Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast.

Marine Pollution Bulletin xxx (2015) xxx–xxx

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Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast Helena A. Kehrig a,b,⇑, Rachel A. Hauser-Davis c, Tércia G. Seixas b,c, Gilberto Fillmann d a

Lab. de Ciências Ambientais, Universidade Estadual do Norte Fluminense, 28013-602 Campos dos Goytacazes, RJ, Brazil Lab. de Radioisótopos Eduardo Penna Franca, IBCCF, Universidade Federal do Rio de Janeiro, 21941-901 Rio de Janeiro, RJ, Brazil Dep. de Química, PUC – Rio, 22453-900 Rio de Janeiro, RJ, Brazil d Instituto de Oceanografia, Universidade Federal do Rio Grande, 96203-900 Rio Grande, RS, Brazil b c

a r t i c l e

i n f o

Article history: Received 1 December 2014 Revised 14 April 2015 Accepted 2 May 2015 Available online xxxx Keywords: Seabird Detoxification Western South Atlantic Ocean Biomonitor Metallothionein Mercury species

a b s t r a c t Magellanic penguins have been reported as good biomonitors for several types of pollutants, including trace-elements. In this context, selenium (Se), total mercury, methylmercury, inorganic mercury (Hginorg), cadmium (Cd) and lead (Pb), as well as metallothionein (MT) levels, were evaluated in the feathers, liver and kidney of juvenile Magellanic penguins found stranded along the coast of Southern Brazil. The highest concentrations of all trace-elements and methylmercury were found in internal organs. Concentrations of Cd and Se in feathers were extremely low in comparison with their concentrations in soft tissues. The results showed that both Se and MT are involved in the detoxification of trace-elements (Cd, Pb and Hginorg) since statistically significant relationships were found in liver. Conversely, hepatic Se was shown to be the only detoxifying agent for methylmercury. Ó 2015 Elsevier Ltd. All rights reserved.

The oceans were previously considered to be a vast reservoir for the safe disposal of pollutants. However, in recent decades, a wide range of chemical contaminants, including trace-elements, have become widely recognized as a source of adverse effects in marine environments (Torres et al., 2008). Contaminants can bioaccumulate over time to reach sub lethal, or even lethal, levels in organisms unless they are excreted or detoxified. This problem is particularly severe for trace-elements in long-lived organisms, such as seabirds. As top predators in marine food webs, seabirds are good biomonitors of pollutants that accumulate along the trophic levels (Furness and Camphuysen, 1997; Dee Boersma, 2008). Penguins are highly specialized for swimming and diving, and therefore reflect regional oceanic variations (such as anthropogenic inputs) more completely than other seabirds (Dee Boersma, 2008). According to Kennish (1997), oceanic birds such as the Magellanic penguin appear to be particularly vulnerable to anthropogenic inputs, since they are a migratory waterfowl that spend a ⇑ Corresponding author at: Lab. de Ciências Ambientais, Universidade Estadual do Norte Fluminense, 28013-602 Campos dos Goytacazes, RJ, Brazil. E-mail addresses: [email protected] (H.A. Kehrig), rachel.hauser. [email protected] (R.A. Hauser-Davis), [email protected] (T.G. Seixas), docgfi[email protected] (G. Fillmann).

considerable amount of time on the sea surface. In general, the main trace-element contamination exposure for seabirds are their ingested prey, varying according to the prey species consumed (Bustamante et al., 1998). Magellanic penguins (Spheniscus magellanicus (Forster, 1781)) are the most abundant species on the coast of South America. They are South American penguin species and not Antarctic species. They are medium-sized (around 71 cm-long and 4 kg weight) (IPCWG, 2005) and can live for about twenty-five years. This penguin species migrates from colonies in the Falkland Islands and Patagonia and reaches the Southern and Southeastern Brazilian coasts during migrations. Cephalopods and pelagic fish are important items in their diet along the Western South Atlantic Ocean (Brandão et al., 2011; Petry et al., 2012). In winter, young Magellanic penguins move along the Brazilian coast to feed on anchovy schools (Brandão et al., 2011; Petry et al., 2012). Although studies regarding contaminants in birds have increased since the 60s, focusing on the toxicokinetics of trace-element compounds and on their use as indicators of trace-element contamination (Monteiro and Furness, 2001), data concerning trace-elements in Magellanic penguin tissues are still scarce and fragmented (Keymer et al., 2001; Vega et al., 2010; Frias et al., 2012).

