n-Alkanes and stable C, N isotopic compositions as identifiers of organic matter sources in Posidonia oceanica meadows of Alexandroupolis Gulf, NE Greece

Marine Pollution Bulletin 99 (2015) 346–355

Contents lists available at ScienceDirect

Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Baseline

n-Alkanes and stable C, N isotopic compositions as identifiers of organic matter sources in Posidonia oceanica meadows of Alexandroupolis Gulf, NE Greece Maria-Venetia Apostolopoulou a,⇑, Els Monteyne b, Konstantinos Krikonis c, Kosmas Pavlopoulos d, Patrick Roose b, Frank Dehairs a a

Analytical, Environmental and Geo-Chemistry and Earth System Sciences Research Group, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Royal Belgian Institute of Natural Sciences, Operational Directorate Natural Environments, ECOCHEM, 3e & 23e Linieregimentsplein, B-8400 Ostend, Belgium c Freelance Statistical and Research Consultant, DatAnalysis, Ioannina, Greece d Department of Geography and Planning, Paris-Sorbonne University Abu Dhabi, P.O. Box 38044, Al Reem Island, United Arab Emirates b

a r t i c l e

i n f o

Article history: Received 2 March 2015 Revised 12 July 2015 Accepted 14 July 2015 Available online 17 July 2015 Keywords: n-Alkanes Posidonia oceanica Carbon and nitrogen stable isotopes Mediterranean Sea Aegean Sea Alexandroupolis Gulf

a b s t r a c t We analyzed n-alkane contents and their stable carbon isotope composition, as well as the carbon and nitrogen isotope composition (d13C, d15N) of sediment organic matter and different tissues of Posidonia oceanica seagrass sampled in Alexandroupolis Gulf (A.G.), north-eastern Greece, during 2007–2011. n-Alkane contents in P. oceanica and in sediments showed similar temporal trends, but relative to bulk organic carbon content, n-alkanes were much more enriched in sediments compared to seagrass tissue. Individual n-alkanes in sediments had similar values than seagrass roots and rhizomes and were more depleted in 13C compared to seagrass leaves and sheaths, with d13C values ranging from 35‰ to 28‰ and from 25‰ to 20‰, respectively. n-Alkane indexes such as the Carbon Preference Index, carbon number maximum, and n-alkane proxy 1 (C23 + C25/C23 + C25 + C29 + C31) indicate strong inputs of terrestrial organic matter, while the presence of unresolved complex mixtures suggests potential oil pollution in some sampled areas. Ó 2015 Elsevier Ltd. All rights reserved.

n-Alkanes are aliphatic hydrocarbons that are well-known long-term biomarkers useful in identifying the origin, source, and fate of organic matter in the environment (Hostettler et al., 1999; Fahl and Stein, 1999; Jansen et al., 2010). They can be both biogenic and anthropogenic in origin, with source-characteristic distribution patterns. For example, terrestrial plants show n-alkane distributions with maximum contributions (Cmax) of nC25 to nC31 compounds (Clarck and Blumer, 1967; Cranwell, 1973; Nishimura and Baker, 1986; Zaghden et al., 2007; Sonibari and Sojinu, 2009), while freshwater macrophytes typically have a Cmax of nC23 and nC25 compounds. Posidonia oceanica seagrasses have their long-chain n-alkanes dominated by nC21 or nC25 compounds, while their short-chain n-alkanes are dominated by nC15 and nC19 compounds (Viso et al., 1993). Planktonic organisms, such as cyanobacteria and algae, are characterized by maximum contributions of nC15 to nC19

⇑ Corresponding author. E-mail addresses: [email protected] (M.-V. Apostolopoulou), [email protected] (E. Monteyne), [email protected] (K. Krikonis), [email protected] (K. Pavlopoulos), [email protected] (P. Roose), fdehairs@vub. ac.be (F. Dehairs). http://dx.doi.org/10.1016/j.marpolbul.2015.07.033 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.

