Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic

MPB-08360; No of Pages 7 Marine Pollution Bulletin xxx (2017) xxx–xxx

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Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic Lucyna Falkowska, Agnieszka Grajewska ⁎, Marta Staniszewska, Iga Nehring, Emilia Szumiło-Pilarska, Dominika Saniewska Department of Marine Chemistry and Environmental Protection, Institute Of Oceanography, University of Gdansk, Al. Marszałka Piłsudskiego 46, 81-378 Gdynia, Poland

a r t i c l e

i n f o

Article history: Received 23 November 2016 Received in revised form 17 January 2017 Accepted 25 January 2017 Available online xxxx Keywords: Mercury PAHs Phenol derivatives Lungs Herring gull Gulf of Gdansk

a b s t r a c t Despite the presence of endocrine disrupting mercury, PAHs, alkylphenols and bisphenol A in inhaled air, scientific literature lacks information on their penetration into the lungs. Large lung capacity in birds makes this route of penetration more significant than in other animals. The studies were conducted on lungs of herring gulls found in the Gulf of Gdansk area. The results were juxtaposed with other tissues, including the intestines, which reflect the main, alimentary penetration route of harmful substances into the organism. It was determined that the capacity of bird's lungs, affects the efficiency with which mercury is absorbed from the air. Birds found to have high mercury concentrations in lungs had low PAHs concentrations, what was determined by the fact that the birds foraged in two different areas, as well as on different trophic levels. The alimentary route of phenol derivatives into the organism was of greater significance than inhalation. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction The air in industrialised and urbanised areas is usually described as polluted, particularly in comparison with non-urbanised, forest or seaside areas. This condition of the environment results from human activity. The most dangerous, toxic, carcinogenic and endocrine disrupting substances penetrate into the air as a result of combustion. Industrial emissions (heavy industries, coal plants, heat and energy plants), transport emissions (burning petrol, diesel fuels, the abrasion of tarmac surfaces and tyres), low emissions, uncontrolled fires, burning wood and grasses are of great significance locally, but often also regionally (Bojakowska, 2003; Oren et al., 2006; Lewandowska et al., 2012). The middle of the previous century saw an expansion of dangerous substances into the atmosphere, driven by technological progress related to the production of solvents, detergents, pesticides, paint additives, textiles, cosmetics and even medicines. The groups of toxic substances included, e.g. mercury, polycyclic aromatic hydrocarbons (PAHs) as well as alkylphenols (APs) - (4-tert-octylphenol- OP and 4nonylphenol- NP) and bisphenol A (BPA). These compounds are the focus of the authors' interest here, as they are on the list of priority pollutants, drawn up by the US Environmental Protection Agency (US EPA) and by the International Agency for Research on Cancer (IARC). PAHs afflict mainly the activity of the immune, reproductive and hormonal systems, and can also initiate a cancer process (Xue and Warshawsky, ⁎ Corresponding author. E-mail address: [email protected] (A. Grajewska).

2005). According to an assessment by the European Environmental Agency, Poland is the country with the highest pollution levels of benzo(a)pyrene (EEA, 2014), which is considered to be the most toxic, carcinogenic and mutagenic for humans and animals. Mercury is the strongest neurotoxin with mutagenic influence, disruptive for the circulatory system and the central nervous system (Rutkiewicz et al., 2011; Falkowska, 2016). In the EU Framework Water Directive of 2000, 4tert-octylphenol, 4-nonylphenol and bisphenol A are on the list of high risk substances. In 2011, alkylphenols were flagged up by the European Chemical Agency (ECHA), as compounds which raise particularly great worries. The main source of mercury, PAHs and phenol derivatives in birds is food. When food is consumed, xenobiotics penetrate through the intestinal barrier into the circulatory system and are distributed with blood to all organs and tissues, where they can then be accumulated (Burgess et al., 2013; Kim et al., 2003; Kannan and Perrotta, 2008; Sturve et al., 2006). Lungs are a less significant route of penetration (Chmielnicka, 1994; Chmielnicka, 2006; Gehle, 2009). Nevertheless, a bird's respiratory system is the largest area of the organism's interaction with air, and probably the most important route of penetration for inorganic mercury. Little is known about the content of PAHs or phenol derivatives in bird organisms, and particularly about their respiratory exposure. The emission of PAHs and phenol derivatives from landfills, as pointed out by the Health Protection Agency, can increase the risk of low weight of birds foraging in such areas (HPA, 2011). The authors of the present paper wanted to determine if the exposure to mercury, PAH and phenol derivatives in the seaside air is

http://dx.doi.org/10.1016/j.marpolbul.2017.01.060 0025-326X/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Falkowska, L., et al., Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.01.060