http://dx.doi.org/10.1016/j.marpolbul.2015.05.006 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kehrig, H.A., et al. Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.006

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H.A. Kehrig et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Metallothioneins (MT) are cysteine-rich low-molecular-weight proteins that bind with high affinity to trace-elements and whose synthesis is mainly induced in response to the presence of certain trace-elements, such as Hg, Cd and Pb (Andrews et al., 1996). MT determinations in biota tissues have been used as a biomarker of previous exposure to a number of trace-elements, since MT levels correlate well with environmental levels (Livingstone, 1993; Andrews et al., 1996). In this context, the present study appraised the accumulation of trace-elements and methylmercury in tissue samples of juvenile Magellanic penguins found stranded along the Southern Brazilian coast. Selenium, total mercury, methylmercury, cadmium and lead were analyzed in feathers, liver and kidney, whilst metallothionein was evaluated in liver as a biomarker of trace-element exposure. The values corresponding to the concentrations of inorganic mercury (Hginorg) were calculated as the difference between the values found for total mercury and methylmercury concentrations. The importance of the liver as a biotransformation, detoxification and enhanced elimination organ was assessed by analyzing relationships with metallothionein levels, as well as relationships with selenium. Moreover, this study also partially addresses the lack of pollutant and metallothionein data for oceanic birds. Magellanic penguin specimens were found stranded during regular beach monitoring along the coast of Rio Grande do Sul State,

Southern Brazil (Fig. 1). According to the penguins’ plumage, i.e. gray-blue backs and one gray blue band between the head and chest, all sampled individuals were classified as juveniles (approximately 1.5 year-old). The body condition of all individuals was poor, presenting reduction of muscle tissues and absence of subcutaneous fat tissue. Feather, liver and kidney samples were collected from 9 females, 6 males and 7 non-sexed individuals of juvenile Magellanic penguins (S. magellanicus (Forster, 1781)). The animals were taken to the laboratory, dissected and, after sampling; the tissues were immediately frozen at 20 °C until analysis. Samples for the determination of metallothioneins (MTs), selenium (Se), total mercury (Hg), methylmercury (MeHg), lead (Pb) and cadmium (Cd) were freeze-dried and kept sheltered from light until analysis. Feather samples were washed in distilled water with EDTA using an ultrasonic cleaner to remove superficial contaminants, dried, and cut into small fragments using stainless steel scissors. For the total mercury analyses, dry samples (50 mg) were digested in a sulphuric–nitric acid mixture. Total mercury was determined by cold vapor atomic absorption spectrometry, using NaBH4 as a reducing agent. A detailed description of the method used is given elsewhere (Kehrig et al., 2009). For selenium (Se), lead (Pb) and cadmium (Cd), the dry samples (100 mg) were digested in nitric acid and element content was determined

N

South Atlantic Ocean

South Pacific Ocean

Brazil

Argentina

Uruguay

Rio Grande do Sul State

Falkland Islands

Fig. 1. Sampling area along the Rio Grande do Sul coast, Southern Brazil.



