FECAL STEROLS

UMA ABORDAGEM M ´ ULTIPLA USANDO BIOMARCADORES PARA IDENTIFICAR FONTES DE POLUIC ¸ ˜ AO EM AMBIENTES COSTEIROS:

USO DE n-ALCANOS, HPAs E ESTER ´ OIS FECAIS

Caroline Kozak^1∗, Aluana Ariane Schleder², Carlos Eduardo Galoski³, Juliane Rizzi⁴, Sandro Froehner⁵, & Juan S´anez⁶

Resumo– Os biomarcadores s˜ao complexas mol´eculas que formam parte da estrutura dos organismos vivos e seus res´ıduos. Podem ser usados na identificac¸˜ao quali-quantitativa de fontes de mat´eria orgˆanica. Adicionalmente, os sedimentos desempenham uma func¸˜ao de sumidouro dessa mat´eria orgˆanica que s˜ao depositados no leito de ba´ıas (por ex.). Neste sentido, o objetivo deste trabalho foi verificar a presenc¸a ou ausˆencia e poss´ıveis fontes de origem, dos biomarcadores n-alcanos, HPAs e ester ´ois fecais em sedimentos marinhos da Ba´ıa de Paranagu´a/PR. Os resultados mostraram que os sedimentos apresentam n-alcanos de fonte terrestre e combust´ıveis, assim como os HPAs encontrados tem sua fonte associada ao uso e queima de combust´ıveis f ´osseis. Adicionalmente foram encontrados ester ´ois fecais, indicando portanto, poluic¸˜ao fecal. A presenc¸a destes biomarcadores est´a associada `as t´ıpicas atividades antr ´opicas desenvolvidas no local, como contaminac¸˜ao fecal e tr´afego de navio no porto (combust´ıveis f ´osseis). Os biomarcadores mostram-se uma ferramenta eficaz na identificac¸˜ao de fontes de poluic¸˜ao orgˆanica ao longo do tempo em sedimentos marinhos.

Palavras-Chave– Ba´ıa de Paranagu´a, poluic¸˜ao orgˆanica, an´alise quali-quantitativa

MULTIBIOMARKER APPROACH TO IDENTIFY POLLUTION SOURCES IN A COASTAL ENVIRONMENT: USE OF n-ALKANES, PAHs AND

FECAL STEROLS

Abstract – Biomarkers are complex molecules that form part of the structure of living or- ganisms and their residues. They can be used in the quali-quantitative identification of organic matter sources. In addition, the sediments play a function as sink of the organic matter deposited in the bed of bays. In this sense, the objective of this work was to verify the presence or absence, as well as possible sources of origin, of the n-alkane, HPAs and fecal sterols in marine sediments of the Paranagua Bay (Parana, Brazil). The results showed that the sediments presented n-alkanes of terrigineousorigin and fuels sources, as well. The found PAHs was associated with the use and burning of fossil fuels. Additionally, fecal sterols indicated potential fecal pollution. The presence of these biomarkers were associated with typical anthropogenic activities, such as fecal contamination from urban areas and ship traffic in the port (fossil fuels). These biomarkers were an effective tool in identifying sources of organic pollution in marine sediments deposited over time.

Keywords– Paranagu´a Bay, multibiomarker approach, quali-quantitative analysis

1∗

PPG em Engenharia Recursos H´ıdricos e Ambiental (PPGERHA) – UFPR. E-mail: carolkozak05@gmail.com

2Programa de P ´os-Graduac¸˜ao em Geologia – UFPR. E-mail: aluana.schleder@gmail.com

3Programa de P ´os-Graduac¸˜ao em Engenharia Ambiental – UFPR. E-mail: EduardoGaloski@hotmail.com

4Doutora em Engenharia de Recursos H´ıdricos e Ambiental - UFPR. E-mail: juliane.rizzi@gmail.com

5Departamento de Engenharia Ambiental – UFPR. E-mail: froehner@ufpr.br

6

INTRODUCTION

Biological molecular markers, or biomarkers, are complex molecules derived from organic compounds. Such compounds are inserted into the environment either by the decay of living organisms or via anthropic activities. Since these molecules are resistant to degradation and persist in the environment, they can be used in the qualitative or quantitative identification of organic matter sources (Netto et al. 2000). Biomarkers can be classified into three main groups: (i) contemporary biogenic markers, which are generally those synthesized by living organisms; (ii) fossil biomarkers, which are biomolecules transformed through diagenetic and/or catagenetic processes; (iii) anthropogenic markers, which are from human activities (Eganhouse 2004).

