Species differences in total mercury concentration in gulls from the Gulf of Gdansk (Southern Baltic)

Journal of Trace Elements in Medicine and Biology 33 (2016) 100–109

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Species differences in total mercury concentration in gulls from the Gulf of Gdansk (Southern Baltic) Emilia Szumiło-Pilarska a , Agnieszka Grajewska a,∗ , Lucyna Falkowska a , Julia Hajdrych a , c ˛ Włodzimierz Meissner b , Tomasz Fraczek , Magdalena Bełdowska a , Szymon Bzoma d a Department of Marine Chemistry and Environmental Protection, Faculty of Oceanography and Geography, University of Gda´ nsk, Al. Piłsudskiego 46, 81-387 Gdynia, Poland b Avian Ecophysiology Unit, Department of Vertebrate Ecology and Zoology, Faculty of Biology, University of Gda´ nsk, Wita Stwosza 59, 80-308 Gdansk, Poland c Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland d ´wierkowa 34/7, 81-526 Gdynia, Poland Waterbird Research Group KULING, S

a r t i c l e

i n f o

Article history: Received 13 March 2015 Received in revised form 3 September 2015 Accepted 28 September 2015 Keywords: Mercury Organic mercury Seabirds Brain Stable isotopes Southern Baltic

a b s t r a c t Aquatic birds occupy a high position in the trophic pyramid of the Baltic Sea. This means that they accumulate the greatest amount of harmful substances, including mercury, in their bodies. This element penetrates into their systems mainly via the alimentary canal. The amount of mercury absorbed from food depends on how badly the environment is polluted with this metal. The aim of this study was to discover the concentrations of total mercury (HgT ) in the contour feathers, muscles, brain, lungs, liver, kidneys, heart and blood of four gull species Herring Gull (Larus argentatus), Common Gull (Larus canus), Blackheaded Gull (Larus ridibundus) and Great Black-backed Gull (Larus marinus) and organic mercury (Hgorg ) in the liver and brain of Herring Gull. The most important characteristic of the results obtained for the studied gulls was the statistically significant differences between the four species, probably resulting from their different diets-confirmed by stable-isotopes analysis (␦15 N and ␦13 C). A logarithmic dependence was found between HgT in the blood and HgT in the brain of the Herring Gull. The authors suggest that among gulls burdened with the greatest mercury load, it is possible that the brain is protected by higher Hg accumulation in the muscles. The percentage share of Hgorg in the brain and liver of the Herring Gull depended on the concentration of HgT in these tissues and was always higher in the brain. In none of the cases, did the mercury levels assayed in the internal gulls’ tissues exceed values associated with adverse health effects. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction Mercury is a highly toxic metal which becomes accumulated in the environment in living organisms, and whose concentration increases with the trophic level. Despite being an element occurring naturally in the environment, as a result of anthropogenic activity its concentration in water and sediments often considerably exceeds the geochemical background [1]. Organisms situated at the top of the trophic pyramid (predatory fish, piscivorous marine mammals and birds) are exposed to the greatest influence of mercury [2]. Mercury enters into the systems of birds mainly by their food [3], and is then transported to the liver with blood, where the organic forms of mercury, particularly methylmercury (MeHg),

∗ Corresponding author. Fax: +48 58 523 66 78. E-mail address: [email protected] (A. Grajewska). http://dx.doi.org/10.1016/j.jtemb.2015.09.005 0946-672X/© 2015 Elsevier GmbH. All rights reserved.

may undergo partial demethylation [4]. In the body of a bird mercury is accumulated primarily in the feathers (only during growth), liver and kidneys. To a smaller degree mercury is also accumulated in skeletal muscles, the heart and the brain. The most effective means of mercury removal is through the growth and exchange of feathers, but it is also excreted with guano and – in the case of females – while laying eggs [5]. The concentration of mercury in the tissues and organs of birds is thus a result of accumulation and excretion, both of which are heavily influenced by environmental and biological factors (taxonomic and trophic classification, and the age and gender). Mercury, particularly in its organic form, is a highly toxic element with an ability to permeate internal barriers of the system, including the blood–brain barrier [6]. Increased mercury accumulation in the brain may impair the visual-kinetic coordination and spatial orientation in birds [7]. Mercury is introduced into the marine environment mainly through atmospheric deposition and surface run-off. The Gulf of

E. Szumiło-Pilarska et al. / Journal of Trace Elements in Medicine and Biology 33 (2016) 100–109

Gdansk is an integral part of the Baltic Sea, which is a shallow, intercontinental sea (average depth 52 m), and the highly urbanized catchment area makes this water basin particularly exposed to the inflow of substances of anthropogenic origin, including mercury. This metal occurs in all elements of the marine environment, resulting in its entry into the trophic chain [8]. The pre-industrial background concentration was determined at 30 ␮g kg−1 for Baltic sediments, however the latest studies in the Gulf of Gdansk have shown a several-fold increase in mercury concentration in sediments, reaching up to 250 ␮g kg−1 [9,10]. Many researchers agree that [10–12] the increase in the concentration of this metal in the surface sediments of the Baltic Sea must therefore be attributed to human activity. It is considered necessary all around the world to effectively monitor the level of mercury contamination of the environment [13], and it is believed that birds, owing to their position in the marine trophic chain, may be good indicators of aquatic environment contamination with this element [14]. Despite the fact that relevant literature abounds in information concerning mercury accumulation in aquatic birds, studies using gulls are widespread only in North America. In Poland, in the Southern Baltic, such studies are sparse and concern mainly Great Cormorants, Mallard Ducks and the Common Merganser [15,16]. Naturally occurring stable-isotope ratios of nitrogen (␦15 N) and carbon (␦13 C) reflect the consumer’s diet at the time of tissue synthesis and are commonly used to investigate trophic relationships and to understand foraging ecology of birds. ␦15 N reflects trophic position and ␦13 C can indicate a geographic foraging area by determining relative contributions of marine and terrestial foods to the diet. Stable isotopes are useful in ecotoxicology studies because they provide continuous variables against which mercury levels can be gauged [17]. The aim of the present paper was to evaluate the exposure of seabirds found around the Gulf of Gdansk. In the years 2009–2012 studies were conducted on:

• determining the concentration level of total mercury in various soft body parts of birds from the Laridae group and comparing the obtained results with those provided in literature from other geographic regions of the Northern Hemisphere, • interspecies similarities and differences in total mercury distribution in various organs, • sex and age differences in the Herring Gull.

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Table 1 The average moisture [%] of internal organs and tissues collected from four gull species found around the Gulf of Gdansk between 2009 and 2012. Statistics

Liver

Muscle

Kidneys

Heart

Lungs

Brain

AM ± SD Min–max

70 ± 3 65–79

72 ± 3 68–81

76 ± 2 71–84

73 ± 3 65–82

80 ± 4 71–89

79 ± 3 69–82

2.2. Material preparation Prior to dissection, the weight was determined with an accuracy of up to ±0.1 g, and the birds’ age was assessed based on their plumage. Three age categories were set out: juvenile, immature, and mature. In the case of the Great Black-backed Gull and the Herring Gull, specimens in their first winter plumage were classed as juvenile, those in their second and third plumage were categorized as immature, and those in the fourth and final plumage were considered mature. In the case of the Black-headed Gull and the Common Gull, specimens were classed as juvenile when in the first plumage, immature in the second, and mature in the final plumage. Due to the sexual immaturity of the birds and the condition of internal organs of some of the collected specimens, the gender was determined in genetic tests through DNA amplification using the PCR method. Contour feathers were collected from the birds’ breast as such feathers tend to reflect the whole-body burden, particularly for mercury [19]. The variability of mercury concentration in these feathers is not as strongly related to the molt pattern as is the case with the flight feathers (remiges and restrices) [20]. During dissection, cause of death was not investigated. Tissues and organs were collected from the carcass, including: the brain, breast muscles (further referred to as muscles), the heart, the blood, the liver, kidneys and lungs. The blood was collected from the heart, often in a congealed form, which is why its moisture is not given. Organs and tissues were rinsed in MilliQ water (17.4 ), and then placed in sterile, labelled ziploc bags and preserved frozen at −20 ◦ C. The samples were homogenized with the use of a stainless steel blade. Having been lyophilized, the biological material was homogenized again in a mortar and underwent chemical analysis within a few days. During the preparation procedure, the moisture of the collected organs and tissues was measured, excluding blood (Table 1). Feathers were washed with 80% acetone, in an ultrasonic bath, then rinsed with MilliQ water and left to dry at room temperature. 2.3. Chemical analysis

Determination of Hgorg level made it possible to initiate discussion on the effectiveness of the demethylation process in the liver and the brain of the Herring Gull.

2. Materials and methods 2.1. Birds collected In Poland all birds from the Laridae family are protected species under a directive by the Environmental Protection Minister (Dz.U. No. 220, Item 2237) [18]. Dead birds were collected between December 2009 and August 2012. Due to the atmospheric conditions and the species characteristics of the birds, 90% of the specimens were found during winter and summertime. Most of the dead birds were collected in Wladyslawowo ( = 54◦ 47 ,  = 18◦ 25 ) and in the “Mewia Lacha” bird sanctuary, situated at the Vistula Estuary ( = 54◦ 21 ,  = 18◦ 57 ). A few species came from the city beaches of the Tri-city and Hel.

The assay of total mercury concentration (HgT ) was carried out using the atomic absorption spectroscopy method (AMA 254 mercury analyzer). Mercury was determined in measured amounts of dried biological material of the following mass: 0.1 g—muscles, heart, lungs and brain; 0.01 g—liver and kidneys; 0.03 g—feathers (accuracy: 0.0001 g). The weighed and prepared material was placed in pre-heated nickel boats, which were automatically inserted into a furnace. The samples were dried at 120 ◦ C for 300 s and mineralized at 550 ◦ C in 180 s. Decomposition products were transported via oxygen as a carrier gas and absorbed on a gold trap. Subsequently, following desorption in a 60 s measurement cycle, absorbancy measurement took place at a wavelength of 253.65 nm. The method used to assay organic mercury (Hgorg ) involved extracting organic mercury from the biological material, and then transferring it onto a hydrophobic carrier [21,22]. Lyophilized material used for the analysis was in the following amounts: brain 0.5 g, liver 1.0 g (accuracy: 0.001 g). Measurement was conducted using an AMA 254 mercury analyzer. The precision and accuracy of the analysis method for HgT and Hgorg were measured using certified standards from the European