by graphite furnace atomic absorption spectrometry, using palladium nitrate as chemical modifier as described by Seixas et al. (2009). The methylmercury (MeHg) analyses (50 mg) were conducted by digesting the samples with an alcoholic potassium hydroxide solution followed by dithizone-toluene extraction. After a series of clean-up steps, MeHg dithizonate was identified and quantified in the toluene layer on a Shimadzu gas chromatograph GC-14 with an electron-capture detector-ECD. A detailed description of the method used is given elsewhere (Kehrig et al., 2009). Quality control was performed by a strict blank control, the analyses of replicates and certified reference materials. Accuracy was assessed through the analysis of certified material DORM-2 (Hg: 4.64 ± 0.26 lg g 1; Se: 1.40 ± 0.09 lg g 1) and TORT-2 (Pb: 0.35 ± 0.13 lg g 1; Cd: 26.7 ± 0.6 lg g 1) from the National Research Council-Canada, and IAEA 350-Tuna fish sample (MeHg: 3.65 ± 0.35 lg g 1) from the International Atomic Energy Agency. Results for DORM-2 were Hg – 4.54 ± 0.13 lg g 1 (N = 12) and Se – 1.47 ± 0.27 lg g 1 (N = 11), while for TORT-2 were Pb – 0.36 ± 0.02 lg g 1 (N = 10) and Cd – 26.6 ± 0.5 lg g 1 (N = 10), and for IAEA 350 (N = 20) were MeHg – 3.59 ± 0.38 lg g 1. The trace-element and methylmercury results were between 97% and 105% of the respective mean certified values. Metallothionein extraction followed the protocol with heat treatment proposed by Erk et al. (2002). Briefly, samples (25 mg) were homogenized in a solution containing Tris–HCl 20 mM pH 8.6, phenylmethylsulphonylfluoride 0.5 mM as an antiproteolytic agent and b-mercaptoethanol 0.01% as a reducing agent. Samples were then centrifuged at 20,000g for 1 h at 4 °C. The supernatants were then separated from the pellet and transferred to new sterile eppendorfs and heated at 70 °C for 10 min. Another centrifugation step in the same conditions was conducted for 30 min. The final supernatants containing the MTs were separated and frozen at 80 °C until analysis. Sample absorbance was evaluated at 412 nm. Concentrations were estimated by using reduced glutathione (GSH) as external standard. MT content was then estimated by assuming the relationship of 1 mol MT = 20 mols GSH. Statistical analyses were performed using STATISTICAÒ 7.0 for Windows (StatSoft, Inc. 1984–2004, USA). Data were tested for normal distributions and non-parametric tests were then applied. The analysis of variance was conducted by the Kruskal–Wallis test-ANOVA followed by a post hoc test (Mann–Whitney U-test) in order to define significant differences in trace-element and methylmercury concentrations among the tissues (liver, kidney and feather). The Spearman correlation test (r) was performed to determine the relationships between body length and all trace-element and MeHg concentrations in internal organs, and also between metallothioneins and all trace-element, Hginorg and MeHg concentrations and selenium and all trace-element, Hginorg and MeHg concentrations (on a lmolar basis) in liver. A Discriminant Analysis was applied to identify and visualize the overall main trends. The significant level was 60.05. Trace-element (Se, Hg, Cd and Pb) and methylmercury (MeHg) concentrations in the feather, liver and kidneys of juvenile Magellanic penguins are summarized in Table 1. Values are presented as the mean ± standard deviation (SD) of dry weights. The tissues of all organisms presented low concentrations which were comparable to those found in previous studies with specimens of Magellanic penguins (Table 1). Males and female Magellanic penguins presented similar trace-element and MeHg concentrations in feather, liver and kidney (U test, p > 0.10). Similar gender-related patterns were also observed by Nam et al. (2005) in great cormorants, a seabird species from Japan. Metallothionein concentrations (mean ± SD) in Magellanic penguin livers varied from 107.3 to 1017 lmol g 1 dry wt. (210.1 ± 137.3). Sex-related difference for metallothionein