Marine sediments of coastal areas may function as a sink for organic matter deposited there from natural or anthropogenic sources, and become part of the global carbon cycle (Guo et al. 2007). Therefore, as the use of biomarkers contained in sediments has an important role in the identification of the organic matter origin (Lu and Zhai, 2006), this paper focuses on the study of n-alkanes, polycyclic aromatic hydrocarbons (PAHs) and fecal sterols as biomarkers.

n-Alkanes

n-Alkanes are straight-chain saturated and unbranched hydrocarbons that are mainly anthropogenic originated from fuel spills by leakage or accident. n-Alkanes can be found in natural sources such as phytoplankton, marine bacteria, biomass combustion and inthe epicuticular wax of leaves in terrestrial plants (Gogouet al. 2000).

Different ratios between n-alkane contents can be used to identify sources of pollution of petrogenic origin, and to differentiate sources of organic matter from a terrestrial or aquatic origin (Prahlet al.1994). Examplesof n-alkanes ratios are the Carbon Preference Index (CPI) and the mean chain length (ACL), which can aid to understand transport processes of organic matter in a river basin into the aquatic environment (e.g., Bray and Evans 1961; Azevedoet al. 2007).

Polycyclic aromatic hydrocarbons - PAHs

The PAHs are molecules of benzene aromatic chains that can be originated from the incomplete combustion of organic materials, such as coal, wood, and petroleum (Liu et al. 2017). Biodegradation resistance of PAHs can cause potential bioaccumulation and have mutagenic and carcinogenic effects, thus PAHs presence represents a major environ- mental concern (Kadri et al. 2017).The Environmental Protection Agency (EPA) and the CONAMA (Brazil 2011) regulate sixteen PAHs. Among them, benzo(a)anthracene, chry- sene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, dibenz(a,h)anthracene and indeno[1,2,3-cd]pyrene are considered carcinogenic substances by the EPA.

Fecal sterols

Sterols are chemical compounds of the alcohol group that can be used as molecular tracers to identify and distinguish sources of fecal pollution (e.g.; Froehner et al. 2008). In addition, sterols can be used to examine (a) the history of sewage addition to a body of water and (b) the distribution and transport of sewage into the environment, as well as the age of

the effluent (Pratt, 2005). Sterols such as dinosterol, cholesterol, campesterol, β−sitosterol, β − sitostanol, cholestanol, and stigmasterol occur in a variety of living organisms. The sterols of exclusively fecal origin are coprostanol, cholestanol, and epicoprostanol (Mudge and Bebiano 1997), being coprostanol the predominant sterol in human feces (Grimaltet al.

1990). Numerous studies have demonstrated the occurrence of coprostanol in environments contaminated with domestic sewage.

The relevance of the biomarkers studies is unquestionable; hence, the aim of this work is to verify the presence or absence of n-alkanes, PHA’s and fecal sterols in marine sediments of the Paranagua Bay (Parana, Brazil) and to relate to human activities in the area.

MATERIALS AND METHODS Study Area and Sampling

The area of study comprised the Paranagua Estuary Complex (Parana), located on southern coast of Brazil, between the coordinates 25^◦16’ and 25^◦34’S and 48^◦17 and 48^◦42’W, total area of 612 km² (Figure 1). The area was divided into two main axes: (i) East-West direction (E-W) with 56 km in length, and (ii) North-South direction (N-S) with 30 km (Rizzi et al. 2016).

Figure 1: Map of the study area and sampling sites.