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L. Falkowska et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

reflected in bird lungs, and what factors condition the xenobiotic burden in the lungs. By comparing results from two different routes of introduction: alimentary and respiratory, an attempt was made to determine the level of exposure for the herring gull (Larus argentatus) and to indicate the significance of food from various trophic levels, as well as potential sources of xenobiotics in the inhaled air. The authors put forward a hypothesis that gulls which limit their foraging area to the coastal zone of the sea, assimilate mercury compounds via two routes (inhalatory and alimentary) to a higher degree than specimens which search for food further away from the sea, the latter being more exposed to organic compounds, chlorine and phenol derivatives. The results of studies on seabirds may constitute important information for humans, who breathe in the same air. In the Pomerania region, N1.5 million people are in danger of developing health problems. What is more, in recent years the incidence of cancers and mortality rates have been observed to be higher in this region when compared to those with heavily contaminated area in Poland (Bartosińska et al., 2005). 2. Sampling area The million-strong agglomeration of the Tri-city (Gdansk, Sopot, Gdynia) is located in the coastal zone of the Southern Baltic, on the Gulf of Gdansk. It is a highly urbanised area with population density exceeding 15,000 people per km2. Tri-city inhabitants are exposed to the emission of toxic substances originating from e.g. car traffic, heat and energy plants, refineries, shipbuilding and harbour industry, domestic stoves/fireplaces or landfills. The monitoring of air quality in the Tricity has indicated exceedances of limits, e.g. mean daily norms of PM10 concentrations in the winter season, but in Gdynia also in summer (N50 μg·m−3) and of mean 8-h ozone concentrations in summer (N120 μg·m−3) (ARMAAG, 2015). According to the EEA (2014), over the Gulf of Gdansk during the summer of 2014 there were 1–15 days on which ozone concentrations exceeded the long-term objective for the protection of health. In addition, Tri-City is located in the vicinity of the Kashubian region characterised by high levels of low emissions. Western winds, which dominates in this region are responsible for transport of particles with adsorbed PAHs to Gulf of Gdansk region (Lewandowska et al., 2012). Studies that accompanied the monitoring showed the air over the Gulf of Gdansk to be characterised by a relatively low concentration of total gaseous mercury (TGM ≤ 3.0 ng·m−3), but in the warm season the increase in the temperature of surface seawater and biological activity influences an increase in the emission of mercury from water into the air. At such times, the level of TMG can increase 6 ng·m− 3 (Bełdowska et al., 2008). Having been emitted into the atmosphere, elemental mercury can become oxidated and undergo conversion. Sea water can also be a source of organic Hg compounds in the air (Schroeder and Munthe, 1998). In the air over the Gulf of Gdansk, the concentration of mercury in aerosols is characterised by high seasonal variability (HgTPM). Increased combustion of fossil fuels (greater demand for heat and electricity) can effect a rise of as much as 100-fold in HgTPM in winter (1963.9 pg·m−3) as opposed to summer (Bełdowska et al., 2007). In winter in the coastal zone of the sea the emission of PAHs also increased: the concentrations of benzo(a)pyrene grew 40-fold on average (in extreme cases even 500-fold). In the winter of 2007/2008 maximum concentrations of B(a)P were observed in Gdynia, reaching 25.02 ng·m− 3 (Staniszewska et al., 2013). It was shown that it is an area with an exceeded norm of mean annual bezo(a)pyrene concentration in PM10 (N1 ng·m−3) (WIOŚ, 2009). The latest studies estimate that as much as 60–96% of PAHs in the heating season are adsorbed onto very small aerosol particles, measuring under 1–2 μm in diameter, inhaled through respiration. Studies so far have shown that in the air over Gdynia mean BPA, OP and NP concentrations in small aerosol fractions PM b 2.5 were within the range of 0.1 to 1.2 ng·m−3. For all the compounds, higher

concentrations were observed in the heating season and were between 2 and 6 times higher than in the warm season. The highest elevation of concentrations in the heating season was observed for BPA (Lewandowska et al., 2012). In addition to atmospheric pollution, the study area is characterised by the presence of endocrine disrupting compounds in the water and sediments of the Gulf of Gdansk. However, mercury concentrations in water and surface sediments did not exceed the limit values considered to be safe for organisms (Murawiec et al., 2007; Jędruch et al., 2015). PAH concentrations also did not exceed Predicted Non-Effect Concentration, although according to Staniszewska et al. (2011), some congeners could have occurred in higher concentrations, particularly in the sediments in the water track for ships entering the ports in Gdynia and Gdansk. The latest research has indicated that only 3% of BPA, OP and NP concentration results were higher than the PNEC guidelines (Koniecko et al., 2014). On the other hand, the results for phenol derivative concentrations in the surface microlayer of the sea were higher (56% - BPA), 69% - OP, 3% - NP) than the limit values determined by PNEC (Staniszewska et al., 2015). 3. Characterisation of the studied species The herring gull (Larus argentatus) is widespread in Northern Europe. These birds inhabit seaside areas, islands, sand bars in river estuaries, but also in the vicinity of in-land water basins. The gulls breeding on the Polish coast are mainly resident, particularly the mature birds. Juvenile birds are also partially resident, and some of them migrate to the west (no further than Germany). In winter, gulls come over to the Polish coast from the eastern part of Scandinavia and the western part of Russia. The herring gull is one of the largest birds and the most numerous species found on the Polish coast in wintertime (Meissner et al., 2007). Herring gulls live on organisms from various trophic levels and their diet may include: fish, invertebrate, shellfish, small amphibians, eggs and chicks of other gulls, as well as offal and communal waste. The latter, supplementary food source is found by gulls on landfills near the Gdansk agglomeration. The total number of gulls there ranges between a few and over 30,000 specimens. 4. Materials and methods 4.1. Biological material for analyses The tests were conducted on 53 dead herring gulls found around the Gulf of Gdansk in all seasons between 2010 and 2012. Most of the birds came from the fishing port area in Wladyslawowo (n = 29) and from the Mewia Lacha bird sanctuary located in the Vistula estuary (n = 15). The remaining birds (n = 9) were found within the Tri-city agglomeration. The age of each bird was determined on the basis of its plumage and three age categories were distinguished between: juvenile specimens (chicks and birds in their first plumage), immature specimens (in their second and third plumage) and mature birds (in the fourth and final plumage). Gender was determined on the basis of DNA using the method of polymerase chain reaction – PCR. The cause of death was not determined, but the cachetic condition of each bird was assessed. 10% of the birds were found to be emaciated, including one male with suspected peritonitis (Falkowska, 2016). All the birds underwent dissection, during which the following were collected: muscles (to assay δ13C, δ15N, HgTOT, HgORG), lungs (HgTOT, HgORG, WWA, BPA, OP, NP), kidneys (HgTOT, HgORG), liver (HgTOT, HgORG), brain (HgTOT, HgORG) and intestines (HgTOT, HgORG, WWA, BPA, OP, NP) (Table 1). Collecting a full set of tissues and organs from each bird was not possible, and sometimes low weight of samples (e.g. brain) made it impossible to carry out all of the assays. The collected material was stored in a freezer (− 20 °C). Material preparation included lyophilisation and