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concentrations in Magellanic penguin livers was not observed (U test; Z = 1.3; p > 0.1). An analysis of all data showed no significant correlations (p > 0.05) between body length and all trace-element and MeHg concentrations in internal organs (liver and kidney) and feathers. The lack of relationships between the concentrations and body length could be related to the similar body length of all the specimens (which varied between 52.1 and 58.7 cm). The same lack of relationships was previously found for Magellanic penguins from two different areas of the Brazilian coast (Vega et al., 2010). According to Furness et al. (1986), total mercury is accumulated and stored in internal seabird tissues throughout their lifetime. However, this metal is mobilized and excreted into the plumage during molt, and therefore does not accumulate with growth, i.e. with increasing body length. On the other hand, cadmium concentrations in internal tissues of seabirds have a tendency to increase with body length (Stewart and Furness, 1998; Nam et al., 2005). The distribution of trace-elements and MeHg in hard (feather) and soft (liver and kidney) tissues of the Magellanic penguins was observed. The Discriminant Analysis for the data on Hg, MeHg, Se, Pb and Cd in tissues samples, feather, liver, and kidney of Magellanic penguins reveals a clear distribution between the concentration of the analyzed elements and methylmercury, indicating different accumulation behaviors by the three tissues (Fig. 2). The variables that contribute with greater weights to explain the variance were selenium, mercury and methylmercury concentrations (p < 0.001), while cadmium and lead have the least contributions (p > 0.05). The Squared Mahalanobis Distances between the groups (feather liver; feather kidney; kidney liver) were high, indicating that there were significant differences between them (F = 23.0; p < 0.001), with a correct classification percentage of 100%. The highest concentrations of all trace-elements and MeHg were found in the liver and kidney, that play an important role in their biotransformation and elimination (Table 1). The present data corroborated with previous studies conducted on Magellanic penguins, in which seabird liver is the soft tissues where total Hg is mainly concentrated, whereas Cd and Pb become firmly bound in the kidneys, and only trace amounts are found in feathers (Keymer et al., 2001; Gil et al., 2006; Vega et al., 2010; Frias et al., 2012). Seabird feathers are particularly convenient for monitoring trace-element pollution in marine food webs (Furness and Camphuysen, 1997; Jakimska et al., 2011; Frias et al., 2012), especially pollutants that are lipid-soluble but have low water solubility, such as MeHg (Furness and Camphuysen, 1997). Since metals may be deposited from atmosphere onto feather surfaces, as well as incorporated into growing feathers from blood, the use of feathers in environmental monitor must take into account that these hard tissues may be responding to a combination of these two processes (Furness and Camphuysen, 1997). Most total mercury and MeHg were found in hepatic tissues, with a relative burden of 63.7% and 50.0%, followed by 27.6% and 33.9% in kidneys, and 8.7% and 16.1% in feathers, respectively (Table 1). Mean concentrations of hepatic total Hg and MeHg in the animals were about twofold and 1.5-fold higher, respectively, than those found in their kidneys (Table 1). It is well known that some trace-elements, mainly Hg, accumulate preferentially in hepatic cells in comparison to other tissues (Thompson, 1990), which is probably related to the role played by the liver in terms of biotransforming pollutants, metabolizing nutrients and essential elements and removing certain non-essential elements and toxins from the bloodstream (Frodello et al., 2000). It is noteworthy that seabirds are more resistant to the effects of Hg because of the detoxification afforded by their molting cycles, the induction of the synthesis and binding


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Table 1 Mean ± SD (minimum–maximum) concentrations of selenium (Se), mercury (Hg), methylmercury (MeHg), cadmium (Cd) and lead (Pb) (lg g 1 dry wt.), and the ratios of methylmercury to Hg (% MeHg) in Magellanic penguin feather, liver and kidneys, and the number of individuals (N) and age class of the analyzed penguins. Tissues

Se (min–max)

Hg (min–max)

MeHg (min–max)

Cd (min–max)

Pb (min–max)

% MeHg

N

Age class

Reference

Feather

0.64 ± 0.32 (0.24– 1.44) –

0.78 ± 0.44 (0.27– 1.78) 0.052 ± 0.035

0.62 ± 0.32 (0.22– 1.27) –

0.13 ± 0.07 (0.04–0.27)

80.1

22

Juveniles

Present study

–

0.14 ± 0.08 (0.05– 0.32) –

–

16

Juveniles

–

0.043 ± 0.033

–

–

–

–

21

Juveniles

–

0.21 ± 0.10

–

–

–

–

21

Older adults

Frias et al. (2012) Frias et al. (2012) Frias et al. (2012)

