Polychlorinated Dibenzo-p-dioxins (PCDDs), Polychlorinated Dibenzofurans (PCDFs), and Polychlorinated Biphenyls (PCBs) in Olivaceous Cormorant (Phalacrocorax brasilianus) from Sepetiba Bay, Rio de Janeiro, Brazil

An expressive number of man-made chemicals have been introduced in the aquatic environment represent the major problems arising in the development worldwide. Many of these chemical contaminants are persistent polyhalogenated aromat ic hydrocarbons known to bioaccumulate and biomagnify as they move through the aquatic food web, effect ing species associated with aquatic systems. Concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) were measured in Olivaceous Cormorant Phalacrocorax brasilianus collected from 2007 to 2011 on Sepetiba Bay, Rio de Janeiro, Brazil. Detectable hepatic concentrations of PCDD/Fs and PCBs were found in all samples analyzed. These data represent some of the first measurements of PCDD/Fs and PCBs in seabirds from this area. While levels of these contaminants in the tested specie currently appear to fall below critical values, continued monitoring is warranted for these compounds, especially in this bay.


Introduction
Oceans cover about 70% o f the earth's surface. Effects of pollution on marine ecosystems have beco me a matter of great concern, especially to coastal states [1]. The oceans cannot supply an infinite sink for anthropogenic wastes but inadequate attention has been given to evaluating the limits of capacity of coastal areas for waste assimilation [2]. Thus, instances of fisheries shortage, spoiled beaches, destroyed coral reefs and wild life habitat , to xic b loo ms and lost coastal ecological co mmunities are extensive, with a corresponding determination of cost benefit [3]. Cu rrent concerns about connectivity of ocean health issues and the relationship to human disease highlight an essential area for research [1,3]. A wareness of the ocean and the impact of human perfo rmance on it can expose the complexity and interdependence of all aspects of the system [4]. Enhanced acquaintance and predictive capabilities are required for more effective and sustained development of the marine environment to obtain associated economic benefits and to preserve marine resources.
Worldwide contamination by dioxin-related compounds, such as polychlorinated dibenzo-p-dio xins (PCDDs), polychlorinated dibezofurans (PCDFs), and coplanar polychlorinated biphenyls (coplanar PCBs), is of great concern due to the persistence, bioaccumulative nature, and toxicity of these compounds [5]. Fo r several decades, these compounds have been produced and extensively used for various purposes. Numerous of these chemical contaminants are persistent polyhalogenated aromatic hydrocarbons known to bioaccumulate and bio magnify as they move through the aquatic food web, effecting species associated with aquatic systems, including humans [6,7].
Due to its position in the marine food chain and their long life length, seabirds congregate significant levels of trace elements. Are good sentinel species because they are observable, sensitive to toxicants, and live in different trophic positions [8,9]. Consequently, studies assessing avian population status, reproductive success, and toxico logical importance of metal exposures can be extrapolated to other wildlife and probably humans [10,11].
Pollution in the marine environ ment has become a subject of enormous apprehension, especially to coastal states. The oceans cannot provide an infin ite sink for anthropogenic wastes but little attention has been given to evaluating the limits of the capacity of coastal areas for waste assimilation [12]. Knowledge of the ocean and of the impact of human activit ies on it can reveal the co mp lexity and interdependence of all aspects of the system [13]. Imp roved acquaintance with these and better forecasting ability are required for mo re effect ive and sustained development of the marine environment to obtain associated economic benefits and to preserve marine resources [14]. Recent concerns about the connectivity of ocean health issues and their relationship to human disease highlight an important area for study.

