2 g of feathers were digested in 9 mL of HNO3 34% (TraceSELECT® Fluka) and 1 mL H2O2 30% (TraceSELECT® Fluka). Afterwards, samples were transferred quantitatively into 15 mL flasks and brought to volume with millipore water. Before analysis, samples were filtered through 0.2 μm PTFE syringe filters (VWR) and, where necessary, diluted. Recovery rates were determined using certified reference material (DORM-3 for Cr and DOLT-3 for all other elements, Natural Research Council Canada). Reference samples comprising 0.2 g (dry weight) of fish protein DORM-3 and dogfish liver DOLT-3 were digested and diluted in the same manner. To determine the detection limits (LOD), analytical blanks were prepared without insertion of a sample. Mercury was detected in the samples immediately after digestion with cold vapor atomic absorption spectrometry (CV-AAS) (FIMS 400 by Perkin Elmer). Zinc was measured with a Perkin Elmer AAnalyst 200 flame atomic absorption spectrometer (F-AAS). For the detection of Cr, Cu, Ag, Cd and Pb a Perkin Elmer PinAAcle 900Z graphite furnace atomic absorption spectrometer (GF-AAS) was used. Recovery rates were within a range from 91.8–104.2%, demonstrating the appropriateness of the method used (Table 1). 2.4. Data processing Data processing and statistics were performed using Microsoft Excel 2010 and SPSS Statistics Version 20. A Kolmogorov-Smirnov-test was performed to check for normal distribution. Differences between hunting grounds and sex were validated using a t-test (if normally distributed) or a Mann-Whitney-U test (if not normally distributed). Comparison between the different tissues was performed with a t-test or a Wilcoxon-test. Results for mercury were checked with a Kruskal-Wallistest, as three kinds of tissues had to be compared. For calculation of the means, randomly chosen values between zero and the LOD were allocated to the samples, if measured concentrations were below the LOD. To compare mercury levels of muscle and liver with feathers, a water content of 72% in tissue (liver & muscle) was assumed following Ristic et al. (2006) to convert wet weight into dry weight. In the results and
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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Fig. 1. Geographical positions of the hunting grounds for mallard sampling in eastern Austria; BE: Bernhardsthal, EB: Ebreichsdorf, FR: Frauenhofen, GR: Groß-Schweinbarth, HA: Haag, LA: Langenschönbichl, LI: Litschau, SA: Saxen.
discussion part, values from other authors, which were available on a dry weight basis, were converted into wet weight, assuming the same water content, to make values comparable. Bioconcentration factor (BCF): The Bioconcentration factors (BCF) were determined using the formula: BCF ðx=yÞ ¼
concentration of trace element in x concentration of the trace element in y
in which the variables x and y stand for matrices that are compared to each other, such as liver, muscle and feathers. To calculate BCF, only values above the LOD were used. 3. Results and discussion Forty female and 37 male mallards with a mean weight of 1214 ± 229 g were sampled. Comparing the sampling regions, for some metal concentrations weak but significant differences occurred, but not coherent regarding the single elements compared in muscle and liver. The sample size differed greatly between hunting grounds; in addition, mallards do not have a steady territory and show opportunistic migration behaviour (Aubrecht and Holzer, 2000). This does not guarantee that sampled specimens fed for a longer period of time in the district they were hunted and thus were exposed to identical environmental
Table 1 Element concentrations in DOLT-3 and DORM-3 (*), certified by NRCC and as detected with GF-AAS(a), F-AAS(b), CV-AAS(c): mean ± SD of six measurements, mean accuracy and detection limits (LOD), all values in mg/kg dw. Element
NRCC (mg/kg) Mean
±SD
Mean (mg/kg)
±SD
Accuracy (%)
LOD (mg/kg)
Cr (a)⁎ Cu (a) Zn (b) Ag (a) Cd (a) Hg (c) Pb (a)
1.89 31.2 86.6 1.20 19.4 3.37 0.32
0.17 1.0 2.4 0.07 0.6 0.14 0.05
1.88 31.43 79.5 1.19 16.68 3.29 0.33
0.11 0.31 6.7 0.09 0.54 0.07 0.01
99.3 100.8 91.8 99.5 101.5 97.8 104.2