5.15 ± 2.82 (1.85– 13.63) –

5.70 ± 3.73 (1.98– 18.07) 5.70 ± 4.10

1.93 ± 0.92 (0.69– 3.76) –

7.25 ± 4.71 (2.52– 22.24) 24.40 ± 23.60

0.58 ± 0.32 (0.20– 1.23) 60.1

36.1

22

Juveniles

Present study

–

35

juveniles

–

3.70 ± 2.10

–

10.90 ± 8.60

60.1

–

12

Juveniles

– –

(1.55–7.24)a –

– –

(9.35–110.70)a 106.67 ± 41.26

(nd–18.33)a 2.78 ± 0.82

– –

13 15

– Adults

Vega et al. (2010) Vega et al. (2010) Gil et al. (2006) Keymer et al. (2001)

8.03 ± 4.48 (2.87– 21.94) – –

2.47 ± 1.42 (0.87– 6.78) (1.28–1.89)b –

1.31 ± 0.73 (0.46– 3.26) – –

46.50 ± 33.55 (15.35– 133.11) (81.57–399.50)b 188.72 ± 99.03

0.55 ± 0.30 (0.19– 1.16) (1.98–8.82)b 1.31 ± 0.75

53.1

22

Juveniles

Present study

– –

13 15

– Adults

Gil et al. (2006) Keymer et al. (2001)

Liver

Kidneys

a b

Wet weight basis concentration was converted to dry weight basis concentration assuming a moisture content of 69.7% (Yang and Miyazaki, 2003). Wet weight basis concentration was converted to dry weight basis concentration assuming a moisture content of 77.3% (Yang and Miyazaki, 2003).

8

Variable 2

4

0

-4

-8 -8

liver

-4

0

kidney

4

feather

8

Variable 1 Fig. 2. Discriminant analysis scatterplot for trace-elements and methylmercury in Magellanic penguin feather, liver and kidneys.

to metallothioneins, as well as demethylation and the formation of a Se-Hg complex (Jakimska et al., 2011). Cadmium and selenium predominated in the renal tissues of Magellanic penguins (relative burden of 86.3% and 58.1%, respectively), while their concentrations in feathers were relatively low (Table 1). Indeed, Cd and Se kidney levels exceed Cd and Se liver levels by a factor of 6.4 and 1.6, respectively. The highest renal cadmium concentrations found are probably related to the filtering and elimination function of the kidneys (Nam et al., 2005). Dynamic interactions have suggested that metallothioneins (MTs) play a major role in Cd detoxification in the internal organs (i.e. liver and kidney) of aquatic birds, since the percentage of the cytosolic Cd bound to MTs can reach almost 100% in these organs (Nam et al., 2005). Thus, the detoxification kinetics that involves MT induction provides Cd accumulation mainly in seabird soft tissues (Stewart and Furness, 1998). Scheuhammer (1987) suggest

that such a situation indicates environmental exposure to chronic low levels of cadmium, since acute exposures result in higher levels in liver than kidney. Concentrations found in the present study for Pb in liver and kidney were similar to levels found in the same organs of Magellanic, Gentoo and Rockhopper penguins from the Falkland Island, South Atlantic Ocean (Table 1) (Keymer et al., 2001). In fact, lead concentrations ranging between 1.0 and 10.0 lg g 1 in kidney and 0.5 and 5.0 lg g 1 dry wt. in liver can be considered low since these are normal background levels for seabirds from uncontaminated areas (Scheuhammer, 1987). Hepatic inorganic mercury (Hginorg) concentrations were twofold higher than MeHg in liver of Magellanic penguins. Methylmercury concentrations correspond to an average of 36% (range of 21% to 47%) of the total Hg found in the liver of all analyzed animals, while Hginorg presented an average of 64% (range of 52% to 79%; 1.29 to 14.31 lg g 1) (Table 1). A previous study with a black-footed albatross suggested that mercury accumulated preferentially in liver of seabirds, mainly as Hginorg (Ikemoto et al., 2004). In seabirds, mercury is taken up from their diet mainly as MeHg and is then transformed into a less toxic form, Hginorg, in their internal organs. This can explain the large fraction of mercury stored at high concentrations in liver and kidneys as inorganic mercury (Thompson, 1990). The relatively low hepatic MeHg fraction and the increase of the Hginorg fraction of all individuals of Magellanic penguin indicate that the liver may act as a mercury demethylation and/or sequestration organ for both the organic and inorganic forms of this element from the body. Thus, demethylation, the transformation of MeHg into the less toxic inorganic form, is believed to be occurring in the liver of these seabirds (Arai et al., 2004). Significant positive linear relationships were found between MT molar concentrations and the analyzed trace-elements (total Hg, Cd, Pb) as well as Hginorg in Magellanic penguin liver (Table 2), which indicates that MT mediated the detoxification of these elements. This would suggest a possible effective physiological mechanism for regulation against trace-element toxicosis (Trust et al., 2000; Nam et al., 2005).