Please cite this article as: Falkowska, L., et al., Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.01.060

L. Falkowska et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

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Table 1 Number of tissues collected from herring gulls [n] for the assays of mercury, selected PAHs and phenol derivatives, with age and gender (Female/Male). Age category

Juvenile n = 19 Immature n = 14 Mature n = 20

Lungs

Intestines

Brain

Kidney

HgTOT

HgORG

PAHs

APs

HgTOT

HgORG

PAHs

APs

HgTOT

HgORG

HgTOT

HgORG

12/6

11/6

4/6

4/5

11/6

5/5

5/6

6/5

10/7

3/0

10/7

5/5

5/9

5/9

2/7

1/6

4/9

2/6

2/6

2/6

5/7

0/2

5/9

3/6

12/8

12/8

8/6

5/5

11/8

8/5

8/5

8/5

11/8

1/0

12/8

11/5

n - Number of samples, F/M - number of females/number of males.

homogenisation of the dried tissues. Until the time of analysis, the samples were kept in an exicator, which maintained the humidity level at 21%. 4.2. Chemical analyses 4.2.1. Total and organic mercury The analysis of total mercury HgTOT was carried out using an AMA254 atomic absorption spectrometer (absorbance measurement at λ = 253.65 nm). For analyses, 0.01–0.1 g of the homogenised biological material (lungs, kidneys, brain) was measured out and placed in prefired nickel boats. In the case of the intestines, cut-out pieces of intestinal walls weighing about 0.1 g were used. The exact parameters of the equipment operations are described by Szumiło-Pilarska et al. (2016). Organic mercury was analysed by extracting this form from the biological material (lungs, intestines, kidneys, brain) and then transferring it to a hydrophobic carrier (Carbonell et al., 2009; Kwaśniak et al., 2012). Further analysis proceeded in the same way as in the case of total mercury. 100-200 μl of the obtained extraction were placed in the nickel boats. Quality control for the method included analysing environmental samples in three repeats. The precision and accuracy of the method of HgTOT and HgORG assays were measured using certified reference materials - BCR463 prepared on the basis of tuna and Dolt-5 based on dogfish liver. Accuracy expressed as mean mercury recovery for BCR463 was determined at 96.7% (HgTOT) and 94.7% (HgORG), while for Dolt-5 at 97.2% (HgTOT) and 92.1% (HgORG). The method's precision expressed as standard deviation was b4% for HgTOT and 8% for HgORG. Limit of quantification for mercury (LOQ) was 0.075 ng Hg g−1 s.m. 4.2.2. Polycyclic aromatic hydrocarbons, bisphenol A and alkylphenols All the reagents (water, acetonitrile and methanol) by Merck were HPLC pure. Mg2SO4, CH3COONa, HCl (VII) and NH4COOH were analytically pure (99.99% supplied by POCh). Standards of individual PAHs, BPA, OP and NP by SIGMA-ALDRICH® were of high purity (N97%). Standards for the preparation of a calibration curve were prepared in methanol. Glass vessels were used, suitably prepared by etching with nitric acid at a concentration of 3.5 mol·dm−3 for 24 h and drying at 200 °C. The extractions of benzo(a)pyrene (B(a)P), fluoranthene (FLA), pyrene (PYR), benzo(a)anthracene (B(a)A) and chrysene (CHR) from 0.2 g samples of bird lungs and intestines were carried out on the basis of a mixture of Mg2SO4 (0.3 g), CH3COONa (0.08 g) and acetonitryle (4 ml) according to the procedure set out by Ramalhosa et al. (2009). In order to assay BPA, OP and NP, 0.1 g sample was taken and extracted with mixture of methanol (8 cm3), NH4COOH (0.01 mol·dm−3 2 cm3) and HCl (VII) (100 μcm3) in an ultrasonic bath (10 min., 20 °C). Extracts were purified on Oasis HLB glass cartridges (5 ml/200 mg) (Waters) according to the method set out by Staniszewska et al. (2014, 2015, 2016a and b). The process of chromatographic separation was conducted on a Dionex UltiMate 3000 liquid chromatograph with a fluorescent detector (for B(a)P λex. = 296 nm, λem. = 408 nm, for FLA,PYR λex. = 270 nm, λem. = 440 nm, for B(a)A, CHR λex. = 275 nm, λem. = 380 nm, BPA and alkylphenols λex. = 275 nm, λem. = 300 nm.), a Thermo Scientific