Source: Adapted from Rizzi (2015)

Samples of superficial marine sediments were collected at seventeensites along the two main axes of the estuary, figure 1. The sites P1 to P13 are located in the E-Waxewith a higher degree of human activity due to the activities of Antonina and Paranagua ports, and the nearby of Paranagua City. The sites P14 to P17 are located in the N-S axe and show a low

level of urbanization,an absence of industrial areas and a lack of shipping vessels traffic.

The collected sediments sampling were stored in aluminum foil and then frozen at –20^◦C.

Finally,the samples were lyophilized and frozen until their further analysis.

Determination of n-alkanes, polycyclic aromatic hydrocarbons and fecal sterols

Briefly, 2 g of dry sediment was sonicated for 40 min with 10 mL of dichloromethane:

methanol (2:1 v/v). Then, the samples were centrifuged and the supernatant was removed and collected in a flask. This process was repeated four times. The extracts were reduced to 1 mL. Sulfur removal was carried out adding copper bars, immersed for 24 h. Later on, the concentrate was transferred to a vial and dried at 40^◦C under a gentle stream of nitrogen gas. Later, the samples were re-dissolved with 1.000µLof n-hexane. A fractionation column was prepared using a Pasteur pipette, filled with silica and alumina, activated at 120^◦C (24 h), and anhydrous sodium sulfate, dried at 120^◦C, 24 h. After conditioning the column, the sample was placed and eluted with 4 mL of n-hexane to separate the n-alkanes. Then, the PAHs were eluted with 3 mL dichloromethane:n-hexane (2:1 v/v). Finally, fecal sterols were eluted with 3 mL of ethyl acetate:methanol (2:1 v/v). The fraction of fecal sterols, previously dried, was silylated with 100 µL of bis(trimethylsilyl) trifluoroacetamide, reacting for 1 h at 70^◦C. Lastly, each fraction was dried and re-dissolved with 100µL of n-hexane for their determination by GC/MS.

Following specific methods of analysis for each group of biomarkers, the samples were injected into a gas chromatograph (Varian 431-GC) coupled to a mass spectrometer (Varian 220-MS), with a ZB-5ms capillary column (30 m, 0,25 mm, 0,25µm ). The n-alkanes C8 to C36, including pristane and phytane, the sixteen PAHs regulated by the American Environmental Agency (EPA) and eight sterols, i.e. coprostanol, epicoprostanol, cholesterol, cholestanol, coprostanone, stigmasterol,β−Sitosteroland, stigmastanol were determined.

RESULTS AND DISCUSSION n-Alkanes

Table 1 exhibits the n-alkanes found in the medium and the highest concentrations for each sample site. These concentrations are relative to C25, C26, and C27. The presence of higher chain n-alkanes (C26–C35) indicates the origin of epicuticular wax from higher plants, and the medium chain n-alkanes (C20-C25) suggest the presence of n-alkanes from submerged aquatic plants (Fickenet al. 2000).

Table 1 also shows CPI values ranged from 0.89 to 3.64, the values are close to unity, indicating petrogenic origin of n-alkanes. However, sites P6 and P7, showed 2.55 and 3.64 for CPI, respectively, indicating a mixture of sources (petrogenic and biotic). Similar results were found for Sojinuet al.(2012). Those low CPI values can be accounted for large microbial activity typical of tributaries of the coastal areas, also could be attributed to degradation of the organic matter in adjacent soils (Moreira-Turcqet al. 2013).

In order to better understand the source of organic matter in sediments, it was applied the ACL index. The ACL index can discrimante petrogenic from biotic sources (Martins, 2001). According to the values showed in Table 1, the ACL values ranged from 25.37 to 27.29, pointing for petrogenic contamination. Considering the area and the intense port

Table 1: Values of the highest concentrations of n-alkanes at each sampling point (ng g⁻¹) and indices to identify the origin of n-alkanes (CPI and ACL).