0.008 0.015 1.5 0.008 0.005 0.008 0.008
conditions. Therefore a separate presentation of results for each hunting ground seemed inappropriate, and results are presented for the whole sample. Mean level, minimum and maximum levels as well as median levels of the analyzed elements in liver and muscle are given in Table 2. For none of the investigated element concentrations was a significant difference between male and female specimens recorded; this is in accordance with the results of Mansouri and Majnoni (2014), who came to identical results for mallards from Southern Iran. Statistical evaluation (Wilcoxon-test, p b 0.05) showed significantly higher levels in liver compared to muscle for all elements under investigation. Mean BCFs ranged from 3.6 for Hg up to 139 for Cd. This agrees with the literature, confirming the liver as the major storage organ for metals and as a major organ for detoxification as well. These properties are due to a content of up to 30% metallothioneins, a group of proteins rich in cysteine, showing a high affinity towards so-called weak metals like Cd, Cu, Ag and Zn (Kaim and Schwederski, 2005). Especially for Cu- and Cd-regulation the involvement of these proteins has been proven for mallards and other aquatic birds by Nam et al. (2005b). In the following the respective metals are discussed briefly. 3.1. Chromium Chromium was well detectable in all samples. Although Vincent (2001) concluded in his review that the element appears to be a trace nutrient for mammals, its essentiality for plants and animals is still under discussion (Markert et al., 2015). Mean levels in liver (1.08 ± 0.66 μg/g ww) were higher but still comparable with those reported by Mateo and Guitart (2003) from Spain, where mean levels from 0.336 to 1.064 μg/g ww were stated in the liver of A. platyrhynchos from different regions. In contrast, Nam et al. (2005b) reported 0.073 ± 0.025 μg/g ww in the liver of mallards from Japan; Eisler (1986) reported mean Cr levels of 0.2 μg/g ww from the liver of various birds, stating that 1.1 μg/g ww should be considered as evidence for chromium contamination. Our results of 0.110 ± 0.046 μg/g ww in muscle are much higher compared to 0.042 ± 0.022 μg/g ww published by Nam et al. (2005b) from Japan. To our knowledge, chromium levels from muscle of mallards from other studies are missing. Whether the elevated chromium levels in the tissues of mallards from Austria are due
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Table 2 Heavy metal concentration in tissues of mallards from Eastern Austria (μg/g ww), n = 77, Med: median, BCFli/mu: mean bioconcentration factor liver/muscle. Liver
Cr Cu Zn Ag Cd Hg Pb
Muscle
BCFli/mu
Mean
±SD
Min
Med
Max
Mean
±SD
Min
Med
Max
1.08 37.0 38.2 0.060 0.228 0.170 0.289
0.657 21.4 11.5 0.071 0.184 0.187 0.801
0.361 2.87 14.2 b0.002 0.016 0.020 b0.002
0.916 35.3 37.1 0.037 0.173 0.123 0.088
4.74 99.3 74.9 0.329 0.762 1.55 6.70
0.110 5.28 7.66 0.002 0.002 0.049 0.177
0.046 2.01 2.90 0.002 0.002 0.040 0.996
0.056 1.27 2.67 b0.002 b0.001 0.007 b0.002
0.103 4.87 7.93 b0.002 0.001 0.040 b0.002
0.458 10.9 13.8 0.012 0.012 0.288 7.89
to elevated Cr levels in the environment, for which there is no evidence so far, or whether they are specific to A. platyrhynchos, remains a subject for further investigations. 3.2. Copper Copper was well detectable in both, muscle and liver. It is an essential trace element which is part of many proteins associated with electron-transfer in respiration and the metabolism of oxygen (e.g. oxidases and oxygenases) (Kaim and Schwederski, 2005). It has been described to be of higher prevalence in liver compared to muscle, which is confirmed in our results displaying a BCF liver/muscle of 8.0. Copper levels in muscle (mean 5.28 ± 2.01 and median 4.87 μg/g ww) in our study are well comparable to values from two different sites in north-western Poland (mean 5.90 ± 1.79 and 6.23 ± 1.81) reported by Kalisińska et al. (2004) and from southern Poland (median 4.21 μg/g ww) reported by Binkowski et al. (2013), but are much higher compared to mallards from western Iran (mean 2.02 ± 0.52 μg/g ww) (Mansouri and Majnoni, 2014). For Cu levels in the