H.A. Kehrig et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx Table 2 Relationships (with their correspondent statistical significance) between MT and cadmium (Cd), lead (Pb), total mercury (Hg), inorganic mercury (Hginorg) and methylmercury (MeHg) concentrations (lmol g 1) in Magellanic penguin liver. Relationship

Statistical significance

[MT] = 6.9 104 ⁄ [Cd] 1.3 102 [MT] = 16.1 104 ⁄ [Pb] 1.3 102 [MT] = 1.6 104 ⁄ [Hg] 1.3 102 [MT] = 2.7 104 ⁄ [Hginorg] 1.6 102 [MT] = 1.7 104 ⁄ [MeHg] + 0.7 102

r = 0.66; r = 0.65; r = 0.66; r = 0.79; r = 0.27;

p < 0.005 p < 0.005 p < 0.01 p < 0.0001 p > 0.1

In fact, many free-living seabirds exhibit relatively high concentrations of trace-elements, Cd and Hg in particular, with apparently little or no evident ill effects. The associations between MT and total Hg and Cd in seabird liver have been previously reported, corroborating the hypothesis of MT-mediated detoxification of these elements which in turn supports the previous affirmation (Kojadinovic et al., 2007). MT concentrations have been shown to increase with total hepatic levels of some elements, such as Cd and Cu in the liver of aquatic birds from Japan, demonstrating that MT are closely associated with metal regulation, especially Cu and Cd, in these birds (Nam et al., 2005). However, MT and MeHg did not show a significant relationship (p > 0.1) in liver of Magellanic penguins (Table 2), suggesting that it is not effective regarding MeHg detoxification. Significant positive linear relationships were found between the selenium (Se) and trace-elements (total Hg, Cd, Pb) and Hginorg molar concentrations in the liver of Magellanic penguins (Table 3). These relationships could be reflecting a direct association between Se and trace-elements in a detoxification mechanism in the liver (i.e. Se detoxifies Hg and other metals, such as Cd) (Arai et al., 2004). In fact, the high correlation between Se and total Hg in liver of seabirds is well known (Ikemoto et al., 2004) and could be reflecting a direct association between these elements in this organ. Selenium, like sulphur, readily complexes with mercury and both of these elements tend to be associated with sulphur in proteins. Thus, it is reasonable to expect that Se and Hg tend to bioaccumulate together in hepatic tissues (Ganther et al., 1972). The chemical forms of mercury and selenium accumulated in the liver of black footed albatross were studied by X-ray absorption fine structure (XAFS) analysis. It was found chalcogenide containing both Hg-Se and Hg-S bonds, which suggests the existence of granules of Hg(Se, S) (Arai et al., 2004). However, total Hg has a million times greater affinity to binds Se, which compromise the biological functions and availability of selenium. Methylmercury molar concentrations showed a clear linear increase with Se molar concentrations ([Se] = 0.35 ⁄ log [MeHg] + 0.75; r = 0.80; p < 0.05) in Magellanic penguin liver. It is noteworthy that the percentage of MeHg to total mercury was negative and poorly correlated to selenium molar concentrations in liver. Some studies have shown that Se may reduce Hg availability, as methylmercury, by blocking it in insoluble compounds

Table 3 Relationships (with its correspondent statistical significance) between selenium and cadmium (Cd), lead (Pb), total mercury (Hg) and inorganic mercury (Hginorg) concentrations (lmol g 1) and the methylmercury to Hg ratios (% MeHg) in Magellanic penguin liver. Relationship