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Community Bureau: BCR463 (prepared on the basis of tuna) and Dolt-5 (prepared on the basis of dogfish liver). The average rate of recovery from BCR465 reference material was 96.7% (HgT ); 94.7% (Hgorg ) and from Dolt-5 was 97.2% (HgT ); 92.1% (Hgorg ) was. The limit of quantification (LOQ) was 0.075 ngHg g−1 dry weight−1 . The achieved precision (variation coefficient) was lower than 4% (HgT ); 8% (Hgorg ). 2.4. Stable isotopes analysis The analysis of stable isotopes ␦15 N and ␦13 C in muscle tissue of the Herring Gull (n = 15), Common Gull (n = 5), Black-headed Gull (n = 5) and the Great Black-backed Gull (n = 5) was carried out using the Sercon 20–22 continuous flow isotope ratio mass spectrometer (CF-IRMS) coupled with Sercon SL elemental analyzer for simultaneous carbon-nitrogen-sulfur (NCS) analysis. Details of the methodology and the method of calculation are given in a paper by Grajewska et al. [23]. The muscles selected for analysis were chosen so as to represent the whole range of total mercury concentration in the samples (maximum, minimum and median). 2.5. Statistical analysis In order to assess the normality of HgT and Hgorg concentrations in the studied biological samples, the Shapiro–Wilks (p < 0.05) test was used (Supplementary material). For further analysis, HgT and Hgorg values for all organs and tissues were transformed to improve normality and stabilize variances. The analysis of the dependence of HgT and Hgorg concentrations on the species and gender of the studied birds were carried out using factor analysis (ANOVA). In order to determine the significance of differences in the percentage share of Hgorg and HgT between the liver and brain of the Herring Gull, the Wilcoxon test was used, while the analysis of the dependence of Hgorg percentage share on age and gender was carried out with a U-Mann–Whitney test. The relative standard deviation of mean values (RSD) was calculated in accordance with the requirements of the International Union of Pure and Applied Chemistry (IUPAC). Quality of model fitting was assessed based on Root Mean Squared Error (RMSE). All statistical analyzes were carried out at a significance level of 5%. The software programs used for the calculations and visualisation of results were Soft Statistica 10 and Microsoft Excel 2007 with the XLSTATA package. 3. Results During the three year period 104 dead specimens of four gull species were collected: the Herring Gull (n = 61) the Common Gull (n = 16), the Black-headed Gull (n = 17) and the Great Black-backed Gull (n = 10), and their age and gender were determined (Table 2). 3.1. Mercury (HgT ) level in organs and tissues The presence of mercury was discovered in all the studied samples, and the range of concentrations for internal tissue was between 15.8 (in the heart of the Black-headed Gull) and 6809.9 ngHgT g−1 dry weight (in the liver of the Great Black-backed Gull). It was found that the distribution of HgT in all organs and tissues of the particular species was not in accordance with normal distribution (the Shapiro–Wilk test). The highest median values were determined in the livers of all species, and only in the case of the Herring Gull was a slightly higher value determined in the kidneys (Table 3). All other species demonstrated high HgT median values in this organ too. The lowest HgT values were found in the brain and the heart. In two mature specimens of the Common Gull much higher HgT values were determined than in other specimens, particularly in the liver-L and kidneys-K (L: 5477.7 and 4477.8 and

K: 4987.1 and 3626.6 ngHgT g−1 dry weight). The obtained results were characterized by high variability in the particular tissues and organs of each species. The lowest mercury concentrations for each studied tissue and organ were found in the juvenile specimens of the Herring Gull (Table 4). In the case of liver and kidneys, HgT medians in immature specimens were higher than in mature specimens. In juvenile and mature specimens the highest HgT concentration was found in the kidneys and blood, while in immature specimens it was in the liver. The level of mercury content in the muscles, heart, lungs and brain of juvenile birds was on a similar level. The greatest differences were determined in the kidneys and liver, and for those organs the variability of HgT concentration in males was the highest. Median concentrations of mercury were the highest in contour feathers, repeatedly exceeding the HgT concentrations obtained for internal tissues (Table 5). The maximum values amounted to 8211.9 ngHgT g−1 dry weight in the Herring Gull, 4180.7 ngHgT g−1 dry weight in the Common Gull, 7270.2 ngHgT g−1 dry weight in the Black-headed Gull and 4339.1 ngHgT g−1 dry weight in the Great Black-backed Gull. Due to the wide range of results and the low number of samples, high RSD values were calculated for Blackheaded Gull and the Great Black-backed Gull, amounting to 959.0 and 2045.0 respectively. 3.2. Distribution pattern of mercury concentration in birds’ system The mercury distribution in bird organs is shown in Table 6. In 53% of the Common Gulls, 51% of the Blackheaded Gulls and 75% of the Great Black-backed Gulls collected in the Gulf of Gdansk, it presented itself as follows: Liver > Kidneys > Lungs > Muscle ≈ Heart > Brain. In Herring Gulls, two groups were identified consisting of 43% specimens each: in one the pattern of distribution was the same as above, while in the other the first and second places were reverted—Kidneys > Liver > Lungs > Muscle ≈ Heart > Brain. In 14% of the Herring Gull specimens, the highest mercury concentrations were determined in the lungs, and mercury distribution was as follows: Lungs > Liver ≈ Kidneys > Muscle ≈ Heart > Brain (7%) and Lungs > Liver > Kidneys > Muscle ≈ Heart > Brain (7%). For 34% of Common Gulls, 37% of Black-headed Gulls and 25% of Great Black-backed Gulls, the highest mercury concentration was found in the kidneys, with the mercury distribution as follows: Kidneys > Liver > Lungs > Muscle ≈ Heart > Brain. 3.3. Organic mercury (Hgorg ) level in liver and brain of Herring Gull The presence of Hgorg was examined in the liver and brain of the Herring Gull. As was the case with total mercury, the distribution of Hgorg and its percentage share in HgT were not in accordance with the normal distribution (Shapiro–Wilk test). The lowest concentrations found in the liver and brain were similar and amounted to 15.5 and 15.9 ngHgorg g−1 dry weight, respectively. In terms of the maximum values, the concentration in the liver was nearly twice as high (1145.7 ngHgorg g−1 dry weight) as that found in the brain (428.5 ngHgorg g−1 dry weight). The medians [Hgorg ] in the livers of mature and immature specimens were similar, while the median in the brains of mature specimens was nearly twice as high as in immature birds. Similar median values of Hgorg were also found in the livers and brains of both sexes. The percentage share of Hgorg in HgT was between 4.9 and 97.0% in the brain and from 5.2 to 70.7% in the liver and in none of the analyzed cases did the median exceed 50%. The percentage share of Hgorg in the brains of all the analyzed bird groups was higher than in the livers (Table 7). In immature