liver, numerous publications are available and our values (mean 37.0 ± 21.4 μg/g ww) are on the higher end of the scale - e.g. mean values of 35.2 μg/g ww were described from Doñana Park, Spain, by Mateo and Guitart (2003), 29.26 ± 20.02 μg/g ww from Poland (Kalisińska et al., 2004) and 35.7 ± 19.3 μg/g ww in mallards from Japan by Nam et al. (2005b). In contrast to these values are those described by Kim and Oh (2012) from the liver of mallards in Korea (11.9 ± 4.0 μg/g ww) and the mean of 3.88 ± 0.38 μg/g ww reported by Mansouri and Majnoni (2014) from Iran. Eisler (2000) suggested that Cu concentrations in birds from areas of high anthropogenic copper use appear to be elevated. This could explain the big differences in copper levels from our study and Poland, compared to Korea and Iran, due to lesser anthropogenic influence in the rural parts compared to European regions. Elevated Cu levels have been reported from Austrian rivers before, amongst others by Jirsa et al. (2008) who found signs of Cu pollution in fish from eastern Austria and in the official report by the Austrian Ministry for Agriculture, Forestry, Environment and Water Management, where 25% of sampling sites showed an increase of Cu concentration in surface water samples between 1998 and 2004 – compared to 1991–1997 (BMLFUW, 2006). 3.3. Zinc As one of the important essential trace elements, Zn was well detectable in both, muscle and liver, displaying a mean BCFliver/muscle of 5.6. Mean levels in muscle from our study (7.66 ± 2.90 μg/g ww) agree well with the results of Kalisińska et al. (2004) for mallards from two sites in north-western Poland (12.25 ± 3.19 and 11.99 ± 1.88 μg/g ww, respectively) and to the results from southern Poland by Binkowski et al. (2013), who reported a median concentration of 7.7 μg/g ww compared to a median of 7.93 μg/g ww from our study. Again these values are higher compared to the results by Mansouri and Majnoni (2014), who report 5.7 ± 0.8 μg/g ww from musculature in mallards from Iran, but the difference is not as pronounced as for copper. Levels in the liver are generally higher compared to muscle, again
10.4 8.0 5.6 25.5 139 3.6 17.3
confirming the importance of the liver for metal transport and storage in the body. Liver concentrations from other publications are in good accordance to our results: we report a mean of 38.2 ± 11.5 μg/g ww, Mateo and Guitart (2003) report a median of 37.6 μg/g ww from the Ebro Delta and 36.5 μg/g ww from Spanish wetlands, Kalisińska et al. (2004) present 39.72 ± 11.46 and 43.32 ± 7.62 from two sites in Poland, and Di Giulio and Scanlon (1984) report 40.3 μg/g ww in mallard livers from Chesapeake Bay. Again, in contrast to these values stand those from Korea reported by Kim and Oh (2012), who found 28.9 ± 13.8 μg/g ww and from Iran reported by Mansouri and Majnoni (2014), who found 18.0 ± 1.8 μg/g ww Zn in their liver samples. The levels in our study appear to be background levels and a sign that Zn pollution is not present; a potential pollution would result in significantly elevated levels both in the liver and muscle, as shown by Gasaway and Buss (1972) during a toxicity test with mallards over 60 days. 3.4. Silver Silver was detectable in only 10% of the muscle samples in very low concentration, resulting in a mean of 0.002 μg/g ww. To our knowledge there are no other reports of Ag levels in the muscle of birds. In contrast, 94.8% of the liver samples showed Ag levels above the LOD. Our mean values of 0.060 ± 0.071 μg/g ww are in perfect accordance with the liver data published by Nam et al. (2005b) for mallards from Japan, who found 0.058 ± 0.076 μg/g ww in their samples. Other data for silver from birds are scarce, e.g. Fredricks et al. (2009) reported Ag in only one sample of 70 above the LOD of 0.050 μg/g ww in white winged doves from Texas, but Custer and Hohman (1994) reported that 10% of liver samples from canvasback ducks Aythya valisineria had detectable Ag levels between their LOD of 0.56 and 3.1 μg/g ww, which are significantly higher levels compared to the studies mentioned before. Silver is not known to have any biological function and has not been in the focus of research