HYPERSIL GOLD C18 PAH chromatography column (250 × 4,6 mm; 5 μm), using a mobile phase gradient: acetonitrile: water. The amounts of organic compounds recovered, determined through a quintuple analysis of each kind of sample containing a known amount of the standard, averaged at 85–99% (PAHs), 85–94% (BPA, APs). The limit of quantification (LOQ) was determined as a tenfold signal to noise ratio for a sample with a very low analyte content and amounted to: 0.10 (individual PAHs), 2.0 (BPA), 0.5 (OP), 0.5 (NP) ng·g−1 d.w. The obtained “background” values for PAHs, BPA, NP and OP were b LOQ. 4.2.3. Stable isotopes Ancillary parameters that enabled the identication of the trophic level were the results for stable isotope proportions in the muscles of 14 birds. The studies of isotopes δ13C, δ15N were carried out on a Sercon 20–22 mass spectrometer (CF-IRMS). The pre-prepared samples weighing 1.0 ± 0.2 mg, with vanadium pentoxide added as a catalyst, were placed in tin capsules. Thiobarbituric acid (δ15N = 0.11 (atmospheric nitrogen), δ13C = 28.35 (PDB-Pe Dee Bellemnite)), was used as a reference material. Detailed information on the calculations are presented in a paper by Grajewska et al. (2015). 4.3. Statistical analysis The results were analysed using the MS Excel and Statistica programs. The normality of distribution among the studied variables was examined using the Shapiro-Wilk test (p b 0.05). The analysis of dependencies between variables was done based on the Pearson correlation (in the case of parametric data) or the Spearmann correlation (nonparametric data). 5. Results The paper is based on the results of the concentrations of: total mercury (HgTOT), organic mercury (HgORG), PAHs, alkylphenols and bisphenol A in lungs and intestines, the organs related to the two routes of pollutant introduction into herring gulls' organisms (Table 2). They are complemented by the results for total mercury (HgTOT) and organic mercury (HgORG) in the brain and kidneys of gulls (Table 3). The description of results and in discussion uses the parameter InHg, as calculated by the subtraction: HgTOT – HgORG. 5.1. Lungs The concentrations of mercury in lungs varied within a wide range. The concentrations of HgTOT, HgORG and InHg were non-parametric (p b 0.05), with most results found in the range of low values (positive skew). The percentage share of HgORG in HgTOT ranged between 6.3 and 100% and decreased as total mercury concentration grew (Spearman correlation: p = 0.000090; r = −0.53; n = 50). The highest HgORG content in HgTOT was observed in a juvenile female which was found to have the lowest HgTOT concentration. Of all the studied PAHs, fluoranthene (40.1 ng·g−1 d.w.) and pyrene (40.6 ng·g−1 d.w.) had the highest mean concentrations, while the lowest median was determined for benzo(a)pyrene (0.4 ng·g− 1 d.w.)

Please cite this article as: Falkowska, L., et al., Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.01.060

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L. Falkowska et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

Table 2 Characterisation of mercury, selected PAHs, APs and BPA concentrations [ng·g−1 d.w.] in the lungs and intestines of herring gulls (Larus argentatus) collected in the coastal zone of the Gulf of Gdansk, 2010–2012. Lungs

Intestines

n

Md

Min–max (noutlier/nextreme)

Rng

n

Md

Min–max (noutlier/nextreme)

HgTOT

52a

317.3

Min–max

49a

205.4

HgORG

51

85.0

Min–184.0

31

72.2

InHg

50

246.0

Min–max

27

175.5

B(a)P

33

0.4

31.4–1104.8 (0/0) 7.9–520.6 (1/1) 0.0–996.2 (0/0) 0.1–25.2 (1/4) 0.1–16.6 (0/3) 0.6–23.5 (0/2) 1.7–40.1 (1/1) 1.5–41.6 (0/2) 4.6–114.6 (0/2) 2.0–27,415.5 (2/2) 0.5–355.3 (1/0) 0.5–4825.4 (0/1)

Min–1.5

31

0.7

26.4–933.2 (1/1) 12.8–164.3 (0/0) 10.7–813.6 (1/1) 0.2–28.9 (1/4) 0.3–15.1 (1/2) 0.4–22.6 (1/1) 0.8–83.2 (2/1) 1.3–143.3 (0/2) 3.5–263.1 (1/2) 4.3–3315.1 (2/2) 9.3–539.7 (2/2) 13.1–31,534.5 (1/3)

CHR

1.2

B(a)A

3.1

FLA

9.6

PYR

10.8

∑PAHs

26.4

BPA

26

174.0

OP

105.7

NP

180.0

Min–2.4

0.9

Min–7.4

2.8

Min–26.1

8.2

Min–22.5

7.9

Min–58.0

26.1

Min–430.8

32

344.1

Min–244.7

49.7

Min–579.7

112.3

a Results published in the paper Szumiło-Pilarska et al., 2016; InHg – inorganic mercury concentration, calculated as the difference between HgTOT and HgORG; n – number of samples – mean value; SD – standard deviation; Md – median value; min – minimum; max – maximum; noutlier – number of outlier values; nextreme – numer of extreme values; Rng - range without outlier and extreme values.