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Table 2 The age and gender of gulls collected around the Gulf of Gdansk in the years 2009–2012 (n—number of specimens sampled). Common name

Herring gull

Age group

n

Female/male

Juvenile Immature Mature

11 29 21

7/3 12/17 13/8

Common Gull

Black-headed Gull

Great Black-backed Gull

n

Female/male

n

Female/male

n

Female/male

1/− 7/5 3/−

1 6 12

1/− 5/1 3/7

1 4 5

1/− 1/3 3/2

1 12 3

Table 3 The statistical characteristics of mercury concentrations [ngHgT g−1 dry weight] assayed in the internal organs and tissues of four gull species (Herring Gull, Common Gull, Black-headed Gull, and Great Black-backed Gull), found around the Gulf of Gdansk in the years 2009–2012. The number of samples did not equal the number of studied birds as in some cases their condition made it impossible to extract all tissues and organs. Species

Statistics

Liver

Muscle

Kidneys

Heart

Lungs

Brain

Blood

Herring Gull

n Median

58 546.9

60 272.3

55 519.9

58 270.6

54 311.8

51 159.4

49 525.1

x RSD Min–max

638.1 110.9 58.1–1764.9

354.5 74.4 42.7–1076.7

662.7 137.2 19.1–1882.6

325.9 67.1 21.5–1043.8

395.2 79.8 31.4–1104.8

210.0 44.4 22.5–687.8

541.9 41.8 15.4–1410.6

n Median

16 524.1

16 156.9

16 499.8

16 162.5

16 231.2

14 154.7

13 459.8

x RSD Min–max

1076.6 822.5 130.7–5477.7

372.1 320.1 28.9–1929.5

981.7 715.2 162.5–4987.1

376.7 307.3 19.1–2001.2

503.8 385.7 62.3–2788.8

326.9 278.4 36.6–1852.6

1163.5 1062.4 97.4–6087.4

n Median

18 346.6

19 142.1

18 203.3

18 155.3

17 204.8

16 136.5

15 251.6

x RSD Min–max

667.8 339.1 53.5–2384.9

270.1 123.5 13.2–872.5

659.2 345.8 35.8–2025.3

308.1 139.0 15.8–987.1

335.6 156.4 18.2–1009.9

235.2 126..9 17.6–924.1

515.8 324.7 21.3–2123.2

n Median

9 2792.2

9 979.5

8 1620.9

9 1144.9

8 1315.1

10 637.1

8 1699.4

x RSD Min–max

2813.5 1323.1 608.8–6809.9

1362.4 1107.3 327.7–5137.5

1942.3 896.4 921.2–4438.9

1232 672 307.5–3232.9

1457 727 559.1–3361.1

717 269 200.8–1567.9

2004 1377 732.7–5987.5

Common Gull

Black-headed Gull

Great Black-backed Gull

Symbols: n—number of samples; x— mean value; RSD—relative standard deviation.

Table 4 The statistical characteristics of mercury concentrations [ngHgT g−1 dry weight] assayed in the internal organs and tissues of the Herring Gull, in particular age groups and in males and females found around the Gulf of Gdansk in the years 2009–2012. The number of samples did not equal the number of studied birds as in some cases their condition made it impossible to extract all tissues and organs. Groups Juvenile

Immature

Mature

Male

Female

Statistics n Median

Liver 9 444.7

Muscle 10 147.2

Kidneys 8 423.4

Heart 9 167.0

Lungs 9 164.4

Brain 8 152.4

Blood 9 490.9

x RSD Min–max

544.4 317.5 58.1–1232.6

283.4 225.3 42.7–951.5

527.8 388.6 57.3–1629.8

244.4 160.3 21.5–577.8

312.7 263.2 31.4–1000.9

179.6 126.3 28.5–412.6

411.9 243.7 20.3–901.3

n Median

28 587.5

29 201.0

26 597.3

28 259.0

25 260.3

24 143.1

22 505.6

x RSD Min–max

651.5 151.2 90.2–1527.2

331.1 108.4 48.2–1076.7

691.9 213.8 19.1–1882.6

329.0 92.3 26.4–820.9

350.6 96.5 43.7–965.3

175.5 45.7 22.5–394.4

549.8 160.9 15.4–1410.6

n Median

21 521.6

21 361.8

21 604.3

21 280.0

20 405.9

19 203.1

18 596.5

x RSD Min–max

660.6 211.1 132.9–1764.9

420.8 125.9 622–1074.6

678.0 225.0 70.9–1816.8

356.6 131.0 59.8–1043.8

488.2 146.9 63.0–1104.8

266.3 94.3 46.7–687.8

597.1 178.0 190.0

n Median

26 644.6

28 287.8

26 605.7

26 301.8

25 307.0

24 162.0

23 673.4

x RSD Min–max

694.0 160.5 90.2–1694.3

339.8 97.0 48.2–766.1

728.1 216.0 70.9–1882.6

347.1 96.4 26.4–861.6

402.0 114.3 43.7–1104.8

241.5 68.7 22.5–635.7

577.0 150.3 15.4–1271.9

n Median

32 440.0

32 262.6

29 455.7

32 259.6

29 316.6

26 137.0

26 492.1

x RSD Min–max

592.8 156.4 58.1–1764.9

367.4 115.6 42.7–1076.7

604.1 180.0 19.1–1872.3

308.7 95.7 21.5–1043.8

389.4 116.0 31.4–1000.9

174.8 59.0 28.5–687.8

510.8 146.4 20.3–1410.6

Symbols: n—number of samples; x— mean value; RSD— relative standard deviation.