PCDDs/Fs and PCBs Contaminati on
The study of PCDDs/Fs and PCBs contamination in aquatic environments has allowed to predict or identify sources of pollution and extensiveness of these pollutants, since they potentially pose a threat to ecosystem balance, being an important instrument to predict the affection to human health and animal [15,16].
There are 75 different PCDDs and 135 PCDFs, wh ich differ fro m each other in the number and positions for the chlorine ato ms [16,17]. Fro m the hu man/biota point of v iew, 17 PCDD/Fs chlorine substitution in the (2,3,7,8) positions are considered to be toxicologically impo rtant [10,18,19]. PCDDs have a planar aromatic tricyclic structure with 1-8 chlorine ato ms as substituents. Some PCBs are called dioxin-like (co-planar/non-ortho-) PCBs. Those congeners do not have any or have only one chlorine atom (mono-ortho-PCBs) in the ortho-position to the carbon-carbon bond between the two benzene rings. Appro ximately 120 of PCBs are present in commercial products such as Aroclor 1254, Aroclor 1260 and Ch lopen A60 [17]. The PCDD and PCDF, commonly called "dio xins", are t wo classes of "quasi-planar" tricycles aro matic ethers with 210 different compounds (congeners) in total. The PCDD/ F have similar physical-chemical properties but different biological potencies [20]. Figure 1 shows the general structure of these classes of compounds.
Most industrial countries have restricted or stopped their use since 1970s, lead ing to decreasing concentrations in long-term environmental surveillance programmes [21,22]. However, co mpounds remained in ecosystems either due to their persistency or transport from developing countries where use in agricultural and industrial purposes is still current. Due to their chemical stability and hydrophobic nature, these compounds are adsorbed onto particles, accumu lated in aquatic organisms, and highly biomagnified through the aquatic food webs [23]. Many organochlorines have been implicated in a broad range of adverse biological effects, including impaired reproduction and immuno suppression [24].
The Olivaceous Cormorant (Phalacrocorax brasilianus) is widespread on both the Atlantic and Pacific coasts of America and in land waters, fro m Panama to Tierra del Fuego in southern Argentina. It feeds in shallow to deep water along lake shores, rivers, estuaries, and beaches [25]. Due to abundance of this specie at study site, it was then chosen for this research. This study aims to provide metal concentrations data of P. brasilianus collected fro m Sepetiba Bay, which constitutes as an important natural breeding ground for mo lluscs, crustaceans, fish, and in the Co roa Grande mangrove swamp, many species of seabird, P. brasilianus being particularly highly. But, due to poor sanitary conditions several environmental problems have been increasing the threat of damage to environmental health [26]. Is situated in the southern Atlantic Coast of Rio de Janeiro State, Brazil ( Figure 2).