(Table 2). Chrysene had the lowest variability of concentrations out of all the studied PAHs. The results for PAHs in the lungs that stood out were those of two birds: a juvenile male and a juvenile female, which displayed the highest total PAH results (male - 100.5 ng·g− 1 d.w., female −114.7 ng·g−1 d.w.). In the case of the male, found in the summer of 2011 in the Vistula estuary (internal haemorrhage, suspected peritonitis), it was characterised by extremely high concentrations of fluoranthene (40.1 ng·g− 1 d.w.), benzo(a)anthracene (18.1 ng·g− 1 d.w.), pyrene (39.3 ng·g−1 d.w.) and extremely low concentrations of HgTOT and HgORG. In the second case, that of the female found in spring 2011 at the Gdynia harbour, its lungs had the maximum (extreme) concentrations of pyrene, benzo(a)anthracene, chysene, as well as a high concentration of benzo(a)pyrene (6.87 ng·g−1 d.w. Only the concentration of fluoranthene in the lungs of this bird was within the range of nonoutlying values (26.1 ng·g−1 s.m.). That same female had a high concentration of 4-nonylphenol and the highest concentration of bisphenol

Table 3 Descriptive statistics of mercury concentrations [ng Hg·g−1 s.m.] in the soft tissues of herring gulls (Larus argentatus), collected in the Gulf of Gdansk region in the years 2010– 2012. n

±SD

Md

Min–max (noutlier/nextreme)

Rng

19.1–1882.6 (1/0) 28.2–315.6 (2/0) 0.0–1568.9 (0/0) 22.5–687.8 (3/0) 52.2–229.3 (0/0) 2.1–253.7 (1/0)

Min–1816.8

51a

Kidneys HgaTOT

669.0 ± 510.6

519.9

35

Kidneys HgORG

134.7 ± 68.8

124.2

Kidneys InHg

562.5 ± 447.6

493.2

48a

Brain HgaTOT

206.7 ± 155.3

155.8

6a

Brain HgaORG

107.9 ± 66.4

88.6

Brain InHg

70.4 ± 94.7

46.0

Symbols as in Table 2.

Min–248.1

A. In the remaining birds, among phenol derivatives the concentrations of OP were the least varied, with standard deviation as much as 85 times lower than the standard deviation for bisphenol A, the range of which was between 2.0 and 27,415.5 ng·g−1 d.w. With one exception - an outlying BPA concentration (669.7 ng·g−1 d.w.) in the lungs of a mature bird - all outlying or extreme values, both for PAH and phenol derivatives, were determined in sexually immature birds. 5.2. Intestines In the intestines of birds the inorganic form of mercury prevailed, and the highest InHg concentrations occurred in gulls with the highest HgTOT. The percentage share of the organic form dropped as total mercury concentrations increased in the samples (Spearman's correlation: p = 0.012814; r = − 0.47; n = 27), while in half of the specimens the proportion of HgORG did not exceed 25%. PAH and phenol derivatives were assayed in all the intestine samples of gulls from the Gulf of Gdansk. As in the case of lungs, the PAHs that reached the highest mean concentrations in the intestines were fluoranthene and pyrene. These concentrations were 3–7 times higher than the mean concentrations of the other PAH compounds, in addition to more diverse pyrene values. The concentrations of benzo(a)pyrene and chrysene were similar, and the lowest. The values for 4-nonylphenol in intestines were markedly higher compared to PAHs where the maximum concentration was two orders of magnitude higher than the total of PAHs (Table 2). 5.3. Other organs

Min–max Min–459.9 Min–max Min–70.7

The results for total and organic mercury and the calculated concentrations for inorganic mercury in kidneys and brain constitute supplementary studies (Table 3). The comparison of obtained results made it possible to determine the following pattern of total mercury distribution in organs: kidneys b lungs b brain b intestines. Additional parameters which were helpful in the interpretation of results were the stable isotopes of nitrogen and carbon. The range of

Please cite this article as: Falkowska, L., et al., Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.01.060