Statistical significance

[Se] = 0.8 ⁄ [Cd] + 1.3 10 1 [Se] = 20.4 ⁄ [Pb] + 0.9 10 2 [Se] = 1.9 ⁄ [Hg] + 1.2 10 2 [Se] = 2.3 ⁄ [Hginorg] + 2.0 10 2 [Se] = 2.2 10 3 ⁄ % MeHg + 0.2

r = 0.89; p < 0.0001 r = 0.81; p < 0.0001 r = 0.91; p < 0.0001 r = 0.87; p < 0.0001 r = 0.40; p < 0.05

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(Sasakura and Suzuki, 1998; Feroci et al., 2005) which, therefore decreases its toxicity. Magellanic penguins presented low concentrations in the hard and soft tissues, which were comparable to those found in previous studies with this seabird. The investigated trace-elements (Hg, Pb, Cd) are mostly located in their soft tissues. Selenium in this organ plays a relevant role in their detoxification, as well as in the detoxification of methylmercury. Metallothionein has also showed to play a role in the detoxification of trace-elements and Hginorg, with the exception of methylmercury, in the liver of Magellanic penguins. Acknowledgements The authors would like to thank the Brazilian National Research Council (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for the financial support. G. Fillmann was sponsored by CNPq (PQ 314335/2009-9 and 312341/2013-0). Special thanks to Daphne S. Cukierman and Julia A. Victorino for several MT analyses. References Andrews, G.K., Fernando, L.P., Moore, K.L., Dalton, T.P., Sobieski, R.J., 1996. Avian metallothioneins: structure, regulation and evolution. J. Nutr. 126, 1317S– 1323S. Arai, T., Ikemoto, T., Hokura, A., Terada, Y., Kunito, T., Tanabe, S., Nakai, I., 2004. Chemical forms of mercury and cadmium accumulated in marine mammals and seabirds as determined by XAFS analysis. Environ. Sci. Technol. 38, 6468– 6474. Brandão, M.L., Braga, K.M., Luque, J.L., 2011. Marine debris ingestion by Magellanic penguins, Spheniscus magellanicus (Aves: Sphenisciformes), from the Brazilian coastal zone. Mar. Pollut. Bull. 62, 2246–2249. Bustamante, P., Caurant, F., Fowler, S.W., Miramand, P., 1998. Cephalopods as a vector for the transfer of cadmium to top marine predators in the north-east Atlantic Ocean. Sci. Total Environ. 220, 71–80. Dee Boersma, P., 2008. Penguins as marine sentinels. Bioscience 58 (7), 597–607. Erk, M., Ivankovic, D., Raspor, B., Pavicic, J., 2002. Evaluation of different purification procedures for the electrochemical quantification of mussel metallothioneins. Talanta 57, 1211–1218. Feroci, G., Badiello, R., Fini, A., 2005. Interactions between different selenium compounds and zinc, cadmium and mercury. J. Trace Elem. Med. Biol. 18, 227– 234. Frias, J.E., Gil, M.N., Esteves, J.L., Borboroglu, P.G., Kane, O.J., Smith, J.R., Dee Boersma, P., 2012. Mercury levels in feathers of Magellanic penguins. Mar. Pollut. Bull. 64, 1265–1269. Frodello, J.P., Romeo, M., Viale, D., 2000. Distribution of mercury in the organs and tissues of five toothed-whale species of the Mediterranean. Environ. Pollut. 108, 447–452. Furness, R.W., Camphuysen, C.J., 1997. Seabirds as monitors if the environment. J. Mar. Sci. 54, 726–737. Furness, R.W., Muirhead, S.J., Woodburn, M., 1986. Using bird feathers to measure mercury in the environment: relationships between mercury content and molt. Mar. Pollut. Bull. 17, 27–30. Ganther, H.E., Goudie, C., Kopecky, M.J., Wagner, P.O.S.H., Hoekstra, W.G., 1972. Selenium: relation to decreased toxicity of methylmercury added to diets containing tuna. Science 175, 1122–1124. Gil, M.N., Torres, A., Harvey, M., Esteves, J.L., 2006. Metales pesados en organismos marinos de la zona costera de la Patagonia Argentina continental. Rev. Biol. Mar. Oceanog. 41 (2), 167–176. Ikemoto, T., Kunito, T., Tanaka, H., Baba, N., Miyazaki, N., Tanabe, T., 2004. Detoxification mechanism of heavy metals in marine mammals and seabirds: interaction of selenium with mercury, silver, copper, zinc and cadmium in liver. Arch. Environ. Contam. Toxicol. 47, 402–413. IPCWG, 2005. International Penguin Conservation Work Group. . Jakimska, A., Konieczka, P., Skóra, K., Namiesńik, J., 2011. Bioaccumulation of metals in tissues of marine animals, Part I: the role and impact of heavy metals on organisms. Pol. J. Environ. Stud. 20 (5), 1117–1125. Kehrig, H.A., Palermo, E.F.A., Seixas, T.G., Santos, H.S.B., Malm, O., Akagi, H., 2009. Methyl and total mercury found in two man-made Amazonian Reservoirs. J. Braz. Chem. Soc. 20 (6), 1142–1152. Kennish, M.J., 1997. Heavy metals. In: Practical Handbook of Estuarine and Marine Pollution. CRC Press Marine Science Series, New York, USA, pp. 253– 328. Keymer, I.F., Malcolm, H.M., Hunt, A., Horsley, D.T., 2001. Health evaluation of penguins (Sphenisciformes) following mortality in the Falklands (South Atlantic). Dis. Aquat. Organ. 45 (3), 159–169.