compounds, with odd-carbon-number alkanes predominating (Clarck and Blumer, 1967; Gogou et al., 2000; Bourbonniere et al., 1997; Meyers and Ishiwatari, 1993). The even nC12 to nC22 alkanes are attributed to bacterial presence and also petroleum residues (Heemken et al., 2000; Gogou et al., 2000; Salas et al., 2006; Gao et al., 2007). Cmax for the nC12, nC14, nC16, nC18, and nC20 n-alkanes are thus considered to indicate petrogenic pollution (Han et al., 1967; Grimalt and Albaiges, 1987; Duan, 2000; Garg and Bhosle, 2004). n-Alkanes in surface sediments have been widely used to detect the presence of refinery products such as crude oil, diesel oil, and higher plant waxes (e.g., Scholz-Böttcher et al., 2008). The presence of unresolved complex mixtures (UCMs; Volkman et al., 1992; Gogou et al., 2000) and alkane indexes (Bouloubassi et al., 2001; Gao et al., 2007) are used to determine n-alkane sources (Table 1). In addition to aliphatic hydrocarbons, the total organic carbon (TOC) content and stable carbon isotope composition (d13C) can be useful in determining the sources and sinks of organic matter (e.g. Hu et al., 2006, 2009). Also the C:N atom ratio is useful as an indicator of organic matter sources. For fresh marine organic matter, this ratio typically ranges between 4 and 10, while higher

347

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355 Table 1 n_Alkane indexes for source identification. Indexes

Petroleum pollution

Other sources

References

Carbon Preference Index CPI = 2x[Rodd(nC22 nC33)]/ Reven(nC24 nC32) + Reven(nC26 nC34) n-Alkane-based proxy 1 Paq 1 = ([nC23] + [nC25])/ (nC23] + [nC25] + [nC29 + nC31])

1

5–10 terrestrial origin

Gogou et al. (2000), Jeng (2006), Heemken et al. (2000) Mead et al. (2005)

1 emergent macrophytes/ terrestrial plants >1 submerged/floating species 1 Emergent macrophytes/ terrestrial plants >1 microbial organisms

n-Alkane-based proxy 2 Paq 2 = ([nC16] + [nC17])/ ([nC16 + [nnC17] + [nC29] + [nC31]) UCM/n-alkane

Positive values

Short chain/long chain n-alkane ratio

1

n-Alkane/C16 C-17/pristane

<15

C18/phytane

<1 presence of degraded hydrocarbon >1 freshly derived hydrocarbons

Terrigenous to aquatic n-alkanes (TAR = (nC27 + nC29 + nC31)/(nC15 + nC17 + nC19)

>1 algae and plankton influence <1 sedimentary bacteria and higher plants >15 <1 presence of degraded hydrocarbon >1 freshly derived hydrocarbons

Viet (2006)

Bouloubassi et al. (2001), Gao et al. (2007) Clarck and Blumer (1967)

Gao et al. (2007) Blumer et al. (1963), Gao et al. (2007)

Gao et al. (2007), Wang et al. (1999)

>1 terrestrial input

Guo et al. (2011)

<1 algal input

Fig. 1. Study area: (A) pristine site (extensive P. oceanica meadows); (B) flood-prone site (extensive P. oceanica meadows); (C) Alexandroupolis Port; and (D) Evros River (P. oceanica meadows are absent here).

– – nC18 278 ± 53.1 nC23 79 ± 13.9 – – nC22 242.2 ± 54 nC16 178 ± 24.7 nC18 18 ± 9.1 nC20 ND nC29 148.8 ± 94.6 nC23 98.3 nC18 15.2 ± 8.4 nC19 96.2 ± 11.6 nC29 113.4 ± 14.6 nC19 42.3 ± 9 nC18 14.3 ± 2.1