Analysis: Identification and Quantification
A total of thirty four specimens (adults) found stranded or dead in areas related to the study site, between March 2007 and December 2011. All fresh carcasses were necropsied following a standardised protocol [27], while putrescent specimens were d iscarded. Livers were collected, weighed, and maintained at -20℃ for later analysis.
Chemical analysis of PCDDs, PCDFs (PCDD/Fs) and coplanar PCBs followed the method described in a previous report, USEPA Method 1668 [28], and USEPA Method 8290 A [29]. Five grams of liver samples were weighed and lyophilised. Dry t issues were inserted in a steel extraction cell and placed in the Accelerated Solvent Extractor (ASE 200, Dionex). This machine using organic solvents operates under high pressure and temperature conditions (10 minutes at 125℃ and 1500psi) and allows the extract ion of the different organic compounds present from the bio logical matrix. After being extracted, the samples were concentrated using Kuderna-Danish, the extract evaporated down to 1 ml, and the solvent was transferred to 10 ml of n-hexane. Fat content was determined gravimet rically fro m an aliquot of the extract [30,31]. Seventeen 2,3,7,8-substituted 13 C-labeled tetra-through octa-CDD and CDF congeners and 12 dio xin-like PCBs (IUPAC Nos. 81, 77, 126, 169, 105, 114, 118, 123, 156, 157, 167, and 189) were spiked. Furthermore, aliquots were treated with sulfuric acid (appro ximately 7-10 times) in a separation funnel. Then the hexane layer with PCDDs/DFs and PCBs was rinsed with hexane-washed water and dried by passing through anhydrous sodium sulphate in a glass funnel.
The solution was concentrated to 2 ml and sequentially subjected to silica gel, alu mina, and silica gel-impregnated activated carbon column chro matography. Extracts were passed through a silica gel-packed g lass column (Wakogel, silica gel 60; 2g) and eluted with 130 ml of hexane. The hexane extract was Kuderna-Danish concentrated and passed through alu mina colu mn (Merck-Alu mina o xide, act ivity grade 1; 5g) and eluted with 30 ml of 2% dichloro methane in hexane as a first fraction, wh ich contained mu lti-ortho-subst ituted PCBs. The second fraction eluted with 30 ml of 50% dichloro methane in hexane, containing non-and mono-ortho-PCBs and PCDDs/DFs, was Kuderna-Dan ish concentrated and passed through silica gel-impregnated activated carbon column (0.5g). The first fraction eluted with 25% dichloro methane in hexane contained mono-and di-ortho-PCBs. The second fraction eluted with 250 ml of toluene containing PCDDs/DFs was concentrated and analyzed using a high-resolution gas chromatograph interfaced with a h igh-resolution mass spectrometer (HRGC/ HRM S).
Identificat ion and quantification of 2,3,7,8-substituted congeners of PCDDs/DFs and dioxin-like PCBs (non-and mono-ortho-substituted congeners) was performed by use of a (i) Shimad zu GC-14B gas chromatograph with AOC-1400 auto-sampler. A procedural blan k including extraction o f b lank Kimwipe and whole purification procedure was run with every batch (normally seven samples). The limit of quantification (LOQ) was set at 2 times the detected amount in the p rocedural blank. Reproducibility and recovery were confirmed through four replicate analyses of an abdominal ad ipose tissue sample with and without standard spiking. The relative standard deviations of concentrations of individual PCDD/F and PCB-congeners were less than 5.8%, and the recoveries were more than 96%. The lipid contents were determined gravimetrically after aliquots of the sample extracts were evaporated to complete dryness. TEQ is the product of the concentration of an individual dio xin -like co mpound (DLC) in an environ mental mixtu re and the corresponding TCDD TEF for that compound. Equation 1 is the formula for calculat ing exposure concentration for n DLCs in a mixture in TCDD to xic equivalence (TEQ). Exposure to the i th individual PCDD, PCDF, or PCB compound is expressed in terms of an equivalent exposure of TCDD by co mputing the product of the concentration of the individual co mpound (Ci) and its assigned TEFi. TEQ is then calculated by summing these products across the n DLCs compounds present in the mixture. The TEQ may be co mpared to the dose-response slope for TCDD and used to assess the risk posed by exposures to mixtures of DLCs.
The different congeners present in the sample were then analysed using a Gas Chro matography equipped with a capillary co lu mn of 40 µm coupled to a High Resolution Mass Spectrometer (GCHRM S). They can be quantified and their concentration calculated when compared to the added internal 13 C standard [32][33][34]. Results are expressed either as pg/g of lipid mass or in terms of toxicity, using WHO TEF for birds [35] as pg TEQ/g, lipid weight.
Statistical analysis was undertaken using an Origin 7.5 software package (Origin Lab Corporation). The average distribution of PCDDs/FS and PCBs was assessed using analysis of variance (ANOVA ). For all the tests, p-values of < 0.05 were used to determine significant differences.

Results
No significant species-related differences in PCB and PCDD/Fs concentrations were found. Concentrations of PCB-congeners with fat percentages are presented (Table 1), and Concentrations of PCDD/Fs-congeners with fat percentages are presented (Table 2).  figure 3 shows the distribution of PCB and PCDD/Fs congeners.  Fat-based log-transformed concentrations were used to determine whether there were significant differences between group geometric means (Tukey test). Null hypothesis (equality of means) was rejected at the 95% significance level (p<0.05). There were no statistically significant d ifferences between mean PCDD/F and PCB-congeners concentrations between the specie. PCB 105 congener accounted for 22.16% o f ΣPCB, PCB 114 congener accounted for 19.32% of ΣPCB, and PCB congeners 118 and 77 accounted for 12.88% of ΣPCB, respectively. PCDD OCDD congener accounted for 53.12% of Σ PCDD/Fs.