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Table 5 The statistical characteristics of mercury concentrations [ngHgT g−1 dry weight] assayed in the contour feathers of four gull species (Herring Gull, Common Gull, Black-headed Gull, and Great Black-backed Gull), found around the Gulf of Gdansk in the years 2009–2012. Statistics n

Herring Gull 61

Common Gull 16

Black-headed Gull 20

Great Black-backed Gull 5

Median

1525.9

1187.0

1234.1

3642.5

x RSD Min-max

1910.7 406.2 131.3–8211.9

1322.9 534.7 124.0–4180.7

1764.2 959.0 325.2–7270.2

3023.2 2045.0 187.9–4339.1

Symbols: n—number of samples; x— mean value; RSD—relative standard deviation. Table 6 Distribution pattern of mercury concentrations assayed in the internal organs and tissues of four gull species (Herring Gull, Common Gull, Black-headed Gull and Great Black-backed Gull), found around the Gulf of Gdansk in the years 2009–2012. Species Herring Gull

% of individuals 43 43 7 7

Distribution patterna Kidneys > Liver > Lungs > Muscle ≈ Heart > Brain Liver > Kidneys > Lungs > Muscle ≈ Heart > Brain Lungs > Liver ≈ Kidneys > Muscle ≈ Heart > Brain Lungs > Liver > Kidneys > Muscle ≈ Heart > Brain

Common Gull

53 34.5 12.5

Liver > Kidneys > Lungs > Muscle ≈ Heart > Brain Kidneys > Liver > Lungs > Muscle ≈ Heart > Brain Lungs > Liver > Kidneys > Muscle ≈ Heart > Brain

Black-headed Gull

51 37 6 6

Liver > Kidneys > Lungs > Muscle ≈ Heart > Brain Kidneys > Liver > Lungs > Muscle ≈ Heart > Brain Lungs > Kidneys > Liver > Muscle ≈ Heart > Brain Lungs > Liver > Kidneys > Muscle ≈ Heart > Brain

Great Black-backed Gull

75 25

Liver > Kidneys > Lungs > Muscle ≈ Heart > Brain Kidneys > Liver > Lungs > Muscle ≈ Heart > Brain

a

Were found differences between muscle and heart for few individuals.

Table 7 The statistical characteristics of organic mercury concentration [ngHgorg g−1 dry weight] and the percentage share [%] of organic mercury in total mercury assayed in the livers and brains of Herring Gull found around the Gulf of Gdansk in the years 2009–2012. The number of samples did not equal the number of studied birds as in some cases their condition made it impossible to extract all tissues and organs. Hgorg

Hgorg /HgT [%]

Tissue

n

Median

x

RSD

Min–max

Median

x

RSD

Min–max

All individuals

Liver Brain

53 34

145.3 64.9

210.8 95.6

58.9 32.7

15.5–1145.7 15.9–428.5

25.6 31.9

30.3 41.3

5.0 10.2

5.2–70.7 4.9–97.0

Adult individuals

Liver Brain

19 12

133.5 93.3

169.0 112.5

57.7 36.7

23.2–463.8 15.9–222.4

21.0 28.8

28.4 33.1

9.6 12.8

5.2–67.2 5.5–70.4

Immature individuals

Liver Brain

33 22

146.1 55.4

234.9 86.3

89.5 48.3

15.5–1145.7 17.2–428.5

32.8 37.1

31.4 46.1

6.1 15.1

6.8–70.7 4.9–97.0

Male

Liver Brain

26 17

143.8 71.8

246.5 111.7

138.6 56.2

15.5–1145.7 17.2–428.5

23.3 37.2

29.3 43.3

9.6 15.1

6.7–70.7 4.9–97.0

Female

Liver Brain

26 17

145.3 58.6

175.2 79.4

72.3 35.7

23.2–627.5 15.9–278.2

27.7 30.4

31.4 39.2

9.2 14.6

5.2–69.9 5.5–97.0

Symbols: n—number of samples; x— mean value; RSD— relative standard deviation.

specimens the median of the percentage share of Hgorg in the liver and the brain was higher than in mature birds.

4. Discussion

3.4. Stable isotopes (ı15 and ı13 C)

4.1. HgT in the contour feathers, tissues and organs of gulls from various regions of the Northern Hemisphere

The analysis of ␦15 N and ␦13 C isotopes was carried out in the muscles of four gulls species. Median value of ␦15 N and ␦13 C for the Herring Gull was amounted to 10.95 and −23.28 respectively, while for the Great Black-backed Gull 13.34 ␦15 N and −21.27 ␦13 C (Fig. 1). The median values of both stable isotopes for Common Gull (␦15 N:10.83; ␦13 C:25.11) and Black-headed Gull (␦15 N:10.72; ␦13 C:24.89) were similar. However, no correlation was found between ␦15 N and the mercury concentration in muscles: R2 = 0.25, p = 0.01. The median value of ␦15 N for mature Herring Gull specimens was 9.70, while for juvenile specimens 10.95, and for ␦13 C, the values for both age groups were similar: mature: −23.97, juvenile: −23.19.