– – – – – – – 2.1 ± 0.9 0.5 ± 0.2 0.6 ± 0.3 3.4 ± 4.2 2.7 ± 1.8 1.3 ± 0.7 ND 4.6 ± 4.9 0.4 ± 0.2 0.4 ± 0.3 2.8 ± 2.1 1.9 ± 0.8 1.4 ± 0.5 ND – – – – – – – 2.1 ± 1.2 0.5 ± 0.4 0.6 ± 0.4 4.2 ± 3.7 2.6 ± 1.9 1.2 ± 0.9 ND 1.7 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 11.2 ± 1.2 1.3 ± 0.2 0.7 ± 0.1 2.1 ± 0.7 1.9 0.6 0.6 1.6 8.3 1.9 0.7 1.5 1.0 1.0 1.6 1.2 1.8 1.5 17.7 ± 12.2 0.3 ± 0.4 0.5 ± 0.3 1.4 ± 0.6 0.1 ± 0.1 2.1 ± 0.8 ND 1.0 0.8 0.9 10.6 1.3 0.8 0.1 1.6 ± 0.6 0.7 ± 0.2 0.8 ± 0.3 1.6 ± 0.2 5.9 ± 7.2 1.8 ± 0.2 1.6 0.9 ± 0.8 1.0 ± 0.0 1.0 ± 0.0 1.6 ± 0.1 0.6 ± 0.0 1.8 ± 0.1 ND 1.1 ± 0.1 1.0 ± 0.0 1.0 ± 0.0 1.7 ± 0.1 1.8 ± 2.1 1.8 ± 0.1 ND 2.0 0.8 0.9 11.2 1.3 0.8 5.4 1.1 0.7 0.8 1.4 13.4 2.0 ND

ND – 6.2

D3 D3

2010 1.0 0.1 19.4 ± 1.5 0.3 10.2 8.7 ± 6.0 2010 2.1 0.3 22.7 6.4 9.4 8.2 ± 10.4

D2 D1

2011 1.7 ± 0.6 0.2 ± 0.1 21.1 ± 0.8 6.6 ± 1.8 10.4 ± 0.7 – 2010 2.8 0.3 25.0 ± 0.8 7.6 12.0 2.8 ± 2.9

D1 C

2011 1.3 ± 1.3 0.2 ± 0.1 20.3 ± 1.1 6.2 ± 1.6 8.9 ± 2.1 4.2 ± 1.7 2010 3.5 ± 0.9 0.3 ± 0.1 19.7 ± 0.4 3.7 ± 0.4 12.0 ± 1.6 15.9

C C

2009 – –

2007 3.2 ± 2.9 0.3 ± 0.3 23.0 ± 2.8 10.6 ± 4.2 12.8 ± 4.3 13.5 ± 8.2

C B

2011 1.6 0.2 18.7 4.9 8.0 3.6 2010 1.8 ± 0.7 0.3 ± 0.0 19.3 ± 1.9 2.9 ± 0.7 7.4 ± 3.7 13.8 ± 3.7

B B

2009 2.7 ± 1.7 0.3 ± 0.1 17.5 ± 3.6 4.1 ± 1.7 9.8 ± 4.5 6.1 ± 0.4 2007 1.2 ± 0.6 0.1 ± 0.0 19.5 ± 4.5 7.3 ± 6.8 10.9 ± 4.2 12.6 ± 0.6

B A

2011 1.4 0.2 20.3 5.4 9.3 2.0 2010 1.0 ± 0.5 0.2 ± 0.1 16.3 ± 1.7 3.4 ± 2.0 6.3 ± 2.3 20.6

A

nC19 83.2 ± 21.1

P. oceanica

nC17 52.2

2011

P. oceanica

1.2 ± 0.2 1.0 ± 0.0 1.0 ± 0.0 1.7 ± 0.1 0.7 ± 0.1 1.7 ± 0.0 ND

2010

P. oceanica

1.0 1.0 1.0 1.7 0.3 1.7 nd

2009

P. oceanica

2009 3.2 ± 1.4 0.4 ± 0.2 18.6 ± 1.4 7.2 ± 5.9 10.2 ± 3.0 9.6 ± 3.2

Sediment, Sediment Sediment, Sediment Sediment, Sediment Sediment Sediment, Sediment Sediment

A

Sample type

A, B C A, B C A, B C D1, D2, D3 A, B C D1, D2, D3

2007 1.1 ± 0.2 0.2 ± 0.1 18.1 ± 2 4.8 ± 4.9 8.7 ± 3.8 13.9

Sampling site

2007

A

Year

Year POC (mg/gdw) PN (mg/gdw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/ gdw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH /nC17 UCM/ Rn_alkanes Cmax Rn_alkanes/ nC16

Table 2 Year, location and sample types.