Discussion
The choice of seabirds for analysis of pollutants, rather than the analysis of pollutants in the abiotic environment, it becomes mo re attractive and pro mising because these indicators can provide precise information on the bioavailab ility of pollutants, their bio-magnification and transfer. Thus, indicating that the magnitude of the observed data, reinforces that there are startling signs regarding the potential risks to public health and these indicators are important to environmental monitoring by being at top the food chain, are sensitive to toxic products, respond to subtle changes in the environ ment, and also because of its high metabolic rate [21,[36][37][38].
The fundamental question to answer is whether the t rophic level is harmfully disturbed when polluted by toxicants. To answer this important question, quantitative understanding of the pollutants behaviour within ecosystems is essential, and therefore researchers develop methods to manage this. The presence of anthropogenic pollutants, such as PCDD/F and PCB-congeners, throughout all co mpart ments of the marine environment has been of international concern for a number of decades [39][40][41]. While a great nu mber of datasets documenting absolute concentrations of persistent organic pollutants in a variety of marine biota are availab le, the bioaccumulat ive nature, to xicity, bio magnification, and the fate of these compounds in the marine ecosystem is still poorly understood. Data on contaminant levels in Brazilian seabirds are limited, and no information exists regarding levels of new o r emerging contaminants.
Reported adverse effects of POPs in wildlife include population declines, increases in cancers, reduced reproductive function, d isrupted development of immune and nervous systems, and also elicit to xic responses which could result in the disruption of the endocrine system [7].
In previous studies, the monitoring of POPs in seabirds has been limited by the availability in organs [42][43][44][45]. This approach can easily be combined with ecological investigations of seabirds, and so this could dramat ically increase the availability of seabird samp les, including repeated sampling on identical birds. Recently, electronic tracking tags have revolutionized our understanding of the large-scale movements and habitat use of mobile marine animals [46].
Increased human activit ies such as industrializat ion, coupled with over-population and increased ambient temperature amongst other factors, have become major environmental issues in recent years. As a result of such actions, additional studies which include the environ ment and their indicators are important because they can show potential impacts that are being reflected, and extending to public health.
It was presented a scientific approach for assessing the ecological condit ion of the Sepetiba Bay and the impacts caused by PCBs and PCDD/FS in a part icular species of bird used as indicator, which has weights throughout Latin America. The key assumptions underlying the approach are: (a) the importance of putting analysis on ecosystems attributes of public importance, (b ) the consistent with scientific understanding of what is important to sustain ecosystems structure and function, (c) measurements in environ mental indicators must be scientifically defensible, and (d) are there implications on health risk to man and along marine trophic chains public health?

Conclusions
The presence of tissue levels of POPs has been associated with bio logical and physiological effects in marine organisms, in specially seabirds. The animals sampled in the current study had PCDD/F and PCB congeners that exceeded the values found in these studies. Wide ranges of POP concentrations were measured in these animals, and our findings indicate that these animals are exposed to POPs levels that may affect their health, and in some classes of to xic POPs that may increase their risk to adverse effects.
The present study confirms the ubiquity of POPs in Phalacrocorax brasilianus, belonging the marine environ ment of Sepetiba Bay, Rio de Janeiro, Brazil. Bio magnification may be the cause of the levels in the species collected and analysed. Further assessments are reco mmended on organisms at higher trophic levels for ecoto xicological impacts. The ubiquity of these pollutants in Sepetiba Bay's marine environment supports the need for a greater awareness of bioaccumulat ion processes, particularly for organisms cultivated (shellfish) or fished locally and destined for hu man consumption.
This research gives reasonable alerts in po llution marine with relevant informat ion that can support the decision-making process and provides a baseline to evaluate future clean up and restoration activit ies at Sepetiba bay. There are clear management decisions that must be made concerning what to clean up, and to what levels.