L. Falkowska et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx

δ15N values in muscles was from 8.30 to 12.06‰ and indicated two trophic levels from which the birds collected their food. The δ13C values, on the other hand, ranged from −26.03 to −21.41‰ and indicated the regional diversity of food origin. 6. Discussion 6.1. The influence of lung capacity on the effectiveness of mercury absorption via respiration Birds need to be capable of quick gaseous exchange, seeing as their oxygen consumption at the time of rest is higher than in other vertebrates and increases multiply during flight. The total capacity of the respiratory system in birds is about twice as large as in mammals of the same size (Gill, 2009; www.britannica.com). The assumption that the capacity of lungs is closely related to the weight of a bird, made it possible to indicate its influence on the effectiveness of gaseous and inorganic mercury absorption from the air (Spearman's correlation: p = 0.028804; r = 0.40; n = 30). 6.2. Food as a source of PAHs and phenol derivatives Despite the presence of PAHs in aerosols and the fact that they can be inhaled, the introduction with food was still more significant for birds. A statistically significant correlation was determined between the concentration of total PAHs in lungs and intestines (Spearman's correlation: p = 0.0004; r = 0.62; n = 28). Taking into account the birds' sex, a similar correlation between PAH concentrations in both the tissues was visible only in male herring gulls (Spearman's correlation: p = 0.00005; r = 0.82; n = 18). In females, laying eggs purifies their organism by building PAHs into the eggs. Moreover, PAHs are quickly metabolised by birds (Naf et al., 1992). It was found, however, that in the case of birds inhabiting an area with high PAH concentrations, the detoxification process may be insufficent (Troisi et al., 2006; Dhananjayan and Muralidharan, 2013; Philips, 1999). BPA and APs also failed to exhibit any correlation with lung capacity. This signifies that the source of phenol derivatives in lungs, as in the case of PAHs, is what is absorbed from food and distributed with blood in the bird's body. This is confirmed by a statistically significant correlation between the concentration of NP in the lungs and intestines of mature birds (Spearmann's correlation: p = 0.0002; r = 0.78; n = 21). When breeding, mature herring gulls search for more valuable food, i.e. fish and seafood. In the breeding period the intestines of birds had higher NP concentrations when compared with the nonbreeding season. In addition, these birds were characterised by BPA and OP concentrations in the intestines that were between 1.5 and 35.0 times higher than in the non-breeding season. A similar tendency was discovered for the lungs. It was found that mature herring gull specimens had between 1.7 and 3.5 times more BPA, OP and NP in the breeding season than the birds collected during the non-breeding period. This may indicate an increased amount of food of marine origin. In herring caught in the Gulf of Gdansk, BPA concentrations were found within the range of 19.7–798.4 ng·g−1 d.w., and NP 5.4–41.9 ng·g−1 d.w. OP concentrations ranged between b0.8 and 89.2 ng·g− 1 d.w. (Staniszewska et al., 2014). Moreover, studies in the same region and season indicated a multifold increase of NP concentrations in zooplankton and mussels (Staniszewska et al., 2016a and b). The linear structure of NP is not insignificant here, as it makes the compound more absorbable and increases its potential for accumulation in the organism (Green et al., 2003). In the breeding season, apart from the fact that birds make the effort to obtain more valuable food for their chicks, the high air temperature accelerates the decomposition of products containing phenol derivatives, which results in an increase of the load introduced to the environment. Furthermore, Staniszewska et al. (2015) state that a large number

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of tourists in summer entails increased consumption of products containing phenol derivatives. 6.3. The influence of habitat on birds' endocrine disruptive compound burden Among the compounds studied in bird lungs, PAHs and InHg were the only ones that in the breeding season were characterised by a reversely proportional correlation (Spearman's correlation: p = 0.008730; r = −0.69; n = 13). The most likely cause of that negative correlation was the fact that the birds spent most of their time in at least two different foraging areas: the coastal zone of the sea and communal landfills. On the basis of PAH concentrations and stable δ15N isotope results (Spearman's correlation: p = 0.004805; r = −0.94; n = 6), the gulls feeding on lower trophic levels (e.g. landfills) were more exposed to PAHs than those that obtained food from higher trophic levels. Food from a high trophic level is obtained in the form of seafood (e.g. crustaceans) or fish snatched off fishing boats. Despite the fact that PAH, mercury and phenol derivatives in the gaseous and aerosol forms are commonly found in the air, their emission at landfills increases dramatically particularly in the warm period (HPA, 2011; Falkowska et al., 2013; Morin et al., 2015). Many researchers have discussed the release of endocrine disrupting phenol derivatives from landfill refuse (Pawlowska and Pawlowski, 2007; Xu et al., 2011; Zhang et al., 2012; Morin et al., 2015), and birds that forage in landfills are therefore exposed to continuous inhalation of the toxic substances released there. On the outskirts of Gdansk, the Szadółki landfill can be occupied by up to 30,000 herring gulls at one time (Meissner et al., 2007). The birds choose this place as it provides easy access to food. Similar observations were made by Blight et al. (2015). On the basis of numerous isotope studies, researchers indicated a decrease in the trophic level of gulls from highly urbanised areas. According to the HPA (2011), organic gases released from landfills during the degradation of refuse constitute only about 1% of emissions. These additional organic pollutants: dioxins, PCB or PAHs, may originate from the combustion of gases emitted from landfills. The presence of PAHs in the lungs of gulls was determined by a pyrogenic origin. That fact was testified to by the factors: FLA/PYR N 1; FLA/(FLA + PYR) 0.4– 0.5 and N 0.5; BaA/CHR N 1 (Yunker et al., 2002; De la Torre-Roche et al., 2009; Akyüz and Çabuk, 2010). The petrogenic origin of PAHs was indicated by results obtained from 10% of the gulls. Mineau et al. (1984), analysing the composition and mutual proportions of PAHs, indicated the exposure of gulls from the Great Lakes of Canada to substances of pyrogenic origin. Custer et al. (2000), on the other hand, pointed to the exposure to petrogenic PAHs in the scaup from the Anatidae family from the Ports of Indiana. Alkylphenols and bisphenol A may come from combustion processes. It is possible that people may conduct uncontrolled burning of plastic waste in Gdynia and in the surrounding cities or villages, where coal-fired furnaces are common. BPA, OP and NP formed in combustion processes (involving plastic debris) undergoes occlusion onto soot and carbon particles, and can thus be transported via the atmosphere and deposited further away from the coastal zone (Lewandowska et al., 2012). 6.4. Alimentary and respiratory exposure of birds to EDCs The muscles, liver and kidneys of herring gulls from the Gulf of Gdansk were the tissues with the highest total mercury concentrations (Szumiło-Pilarska et al., 2016), but about 14% of birds had higher concentrations of mercury in their lungs than in other tissues. Respiration is the penetration route mainly for gaseous mercury and inorganic mercury in aerosols, while what is introduced from food through the intestinal walls is methylmercury. Neither the lungs nor the intestines accumulate mercury, which is the reason why they are better than other organs at indicating the significance of a given Hg penetration route. Our studies have proven that inorganic mercury is transported