Site

ratios of 20 and larger indicate the presence of organic matter derived from terrestrial vascular plants (Redfield et al., 1963; Meyers, 1994), or petroleum hydrocarbons (Friligos et al., 1998; Friligos and Krasakopoulou, 2001). Stable isotope (SI) analysis has been used directly to identify sources of pollution as well as to confirm or constrain results obtained by other techniques for source identification (e.g. Schubert and Calvert, 2001; Sulzman, 2007). The d13C composition of primary producers is influenced by the source of CO2 (this sets the d13C of the substrate), the photosynthetic pathway used (C3, C4, CAM; Fry and Sherr, 1984), and also specific growth conditions (e.g. diffusion limitation). The d13C values of terrestrial C3 plants range from 22‰ to 33‰ and those of C4 plants range from 9‰ to 16‰ (see e.g. Pancost and Boot, 2004; Guo et al., 2006; Hu et al., 2009). The d13C values in seagrass range from 23‰ to 3‰, with the most frequent seagrass d13C composition around 10‰ (see e.g., Hemminga and Mateo, 1996; Dauby, 1989; Marguillier et al., 1998; Lepoint et al., 2004). The d13C values are higher in seagrass than phytoplankton, due to the ability of seagrass to use bicarbonate as an inorganic carbon source and due to a thicker diffusion boundary layer surrounding the leaves, controlled by turbulence in the water (Osmond et al., 1981; Maberly et al., 1992; Keeley and Sandquist, 1992). Furthermore, the d13C values of seagrasses are often related to depth and may vary with season, location, and community structure (Lepoint et al., 2004). In a previous study we used P. oceanica seagrass as a bioindicator for spatiotemporal trends of polycyclic aromatic hydrocarbons (PAH) in the Mediterranean Sea, and seagrass leaves were found suitable to represent the PAH pollution status of seawater (Apostolopoulou et al., 2014). In the present study, we analyze n-alkanes and stable C & N isotopic composition to evaluate the potential of P. oceanica seagrasses as bio-indicators for aliphatic hydrocarbons. Therefore, we assessed the composition and sources of n-alkanes sediments and different tissues of the seagrass P. oceanica from the North East Aegean Sea and Evros River. The study sites in the coastal waters of Alexandroupolis Gulf and in Evros River are presented in Fig. 1 and in the Supplementary Material 1. The sampling is detailed in Table 2 and the treatment of samples in Supplementary Material 1. Samples were Soxhlet-extracted with hexane/acetone (3:1) and, to verify the efficacy of the extraction procedure, some samples were subjected to pressurized liquid extraction (Dionex Thermo Scientific ASE 200; see Supplementary Material 2). n-Alkanes were measured with a GC system coupled to an ion trap mass spectrometer (Thermoquest Trace GC–MS, Thermo Finnigan, Austin, US) while a GC-C-isotope ratio mass spectrometer (IRMS; Thermo Delta V) was used for d13C analyses of the n-alkanes (see Supplementary Material 3). An elemental analyzer (Flash 1112, Thermo) connected to an IRMS system (Delta V, Thermo) via a ConFlo III interface was used for determining the TOC, TN contents and d13C, d15N compositions of bulk organic matter (see Supplementary Material 4).

2011 1.7 ± 0.5 0.2 ± 0.1 19.9 ± 1.0 7.2 ± 1.3 9.8 ± 0.3 –

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355 Table 3 Average values for particulate organic carbon (POC), particulate nitrogen (PN), C: N atom ratio, carbon and nitrogen isotopic composition, and n-alkane indexes for sediments of Alexandroupolis Gulf (A, B, C locations) and Evros River (D1, D2, D3 locations) for 2007, 2009, 2010 and 2011 (ND: not detected, – means no data).