Mercury concentration in the body of birds is closely related to their diet, food being the main supply. The gulls found around the Gulf of Gdansk use both natural and anthropogenic food sources. Specimens inhabiting the vicinity of fishing harbors in Hel, Wladyslawowo and Swibno follow fishing boats and snatch the fish intestines thrown on board during gutting. In winter, when access to fish and other natural resources is limited, the birds use anthropogenic sources from landfills. At the largest landfill in the area, located in Gdansk–Szadolki, the yearly wintertime bird count showed that Herring Gulls were the most numerous (about 30 000 specimens), and the proportion of Great Black-backed Gulls was no higher than 7% [24]. All of the species are migratory birds, which

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Fig. 1. Differences in ␦15 N (A) and ␦13 C (B) [‰] in muscles of four gull species (HG—Herring Gull, CG—Common Gull, BHG—Black-headed Gull, and GBBG—Great Black-backed Gull) found around the Gulf of Gdansk in the years 2009–2012.

differ in terms of the duration and direction of their journeys. Ornithological studies (unpublished data) have shown that some specimens of the Herring, Black-headed and Common Gull remain around the Gulf of Gdansk all year and the migration of the remaining birds is directed towards the North-Eastern coast of the Baltic (Scandinavia). The mercury accumulated in the tissues of birds reflects the pollution of the environment from which their food originates. The Baltic Sea, including the Gulf of Gdansk, is particularly exposed to pollution of anthropogenic origin, and reports of the Baltic Marine Environment Protection Commission-Helsinki Commission [25] indicate considerable mercury contamination in this area. Many regions of the world where studies into birds have been conducted are characterized by a relatively large proportion of anthropogenic mercury emission sources such as those found along the Southern Baltic coastline. Despite this, HgT in the internal tissues of gulls from the Gulf of Gdansk was similar to or lower than in birds from other regions of the world such as the Barent Sea [26], Spitsbergen [27], Greenland [28], Northeast Siberia [29], Jade Bay-Germany [3], and New Brunswick, Canada [30]. As was the case with tissues and internal organs, the mercury concentrations found in the feathers of birds from the Gulf of Gdansk were similar or lower compared to the contour feathers of birds from other areas, such as Jade Bay, Germany [3], the Portuguese Atlantic [31], New Brunswick, Canada [30], and the Canadian Arctic [32]. Monteiro et al. [31] proved a strong relation between the level of mercury in the body of the prey and in the feathers of the predatory bird. The obtained results have shown that the diets of birds inhabiting the Gulf of Gdansk are not heavily burdened with mercury. However, on the basis of isotope studies, a change in the trophic level of gulls from highly urbanized areas has been observed for many years [33]. These birds tend to prefer anthropogenic food, and this may result in the lowering of mercury levels in their systems. Isotope studies carried out in the present paper showed that the trophic level of the Herring Gull (median: 10.95 ␦15 N and −23.32 ␦13 C), Common Gull (median: 10.83 ␦15 N and 25.11 ␦13 C) and Black-headed Gull (median: 10.72 ␦15 N and 24.89 ␦13 C) is lower than that of the Great Black-backed Gull (median: 13.44 ␦15 N and −21.92 ␦13 C), which prefers food of marine origin [34]. This explains the significant differences in the level of mercury present in the organs of these species. However, during the breeding season in particular, the Herring Gull exploited the marine food source, which was evident from the composition of pellets found in breeding colonies (authors’ own observations). In the case of the four gull species found in the Gulf of Gdansk, in none of the analyzed internal organs were concentrations found

that could be dangerous for the birds’ health: >5000 ngMeHg g−1 wet weight in brain and >20 000 ngMeHg g−1 wet weight in liver [35]. However, in the contour feathers of three Herring Gull specimens (6031.8 ngHgT g−1 dry weight mature, 6181.3 ngHgT g−1 dry weight juvenile and 8211.9 ngHgT g−1 dry weight immature) and one Black-headed Gull specimen (7207.2 ngHgT g−1 dry weight mature) mercury concentrations were found to exceed the critical value of 5000 ngHgT g−1 dry weight [36]. 4.2. Inter- and intra-specific differences in the accumulation and redistribution of HgT A species indicates the position of an organism in the trophic chain and the proportion of anthropogenic food in the diet, while the age and sex reflect the time of their exposure to mercury contained in food and possible means of its removal from the body. Using factor analysis, it was investigated whether HgT concentration in tissues and organs was significantly dependent on the species, age and sex of the studied birds. In this, species was found to be a significant factor (liver-ANOVA: F96.481 = 5869, p < 0.0001; kidneys-ANOVA: F106.13 = 3690, p < 0.004; musclesANOVA: F120.92 = 4718, p < 0.001; heart-ANOVA: F118.97 = 3999, p < 0.002; lungs-ANOVA: F99.75 = 4887, p < 0.001; brain-ANOVA: F83.31 = 4708, p < 0.001; blood-ANOVA: F101.02 = 3265, p < 0.01) but the effects of age and gender were found to be insignificant. On the basis of HgT assayed in the tissues and organs of gulls found around the Gulf of Gdansk, with the use of cluster analysis, two groups of organs with a similar mercury concentration were singled out. The first group consisted of liver, kidneys and blood, which are all related to the distribution of mercury in the system, its transformation and removal (Fig. 2). Blood is pumped through the whole body, which means that internal organs are in constant contact with the mercury in blood. Birds’ liver and kidneys are crucial for mercury demethylation and excretion [28,29,37,38], and this is confirmed by similarly high HgT levels in these organs and in the blood in all of the studied species. The second group consisted of organs related to the accumulation of mercury in the system, such as muscles, the heart and the brain. In the muscles and heart the dynamics of mercury accumulation and removal processes are much slower than in the liver or the kidneys. This means that the muscle tissue reflects the long-term accumulation process of the mercury supplied with food, which is confirmed by the high correlation between HgT in the liver and the muscles: Herring Gull,