Please cite this article as: Falkowska, L., et al., Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.01.060

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from the lungs to the kidneys and that these are the main recipients (Spearman's correlation InHgLUNGS: InHgKIDNEYS, r = 0.74; p = 0.000000, n = 35). Mercury, removed from the kidneys, is excreted with guano (Ritchie et al., 1994). The intestines of birds, like in other vertebrates, e.g. fish (Boudou and Ribeyre, 1985), do not accumulate mercury either. Hg concentrations in the intestines are lower compared to the concentrations in blood and in other tissues (Szumiło-Pilarska et al., 2016). However, mercury concentrations in the intestines may reflect alimentary exposure (Spearman's correlation HgORG: δ15N, p = 0.028883, r = 0.68, n = 10; Spearman's correlation HgORG: δ13C, p = 0.025097, r = 0.70, n = 10). Food originating from higher trophic levels, e.g. of marine origin, is considered to be more burdened with organic Hg than other food from land sources. This statement is confirmed by the correlation between HgORG concentration in the intestines and InHg in the lungs of herring gulls during the breeding season (Spearman's correlation: r = 0.60; p = 0.018050, n = 15). It shows that a high level of HgORG in the intestines is linked to marigenic food, while a high level of inorganic mercury in the lungs is related to a prolonged period of time spent in the coastal zone of the sea. Several publications have indicated the marine origin of gaseous mercury, which manifests itself in the warm season in the very productive coastal zone of the Gulf of Gdansk (Schroeder and Munthe, 1998; Bełdowska et al., 2003, 2006). Mercury emission from the sea into the air could have contributed to the higher inorganic mercury in the lungs of some of the birds compared to those gulls that preferred food from landfills. However, the marine origin of Hg is only an addition to the main load of gaseous and aerosol mercury introduced into the atmosphere by e.g. the shipbuilding industry, transhipment in ports, marine transportation or from chemical storage. The PAH and phenol derivatives burden was similar in the lungs and intestines of birds. However, mature birds tended to have higher PAH concentrations in their lungs, and more phenol derivatives in the intestines, which could have been related to their migration in-land in the search for food. At landfills, in addition to the alimentary exposure, the inhalation of these pollutants increases, mainly during the warm season when the emission of organic compounds from refuse is higher. While not trying to question the importance of the main alimentary route of pollutant introduction into bird organisms, the authors of the discussed results point to the fact that the levels of xenobiotic presence are comparable in the lungs and intestines, and at the same time indicate the dangers that may be posed by inhalation. Elemental gaseous mercury penetrates easily through the lungs into the brain (Ritchie et al., 1994; www.britannica.com). Strong correlation between the concentrations of inorganic Hg in the lungs and total Hg in the brains of herring gulls showed that the penetration of mercury from the air has a statistically significant influence on the central nervous system of the bird, where mercury is accumulated in the long term (Spearman's correlation: p = 0.000000; r = 0.79; n = 44). 7. Summary The lungs of birds can be considered to be an indicator of air quality. Inorganic and organic mercury, as well as PAHs and phenol derivatives were detected in the lungs of all the studied gulls. Seeing as the organ distribution of mercury in most of the birds was as follows: kidneys N lungs N brain N intestines, the respiratory route ought to be considered a significant route of mercury penetration into the organisms of herring gulls inhabiting the Southern Baltic region. That was confirmed by the strong correlation between inorganic mercury in lungs and in other tissues. The study results indicated a correlation between inhalation and the trophic level of the gulls' food. The lungs of the birds foraging on low trophic levels, probably in communal landfills, were more exposed to PAHs of pyrogenic origin compared to the gulls which remained mainly in the coastal zone of the sea. The PAH concentration in the lungs of gulls was reversely proportional to the trophic level occupied by the birds. On