Sample Site C25 C26 C27 P

Ci CPI ACL

P1 305 0,00 202 3,469 0,89 27,3

P2 2.393 1.910 2.217 16.397 1,13 26,8 P3 9.379 9.580 9.955 71.780 1,04 27 P4 2.477 1.923 2.316 16.865 1,2 26,8 P5 1.652 1.396 1.526 9.888 1,06 26,5 P6 1.686 195 1.528 11.616 2,55 27,1

P7 716 60,4 389 2.885 3,64 25,9

P8 774 121 385 3.676 1,78 25,4

P9 6.253 5.698 5.418 39.594 1,04 26,4

P10 0,00 0,00 0,00 8 NA NA

P11 571 259 431 2.889 1,4 26,1

P12 2.286 1.930 1.631 12.449 0,97 25,8 P13 3.323 3.119 3.205 21.562 1,04 26,6

P14 576 265 335 2.919 1,19 25,6

P15 704 456 432 3557 1,05 25,7

P16 3.707 3.258 3.005 20.234 1,03 26,1

P17 172 0,00 8,07 948 1,09 25,5

PCi =Sum from C8 to C36; CPI: Carbon Preference Index; ACL: Average Chain Lenght;

CPI= ¹₂(^C_C¹⁷₁₆^+C_+C¹⁹₁₈^+C_+C²¹₂₀^+C_+C²³₂₂^+C_+C²⁵₂₄^+C_+C²⁷₂₆^+C_+C²⁹₂₈^+C_+C³¹₃₀^+C_+C³³₃₂ + ^C_C¹⁷₁₈^+C_+C¹⁹₂₀^+C_+C²¹₂₂^+C_+C²³₂₄^+C_+C²⁵₂₆^+C_+C²⁷₂₈^+C_+C²⁹₃₀^+C_+C³¹₃₂^+C_+C³³₃₄) ACL= ^(23.C23+25.C_C23+²⁵_C^+27.C_25+C²⁷₂₇^+29.C_+C29+²⁹_C^+31.C_31+C33^31+33.C33)

activities, this scenaryo was expected as observed in previous work (Martins, 2001; Rizzi, 2015). Also, this area suffers from discharge of untreated sewage. Biotic contribution could be from erosion (Prahlet al. 1994). It is not discharged the possibility of erosion represented by n-alkanes from biotic source.

Polycyclic aromatic hydrocarbons

The distribution of total PAHs, such as those of heavy and light molecular weight in the marine sediment, is shown in Table 2. The highest value of total concentrations of PAHs were found in the sediments of sites P4 (49.150 ng g^–1) and P7 (12.907 ng g^–1). In contrast, the lowest values were found at sites P15 (129 ng g^–1) and P17 (1.255 ng g^–1). Anthracene (A) (P4, 2.683 ng g^–1), benzo[a]anthracene (P4, 219 ng g^–1), fluoranthene (FA) (P4, 7.292 ng g^–1), naphthalene (NA) (P7, 1.225 ng g^–1), phenanthrene (P4, 13.710 ng g^–1), pyrene (PY) (P4, 9.286 ng g^–1), indeno [1,2,3-cd] pyrene (P7, 2.444 ng g^–1) were the predominant compounds. These values exceed those established by CONAMA 344/2004 (Brazil, 2004); thus, the sediments could be classified as polluted. Site P4 is located near the port of Paranagua, with intense navigation of cargo ship, and Paranagua city, where there is a booming urban expansion, which could be responsible for high values of PAHs concentrations. However, sites P15 and P17 are located in a region of the estuary with little anthropic interference and hold

Table 2: Distribution of PAHs in marine sediment (ng g^–1of dry sediment) Sample Site P