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Euclidean distances 16

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Fig. 2. Cluster analysis of mercury concentrations in selected tissues and organs of four gull species (Herring Gull, Common Gull, Black-headed Gull, and Great Black-backed Gull) found around the Gulf of Gdansk in the years 2009–2012.

y = 0.6748x + 2.4934, R2 = 0.68; Common Gull, y = 0.8375x + 2.0471, R2 = 0.86; Black-headed Gull, y = 0.9436x + 0.9619, R2 = 0.87; Great Black-backed Gull, y = 0.7578x + 2.54, R2 = 0.77). Taking into consideration the three age groups (juvenile, immature and mature), a significant effect of age was found only in the muscles (ANOVA: F33.65 = 5090, p < 0.006), lungs (ANOVA: F40.690 = 2690, p < 0.025), brain (ANOVA: F32.118 = 2873, p < 0.019) and blood (ANOVA: F46.985 = 2775, p < 0.025) of the Herring Gull. At present there are no available methods that would allow researchers to determine the age of a bird after it has reached sexual maturity. The average life duration of the Herring Gull is about 17 years and so, when examining a mature specimen, the time over which mercury has accumulated in its body is unknown. Birds in the first year of their lives had considerably lower HgT concentration than other birds. However, the difference between the HgT concentration in the organs and tissues of mature and immature specimens was ambiguous. A similar situation was observed by Riget and Dietz [39], studying HgT concentration in the liver of the Glaucous Gull. Mature and immature specimens differ not only in terms of mercury accumulation in their bodies, but also in their feeding habits. Among gulls found around the Gulf of Gdansk there is inter- and in-species competition which results in different food being consumed by the different age groups. On the basis of isotope studies, it was determined that mature specimens occupy a lower trophic position than juvenile specimens. This may explain the lack of statistically relevant differences between mercury concentrations in the liver, kidneys and heart of both age groups. In addition, in the livers of juvenile specimens the median of mercury concentration was higher (Table 4). It seems that, in order to explain the suggested dependence, a larger data set is necessary because the available HgT concentration results did not show statistically significant differences. Among the Herring Gulls, higher HgT concentrations were found in males than in females but the differences were not statistically significant. This would suggest similar feeding habits and metabolism of both genders. In a group of 13 females, only two were found during the breeding period, the rest being found in winter. Literature concerning the effectiveness of toxin elimination from the body of a female into the egg is very scarce. Studies involv-

ing Spheniscus demersus showed that a female can remove no more than 5% of mercury supplied with food [40]. In other papers the conclusions were ambiguous, showing both significant differences between the genders [27,41] and similar HgT concentrations in the tissues of males and females [42].

4.3. Organic mercury Seabirds found around the Gulf of Gdansk are, in their diet, exposed predominantly to organic mercury, it being the only form that undergoes accumulation in organisms and transgresses system barriers, including the blood–brain barrier. However, certain amounts of Hgorg (mainly methylmercury), can be demethylated [7,43]. As a result, inorganic mercury may constitute a significant percentage of total mercury, particularly in the liver, kidneys and brain. With the use of one factor analysis (ANOVA), it was found that Hgorg concentration in the liver and brain was not dependent on gender or age in the two age categories set out: mature and immature (gender: liver-ANOVA: F17.55 = 2010, p < 0.055; brain: ANOVA: F11.52 = 11,380, p < 0.06; age: liver-ANOVA: F13.65 = 6020, p < 0.09; brain-ANOVA: F23.62 = 8320, p < 0.051). The fact that there were no statistically significant differences between Hgorg in the liver and in the brain of specimens at different ages and of different sexes may indicate that there are other factors influencing the levels of Hgorg ´ et al. [16] suggest, on the basis of in the systems of birds. Kalisinska histopathological research into goosanders, that HgT and Hgorg in the muscles is related mainly to the birds’ condition, which directly influences the amount and kind of consumed food. According to the suggestions put forward by Ikemoto et al. [43] and Scheuhammer et al. [7], bird livers and brains are where demethylation takes place, a process which reduces the toxic effect of mercury. The results of the present study demonstrate that there is a statistically significant relationship between Hgorg in the liver and the brain of the Herring Gull (Fig. 3). No correlation, however, was found between the percentage share of the organic fraction in these organs (y = 0.3228x + 37.694; R2 = 0.05). In addition, statistically significant differences were observed between the percentage share of Hgorg in the liver and brain (Wilcoxon test, p = 0.01).

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70 60 50 40 30 20 10 0

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Fig. 3. Linear dependence between the concentration of organic mercury ln [ngHgorg g−1 dry weight] in the liver and brain of the Herring Gull found around the Gulf of Gdansk in the years 2009–2012 (y = 0.507x + 1.9199, R2 = 0.51).

100.0

20 0.0

300.0 400.0 50 0.0 ngHgT g d.w. -1 in brain

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Fig. 4. Logarithmic dependence between the concentration of total mercury and the percentage share of organic mercury in the brain of the Herring Gulls found around the Gulf of Gdansk in the years 2009–2012 (y = −26.86 ln(x) + 186.75).