the other hand, the mercury burden in the lungs was directly proportional to the trophic level of the birds' food obtained in the coastal zone of the sea. However, the marine source of mercury in the air cannot be the only one influencing the burden on the gulls' organisms. In the industrialised and urbanised seaside region, the highest mercury load is introduced into the atmosphere by shipyards, sea transportation, fishing vessels, transshipment in ports, as well as port storages of chemicals. The correlation between the concentration of inorganic mercury in the lungs with that in the kidneys proves that kidneys are one of the main recipients of InHg that penetrates into the lungs. The alimentary route is the main way by which phenol derivatives penetrate into the body, while inhalation is rather of secondary importance. In spite of that fact, studies on alimentary and respiratory exposure to endocrine disruptive compounds can provide information on the absorption and distribution of pollutants in the body. The knowledge of xenobiotic burden in wild birds can be used as a system of pre-warning for the entire ecosystem. Monitoring programmes are based on the observation and examination of predatory birds. Acknowledgements The authors would like to thank Włodzimierz Meissner and Szymon Bzoma for the samples of herring gulls. These studies were financed by the Polish Ministry of Science and Higher Education, project no. NN304 161637. References Akyüz, M., Çabuk, H., 2010. Gas–particle partitioning and seasonal variation of polycyclic aromatic hydrocarbons in the atmosphere of Zonguldak, Turkey. Sci. Total Environ. 408, 5550–5558. ARMAAG, 2015. http://armaag.gda.pl/files/101/184/1_raport_2015. Bartosińska, M., Ejsmont, J., Zaborski, L., Zagożdżon, P., 2005. Analiza umieralności z powodu nowotworów złośliwych w województwie. 35. Pomorskim Annales Academiae Medicae Gedanensis, pp. 85–95 (in polish). Bełdowska, M., Falkowska, L., Lewandowska, A., 2006. Airborne trace metals [Hg, Cd, Pb, Zn] of the coastal region, Gulf of Gdansk. Oceanol. Hydrobiol. Stud. 35 (2), 159–169. Bełdowska, M., Falkowska, L., Marks, R., 2003. Total gaseous mercury over the coastal zone of the Gulf of Gdansk. Oceanol. Hydrobiol. Stud. 22 (3), 3–18. Bełdowska, M., Falkowska, L., Siudek, P., Gajecka, A., Lewandowska, A., Rybka, A., Zgrundo, A., 2007. Atmospheric Mercury over the coastal zone of the Gulf of Gdańsk. Oceanol. Hydrobiol. Stud. 36 (3), 9–18. Bełdowska, M., Zawalich, K., Falkowska, L., Siudek, P., Magulski, R., 2008. Total gaseous mercury in the area of southern Baltic and in the coastal zone of the Gulf of Gdańsk during spring and autumn. Environ. Prot. Eng. 4, 130–139. Blight, L.K., Hobson, K.A., Kyser, T.K., Arcese, P., 2015. Changing gull diet in a changing world: a 150-yearstable isotope (δ13C, δ15N) record from feathers collected in the Pacific Northwest of North America. Glob. Chang. Biol. 21, 1497–1507. Bojakowska, I., 2003. Charakterystyka Wielopierścieniowych Węglowodorów Aromatycznych i ich występowanie w środowisku. 405. Biuletyn PIG, pp. 5–28 (in polish). Boudou, A., Ribeyre, F., 1985. Experimental study of trophic contamination of Salmo gairdneri by two mercury compounds (HgCl2 and CH3HgCl) – analysis at the organism and organ levels. Water Air Soil Pollut. 26, 137–148. Burgess, N.M., Bond, A.L., Herbert, C.E., Neugebauer, E., Champoux, L., 2013. Mercury trends in herring gull (Larus argentatus) eggs from Atlantic Canada 1972-2008: temporal change or dietary shift? Environ. Pollut. 172, 216–222. Carbonell, G., Bravo, J.C., Fernandez, C., Tarazona, J.V., 2009. A new method for Total mercury and methyl mercury analysis in muscle of seawater fish. Bull. Environ. Contam. Toxicol. 83, 210–213. Chmielnicka, J., 1994. Metale i metaloidy. In: Seńczuk, W. (Ed.), Toksykologia. Podręcznik dla studentów farmacji 49. Wydawnictwo Lekarskie PZWL, Warszawa, pp. 301–353 (in polish). Chmielnicka, J., 2006. Toksyczność metali i półmetali (metaloidów). In: Seńczuk, W. (Ed.), Toksykologia współczesna. Wyd. Lekarskie PZWL, Warszawa, pp. 360–446 (in polish). Custer, T.W., Custer, C.M., Hines, R.K., Sparks, D.W., 2000. Trace elements, organochlorines, polycyclic aromatic hydrocarbons, dioxins, and furans in lesser scaup wintering on the Indiana Harbor Canal. Environ. Pollut. 110 (3), 469–482. De La Torre-Roche, R.J., Lee, W.Y., Campos-Díaz, S.I., 2009. Soil-borne polycyclic aromatic hydrocarbons in El Paso, Texas: analysis of a potential problem in the United States/ Mexico border region. J. Hazard. Mater. 163, 946–958. Dhananjayan, V., Muralidharan, S., 2013. Levels of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides in various tissues of white-backed vulture in India. Biomed. Res. Int. 2013 (9s). http://dx.doi.org/10. 1155/2013/190353. EEA, 2014. Air Quality in Europe — 2014 Report, EEA Report No 5/2014. European Environment Agency, Copenhagen.

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Please cite this article as: Falkowska, L., et al., Inhalation - Route of EDC exposure in seabirds (Larus argentatus) from the Southern Baltic, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.01.060