for a long time. The enhanced anthropogenic use as an antibacterial agent not only in medical products but increasingly in household products, especially in the form of nanoparticles, has led to rising concern for this metal in the environment (Yu et al., 2013). Although the mechanism of Ag-nanoparticle toxicity in the body of vertebrates including humans is still under discussion, it is suggested that the effects induced by these particles are mediated via Ag+, which is released from the particle surface (Hadrup and Lam, 2014). Studies on Ag toxicity in aquatic birds are still missing, but the adverse effects of Ag-nanoparticles have been demonstrated for aquatic invertebrates and fish, e.g. Choi et al. (2010) and Girilal et al. (2015). Therefore we strongly suggest including Ag analyses in environmental samples to monitor the trend of its distribution in the aquatic environment in the future. 3.5. Cadmium Cadmium was above the LOD of 0.001 μg/g ww in 36 (46.8%) of the muscle samples; only one sample contained N0.010 μg/g ww, specifically 0.012 μg/g ww. In contrast to that, Cd was well detectable in all liver samples, reaching up to 0.762 μg/g ww. The mean BCFliver/muscle of 139
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was the highest for all metals under investigation. Compared to other studies the Cd levels in muscle from our study are rather low: Kalisińska et al. (2004) found similar mean levels of 0.005 ± 0.009 μg/ g ww in mallards from one site in Poland, but the other site in their publication shows elevated levels of 0.027 ± 0.029 μg/g ww. Even more elevated levels are reported by Mansouri and Majnoni (2014) with 0.14 ± 0.02 μg/g ww from Iran, and Binkowski et al. (2013) give a median of 0.27 μg/g ww from southern Poland. Cd concentrations in the liver in our study (mean 0.228 ± 0.184 μg/g ww) are in good accordance with the results of Mateo and Guitart (2003), who found a mean of 0.182 μg/g ww and 0.15 μg/g ww in mallards from two regions in Spain, respectively, by Kalisińska et al. (2004) who reported 0.282 ± 0.161 μg/g ww from the less polluted site in Poland, as well as by Kim and Oh (2012) who found 0.22 ± 0.28 μg/g ww in mallard livers from Korea. Similar concentrations were reported by Nam et al. (2005a), noting 0.253 ± 0.109 μg/g ww from Japan. More elevated levels were reported by Mansouri and Majnoni (2014) (0.51 ± 0.06 μg/g ww) from Iran, Binkowski et al. (2013) who found a median concentration of 0.969 μg/g ww in liver from Poland, and by Kalisińska et al. (2004) (0.869 ± 0.682 μg/g ww) from the more polluted site within their study from Poland. Regarding human consumption, note that 6 of the sampled birds had liver concentrations N 0.5 μg/g ww, which is the maximum allowance for poultry liver set by the European Commission (EU, 2001a). Cd is one of the metals of major concern in the EU and worldwide. Although its use as a material itself has a short history of about 80 years, even before that Cd was released into the environment through the processes involved with Pb and Zn production. Cd toxicity is well reported and this metal has no known biological function – with one exception, where Lane and Morel (2000) showed that Cd can replace Zn in functioning enzymes in marine diatoms. Its toxicity for most other biota is believed to have exactly the same reason: it replaces Zn in enzymes, but blocks their function by doing so. Cd is a common contaminant in phosphate fertilizers (Tarras-Wahlberg et al., 2002), but levels of Cd in the different phosphorites differ greatly from region to region. Agricultural activities in catchment areas therefore doubtlessly represent a source of elevated Cd levels in inland waters; additionally, atmospheric deposition of Cd has been reported to be of great importance for pollution with this element (Alloway, 1994). Rising Cd concentrations in eider ducks from Canada over the last 20 years, reported by Mallory et al. (2014), certainly demonstrate still increasing levels of this toxic element in the environment.
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Fig. 2. Total mercury concentrations in different compartments of mallards Anas platyrhynchos.