Houserova et al. [38], who indicate the trophic level as being a factor which determines the Hgorg share in the body of the predator. The results presented in the current paper show that the percentage share of Hgorg in HgT in the liver and brain varied considerably (Fig. 3 and Table 7). According to data found in literature, Hgorg in the liver of birds is the predominant form if the HgT concentration does not exceed ∼10 ␮g g w.w.−1 [35,43]. In the case of the Herring Gull from the Gulf of Gdansk, HgT in the liver per wet mass never exceeded that value, and the percentage share of Hgorg ranged from 5 to 70%, while in the brain the range was between 5 and 97%. However, the percentage was higher for specimens with lower total mercury concentration both in the liver and in the brain. What is more, an inversely proportional logarithmic dependence between total mercury concentration and the percentage share of organic mercury was found in the brain (Fig. 4) . In the liver no similar dependence was noted, yet specimens with minimum HgT concentrations had the highest Hgorg share. A reverse dependence could be observed in the liver, at maximum HgT concentrations. It was probably a varied diet (from anthropological and natural sources) of some of the studied birds that determined the lack of a statistically significant dependence. Such is the suggestion of

4.4. Brain protection Being a powerful neurotoxin, mercury disrupts the functioning of the central and peripheral nervous system [7]. HgT concentration in the brain was the lowest compared to other tissues in all the gull species. Only in the specimens of the Herring Gull, however (probably owing to the most numerous data), was an exponential dependence found between mercury concentrations in the blood and the brain (Fig. 5). This testifies to the high sensitivity of the brain to a concentration increase of mercury delivered with food. In all gull species with high HgT level in internal tissues a protective mechanism could have occurred, resulting in a higher HgT rise in the muscles and heart as opposed to brain tissue (Fig. 6). A similar protective mechanism of the nervous system was observed in large Baltic Cod (>80 cm length) [44]. These specimens carried the greatest mercury burden, and HgT in their brains was closely correlated with HgT in their muscles r = −0.82 (p = 0.00000). In the group of juvenile fish the correlation was linear (r = 0.76; p < 0.05) while in adult cod less than 80 cm in length, HgT concentration in

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ln ngHgT g d.w.-1 in blood Fig. 5. Dependence between mercury concentration in the blood and the brain of four gull species found around the Gulf of Gdansk in the years 2009–2012 (blue color) Herring Gull, y = 2.2943e0.2325x; (red color) Common Gull, y = 0.7786x + 0.3967, R2 = 0.86; (green color) Black-headed Gull, y = 0.8318x + 0.2952, R2 = 0.87; (violet color) Great Black-backed Gull, y = 0.6207x + 1.8323, R2 = 0.45. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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between juvenile, immature and mature specimens. No statistically significant differences, were found between the female and male Herring Gulls in any of the analyzed cases, despite the fact that males always had higher HgT . Therefore gender, in the case of Herring Gulls found around the Gulf of Gdansk, is not a factor in determining HgT in internal tissues. Organic mercury concentration assayed in the brain and liver of the Herring Gull also failed to show statistically significant differences between specimens of both genders, but also between specimens of differing age. The percentage share of Hgorg was probably connected mainly with HgT concentration, whereas the body burden depended primarily on the diet of the studied birds. It is probable that in gulls found around the Gulf of Gdansk there are two processes that take place in order to reduce the toxic influence of mercury on the brain: while the organic form becomes demethylated, gulls burdened with higher concentrations of total mercury accumulate it in the muscles and the heart. Acknowledgement Fig. 6. A scatter diagram of mercury concentration in the brain to its concentration in the muscles and heart, expressed in ln [ngHgT g−1 dry weight] for four gull species found around the Gulf of Gdansk in the years 2009–2012.

the brain was characterized by exponential growth in relation to HgT in the muscles (r = 0.95, p < 0.05). In birds found around the Gulf of Gdansk, the fact that the rise in mercury concentration was higher in muscles and in the heart than in the brain is confirmed by the logarithmic function (muscles–brain: y = 3.8781 ln(x) − 1.3179; heart–brain: y = 3.7078 ln(x) − 1.0202). With the exception of two cases (1852.6 ngHgT g−1 dry weight Common Gull and 1567.9 ngHgT g−1 dry weight Great Black-backed Gull), mercury concentration in gulls’ brains did not exceed 1000 ngHgT g−1 dry weight It appears that the mechanism protecting the brain in gulls, when compared to the Baltic cod, does not depend on age, only on diet. Despite the lack of statistically significant dependencies between the percntage share of Hgorg in the brains and livers of gulls in the three different age groups, in a few juvenile and mature specimens certain mechanisms were observed. In 5 juvenile specimens, whose systems were less burdened with mercury (brain: 22–139 ngHgT g−1 dry weight; liver: 82–570 ngHgT g−1 dry weight), regardless of gender and habitat, high Hgorg proportional values were found in the brain (from 90 to 98%), while in the same individuals the proportion of Hgorg was relatively low in the liver (from 17 to 30%). In 6 mature specimens, characterized by relatively high HgT concentrations in both the tissues (brain: 160–950 ngHgT g−1 dry weight; liver: 690–1690 ngHgT g−1 dry weight) the Hgorg proportion was found to be low and amounted to 9–28% in the brain and 6–19% in the liver. These dependencies indicated greater effectiveness of demethylation in both the tissues, mainly in mature specimens, while limited demethylation in the brain was indicated only in juvenile specimens. 5. Conclusions Mercury accumulation in the tissues and organs of final accumulation(muscles, heart & brain) depends on metabolic transformations in the liver and mercury distribution via blood. This is suggested by a high correlation between total mercury concentration in the liver and the muscles. As birds grow older, the period in which they have been exposed to mercury in food extends. As a result, older specimens have higher concentrations of this metal in tissues and internal organs. However, only in the case of the muscles, heart, lungs and blood of the Herring Gull were statistically significant differences in HgT found

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