Total P

LMW P

HMW _IP+^IP_{BghiP BaA}^BaA_{+CHR AN}^AN_{+PHE HMW}^LMW _FL^FL_+PY

P1 1.674 1.674 - - - 0,62 - -

P2 1.434 1.378 - - - 0,84 - -

P3 2.226 1.848 378 - - 0,60 4,89 -

P4 49.150 37.910 11.240 0,48 0,42 0,08 3,37 0,00

P5 3.950 3.483 467 0,53 0,37 0,38 7,46 -

P6 3.918 2.931 987 0,43 0,18 0,50 2,97 -

P7 12.907 8.715 4.192 0,76 0,37 0,27 2,08 0,27

P8 2.873 2.225 648 0,32 0,26 0,60 3,43 0,43

P9 1.721 1.650 71 - - 0,62 23,2 0,34

P10 1.364 1.364 - - - 0,82 - -

P11 4.357 4.155 202 - - 0,26 20,6 0,48

P12 1.650 1.650 - - - -

P13 5.771 4.679 1.092 0,41 0,13 0,28 4,28 -

P14 1.558 1.488 70 - - 0,98 21,3 -

P15 129 129 - - - -

P16 7.214 7.038 176 - - 0,72 40,0 -

P17 1.255 1.219 36 - - 0,87 33,6 -

PTotal,P

LMW andP

HMW in (ng g^–1)

sediments that are predominantly composed of sand (Rizzi 2015).

PAHs commonly have two major sources: pyrogenic or petrogenic (Yunker 2002).

The identification of PAH sources can be interpreted by ratios among compounds. For instance, lower molecular weight compounds (LMW) are abundant in petroleum, whilst higher molecular weight compounds (HMW) have pyrogenic origin. Hence, HMW/LMW>1 values suggests a pyrogenic origin, whereas HMW/LMW < 1 denotes a petrogenic origin (Barakatet al. 2011). In this study HMW/LMW ratio values were above 1; therefore, they suggest a petrogenic origin mainly.

Other ratios are helpful such as AN/(AN+PHE), IP/(IP+BghiP) and BaA/(BaA+CHR).

The FL/(FL+PY) ratio shows that, in most of the sediments, the PAHs have a pyrogenic ori- gin (combustion of petroleum derivatives), except site P9 (0.34), which source is petrogenic.

The petrogenic origin of PAHs in this site was also confirmed by the AN/(AN+PHE) and IP/(IP+BghiP) ratios. However, the BaA/(BaA+CHR) ratio shows a mixture of sources, i.e., pyrogenic and petrogenic (Maioliet al. 2012).

Fecal sterols

For sterols, presence (+) or absence (–) was determined at this stage of the present work.

Recognizable sterols from plant origin, i.e. stigmasterol,β-sitosterol and stigmasterol, were found in almost all samples. Fecal sterol coprostanol, epicoprostanol and cholestanol were present mainly in those sites near urban areas and port activities, i.e. sites P1 to P13 (Table 3).

Epicoprostanol is formed by diagenic process in sediments and sewage treatment. Normally

Table 3: Presence of sterols in sample sites, in italic those of fecal origin.

Compound P1 P3 P5 P6 P7 P8 P10 P11 P12 P13 P14 P15 P17

Coprostanol + + + - + - - - -

Epicoprostanol - + - - + + - - + + - - -

Cholesterol - + - + + + + + + + + + -

Cholestanol - + - + - + - + + + + + -

Coprostanone - + - + + + + + + + + + +

Stigmasterol - + - + + – + - + + + + +

β-Sitosterol - + + + + + + + + + + + +

Stigmastanol - + + + + + + + + + + + +

the ratio coprostanol and epicoprostanol is used to know if the sewage is treated or not.

Interestingly, in the present work, in some sites, epicoprostanol was only present without coprostanol, sites P8, P12 and P13. Such observation can be associate with old sewage.

CONCLUSION

The presence of n-alkanes, PAHs and fecal sterols confirmed the main source of con- tamination is oil and ilegal sewage inputs. The epicoprostanol and coprostanol ratio also confirm that sewage presented in sediments were untreated. Finally, the n-alkanes revealed two sources for organic matter: oil derivatives, due the port activities and biotic source, possibly from erosion process.

ACKNOWLEDGEMENTS

The authors thank to the Coordination of Personal Improvement of Higher Education (CAPES), the Araucaria Foundation for Support to Scientific and Technological Develop- ment, and the National Council for Scientific and Technological Development (CNPq) for the financial assistance to the Graduate Programs in Water Resources Engineering and Environ- mental Engineering (PPGERHA). The authors also thank to the Laboratory of Hydrogeology Research (LPH) of the Geology Department for the help with the analysis, and the Federal University of Parana for technical and intellectual support.

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