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Two different avipoxviruses associated with pox disease in Magellanic penguins (Spheniscus magellanicus) along the Brazilian coast.

Variations in the morphology of Rhizomucor pusillus in granulomatous lesions of a Magellanic penguin (Spheniscus magellanicus).

The eye of the magellanic penguin (Spheniscus magellanicus): structure of the anterior segment.

Complete Genome Sequence of Enterococcus faecalis Strain P8-1 Isolated from Wild Magellanic Penguin (Spheniscus magellanicus) Feces on the South Coast of Brazil.

Comparative study between photodynamic and antibiotic therapies for treatment of footpad dermatitis (bumblefoot) in Magellanic penguins (Spheniscus magellanicus).

Anisakids of seals found on the southern coast of Baltic Sea.

The vocal repertoire of the African Penguin (Spheniscus demersus): structure and function of calls.

Diversity in the Toll-Like Receptor Genes of the African Penguin (Spheniscus demersus).

Valvular dysplasia and congestive heart failure in a juvenile African penguin (Spheniscus demersus).

Cloacolithiasis and intestinal lymphosarcoma in an African black-footed penguin (Spheniscus demersus).

Magellanic penguin telomeres do not shorten with age with increased reproductive effort, investment, and basal corticosterone.

Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

Assessment of microsatellite and SNP markers for parentage assignment in ex situ African Penguin (Spheniscus demersus) populations.

Bioactive Compounds Found in Brazilian Cerrado Fruits.

Medicinal species with gastroprotective activity found in the Brazilian Cerrado.

Stranding Events of Kogia Whales along the Brazilian Coast.

Brown pelicans: improved reproduction off the southern California coast.

Identification of Cyanobacteria in a Eutrophic Coastal Lagoon on the Southern Baltic Coast.

Influence of water salinity on corrosion risk-the case of the southern Baltic Sea coast.

Marine debris ingestion by Chelonia mydas (Testudines: Cheloniidae) on the Brazilian coast.

Influence of dietary protein levels on the fate of methylmercury and glutathione metabolism in mice.

Evaluation of zinc effect on cadmium action in lipid peroxidation and metallothionein levels in the brain.

Estrogenic evaluation and organochlorine identification in blubber of North Sea harbour porpoise (Phocoena phocoena) stranded on the North Sea coast.

Plant selection for ethnobotanical uses on the Amalfi Coast (Southern Italy).