0.847 μg/g. These values are much higher compared to the levels published by Zolfaghari et al. (2009) in mallards from Iran (0.30 ± 0.14 μg/g) but they reflect well the levels Doi et al. (1984) reported from a mercury polluted area in Japan (0.9–1.9 μg/g). Feathers have proven to be a suitable tool for monitoring dietary exposure to MeHg in birds, as Hg is deposited during feather growth and bound strongly to the feather matrix (Scheuhammer, 1987). Even if Hg levels found in our study do not sound too alarming regarding human consumption of mallards, it should be borne in mind that chronic Hg poisoning, even in very low concentrations (e.g. 0.040 μg/g ww in feathers), has been shown to reduce reproductive success in the aquatic bird species common loon Gavia immer by Evers et al. (2008), who conducted a long term investigation. In contrast, Heinz et al. (2010) did not find a high sensitivity of mallards towards MeHg in their food, and the relatively high concentrations in food (up to 4 μg/g ww) and in their eggs did not significantly reduce reproductive success and short term survival under experimental conditions. Until long term data become available for mallards, the elevated Hg concentrations in the birds of our investigation should at least be considered an additional stressor for the development of mallard populations. 3.7. Lead
3.6. Mercury Mercury was well detectable in all samples of muscle, liver and feathers; a statistical comparison of the three matrices is given in Fig. 2. Although Hg has been under investigation in many biota, data for muscle and liver levels from mallards are scarce: Rothschild and Duffy (2005) reported a mean of 0.089 μg/g ww (n = 3) from western Alaska, which is comparable to the levels we found (0.049 ± 0.040 μg/g ww). Four of the mallards in our study had levels N0.1 μg/g ww, which corresponds to the closely related species Anas clypeata that was reported to have 0.132 μg/g ww from the Gulf of California (Ruelas-Inzunza et al., 2009). Interspecific comparisons require caution because especially the Hg content in all biota depends highly on the composition of their diet; especially piscivorous birds have been shown to have elevated levels in all tissues compared to omnivorous or herbivorous species (Becker et al., 2002; Zolfaghari et al., 2009), as Hg has the property of biomagnification in its methylated form (MeHg), which presents the major chemical species in aquatic food chains (Driscoll et al., 2007). The mercury content in livers was significantly higher compared to muscle, and 10 samples contained N 0.3 μg/g ww, a level critically seen for human consumption of fish by the US environmental protection agency (EPA, 2016). Levels in feathers were higher compared to muscle and liver; we measured between 0.070 μg/g dw and 4.38 μg/g dw, resulting in a mean value of 1.031 ± 0.948 μg/g dw, with a median of
Lead levels in the muscle were below the LOD of 0.002 μg/g ww in 75.3% of the samples and two samples contained exceptionally high concentrations of 3.84 and 7.89 μg/g ww, respectively, clearly pointing to lead poisoning. Our mean value of 0.177 ± 0.996 μg/g ww corresponds well to an earlier investigation on lead levels in mallards from Austria in which a mean of 0.145 ± 0.077 μg/g ww was reported (Ferstl, 1993) – in her work 8 of the 30 samples were below the LOD. In liver, lead concentrations were above the LOD of 0.002 μg/g ww in 96.1% of the samples, displaying moderate values up to 1 μg/g in 94.8% of the samples; four samples were above 1 μg/g, of which one specimen had a liver concentration of 6.7 μg/g Pb, which can be classified as clinical poisoning (Franson, 2011 and references therein). Our mean level of 0.289 ± 0.801 μg/g ww is lower than the concentrations measured by Ferstl (1993), who reported 1.537 ± 1.585 μg/g ww Pb. Guitart et al. (1994) found a median concentration of 0.822 μg/g ww (0.059– 21.60 μg/g ww) in mallards from the Ebro delta in Spain, with 25% of the birds having N 1.5 μg/g Pb in the liver, which these authors considered as lead-intoxication. Taking various levels seen as a sign for lead poisoning in consideration, at least 3.9% of the specimen in our study must be classified as poisoned. Comparing the numerous publications on lead in birds, it has to be pointed out that all mallards sampled for our study were able to fly, as they were shot during flight; any specimen unable to fly due to any reason, including potential lead poisoning, was
Please cite this article as: Plessl, C., et al., Heavy metals in the mallard Anas platyrhynchos from eastern Austria, Sci Total Environ (2016), http:// dx.doi.org/10.1016/j.scitotenv.2016.12.013
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not included. Accordingly, our results may well be an underestimation of lead poisoning cases. Future investigations will have to show if the ban on lead shotgun pellets for hunting water fowl has been a sufficient measure to reduce lead poisoning cases in aquatic bird species. 4. Conclusions Mean heavy metal concentrations in Austrian mallards are generally low, but there are definite signs for pollution, as some specimens displayed elevated concentrations of Cr, Cu, Cd, Hg and Pb. Some of these concentrations must be seen as critical for human consumption of the birds, but more importantly also as signs for persisting metal pollution in Austrian rivers. For Pb, three specimens showed elevated concentrations in muscle as well as in the liver, pointing to an individual poisoning with lead. Mercury concentrations reflect the trend of rising environmental concentrations. Nonetheless, higher levels in feathers compared to muscle and liver also confirm feathers as an important pathway for excretion of this element and point to the biological advantages of birds in contrast to e.g. fish, where this excretion pathway does not exist and mercury is accumulated to a much higher degree in muscle. The concentrations of silver are a first record for this bird species from Europe and should lead to rising awareness for this metal because the consequences of rising Ag levels in animals due to increasing use in consumer products are widely unknown. Acknowledgements We cordially thank Anja Joachim from the University of Veterinary Medicine, Vienna, for providing lab space for dissection and space in deep-freeze chambers. Michael J. Mühlegger is thanked for the dissection of the birds. In addition the cooperation with the confederations of hunters from Lower Austria and Upper Austria, mediated by Alois Gansterer, is highly acknowledged. References Alloway, B.J.E., 1994. Heavy Metals in Soils: Springer. Aubrecht, G., Holzer, G., 2000. Stockenten: Biologie, Ökologie, Verhalten. Österr. Agrarverlag, Leopoldsdorf. Avery, D.W., 2009. R.T. Regulation of lead-based ammunition around the world. In: Watson, R.T.F.M., Pokras, M., Hunt, W.G. (Eds.), Ingestion of Spent Lead Ammunition: Implications for Wildlife and Humans. The Peregine Fund, Boise, Idaho. Bauer, H.-G., 2005. Kompendium der Vögel Mitteleuropas. Vol 1. Wiesbaden, AulaVerlag. Becker, P.H., Gonzalez-Solis, J., Behrends, B., Croxall, J., 2002. Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age. Mar. Ecol. Prog. Ser. 243, 261–269. Berlakovich, N., 2011. Verwendung von Bleischrotmunition bei der Jagd auf Wasservögel. Bundesministerium für Land- und Forstwirtschaft, Jahrgang 2011. Republic of Austria. Binkowski, L.J., Stawarz, R.M., Zakrzewski, M., 2013. Concentrations of cadmium, copper and zinc in tissues of mallard and coot from southern Poland. J. Environ. Sci. Health B 48, 410–415. BMLFUW, 2003. Wassergüte in Österreich - Jahresbericht 2003. Bundesministerium für Land- und Forstwirtschaft, Wien, p. 178. BMLFUW, 2006. Wassergüte in Österreich - Jahresbericht 2006. Bundesministerium für Land- und Forstwirtschaft, Umwelt und Wasserwirtschaft & Bundesumweltamt, Wien, p. 186. BMLFUW, 2011. Wassergüte in Österreich - Jahresbericht 2011. Bundesministerium für Land- und Forstwirtschaft, Umwelt und Wasserwirtschaft & Umweltbundesamt, Wien, p. 94. Choi, J.E., Kim, S., Ahn, J.H., Youn, P., Kang, J.S., Park, K., et al., 2010. Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish. Aquat. Toxicol. 100, 151–159. Custer, T.W., Hohman, W.L., 1994. Trace elements in canvasbacks (Aythya valisineria) wintering in Louisiana, USA, 1987–1988. Environ. Pollut. 84, 253–259. Di Giulio, R.T., Scanlon, P.F., 1984. Heavy metals in tissues of waterfowl from the Chesapeake Bay, USA. Ecological and Biological. Environmental Pollution Series A 35, pp. 29–48. Doi, R., Ohno, H., Harada, M., 1984. Mercury in feathers of wild birds from the mercurypolluted area along the shore of the Shiranui Sea, Japan. Sci. Total Environ. 40, 155–167. Driscoll, C.T., Han, Y.J., Chen, C.Y., Evers, D.C., Lambert, K.F., Holsen, T.M., et al., 2007. Mercury contamination in forest and freshwater ecosystems in the northeastern United States. Bioscience 57, 17–28.
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