348

349

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355

Table 4 Average values for particulate organic carbon (POC), particulate nitrogen (PN), C: N atom ratio, carbon and nitrogen isotopic composition, and n-alkane indexes at site A for leaves, sheaths, roots, and rhizomes (nd: not detected, – means no data). A

Leaves

Sheaths

Roots

Rhizomes

2007 POC (mg g 1 dw) PN (mg g 1 dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

319.1 ± 27.1 8 ± 1.8 14.2 ± 1 3 ± 0.2 46.6 ± 8.6 – – – – – – – – – –

611.5 9.4 14.1 15.9 75.7 2.2 2.7 1.0 1.0 1.3 – 0.8 ND nC17 –

219.7 ± 92.9 5 ± 2.6 13.6 ± 0.7 1.7 ± 0.2 53.2 ± 6.5 – – – – – – – – – –

429.7 ± 7.3 6.5 ± 0.8 13.2 ± 0.1 2.9 ± 0.9 78 ± 8.3 118.7 3.8 1.0 0.8 7.8 2.8 0.2 ND nC19 98.2

2009 POC (mg g 1 dw) PN (mg g 1 dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

294.6 ± 29.0 13.2 ± 1.7 13.2 ± 1 5.2 ± 2.7 26.4 ± 4.6 34.6 ± 14.5 3.6 ± 2.5 0.8 ± 0.1 0.9 ± 0.1 0.5 ± 0.2 1.6 ± 1.8 2.0 ± 0.5 ND nC19 116.5 ± 33.1

543.2 21.3 12.6 18.1 29.8 2.8 8.1 1.0 1.0 0.1 – 9.1 ND nC21 –

– – – – – 4.5 1.0 0.7 0.8 – – – ND nC17 16.0

375.7 ± 87.4 19 ± 10.4 13.2 ± 0.8 5.1 ± 1.9 34.2 ± 31.6 29.7 7.8 0.9 0.9 – – – ND nC19 162.2

2010 POC (mg g 1 dw) PN (mg g 1 dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

281.7 ± 21.4 20.7 ± 10.6 14.9 ± 0.3 5.7 ± 0.7 21.1 ± 6.7 9.3 ± 2.1 1.2 ± 0.1 0.9 ± 0.1 1.0 ± 0.1 2.0 ± 0.1 1.2 ± 0.0 1.3 ± 0.0 ND nC19 13.2 ± 3.0

398.8 6.8 14.6 13.8 68.6 80.5 ± 22.6 – – – – – – – – –

188.2 12.2 13.8 9.3 18.0 77.8 1.2 0.9 1.0 1.6 0.7 1.8 ND nC17 10.1

382.6 ± 6.9 12.77 ± 3.27 14.3 ± 0.4 12.1 ± 3.8 27.8 ± 19.4 80.5 ± 22.6 0.7 ± 0.7 1.0 ± 0.1 1.0 ± 0.0 1.0 ± 0.7 1.8 ± 0.7 2.5 ± 1.3 ND nC19 9.8 ± 0.6

2011 POC (mg g 1 dw) PN (mg g 1 dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

344.8 6.0 14.2 14.4 66.6 44.8 ± 26.9 4.8 ± 2.5 0.9 ± 0.0 0.9 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 8.3 ± 4.2 ND nC19 102.3 ± 35.4

761.6 13.2 13.3 14.7 67.1 50.1 11.2 1.0 0.8 2.7 1.3 0.4 ND nC17 346.8

152.4 18.6 15.2 11.7 29.7 1.5 2.1 0.6 0.7 2.0 – 0.5 ND nC17 24.5

792 18.6 12.6 11.7 49.7 19.3 5.3 0.9 0.4 – – – ND nC19 195.2

For n-alkane identification, 10 certified n-alkane standard mixtures (nC8 to nC40; AccuStandard, Inc.; certified error of ±0.5%) were analyzed during each GC–MS run, and 5 standard mixtures during each GC-C-IRMS run. The precision and accuracy of the extraction methods were evaluated from GC–MS and GC-C-IRMS measurement of five n-alkane standards, and by repeated analyses of collected

samples. The obtained recoveries varied from 98% to 120% for the GC–MS and GC-C-IRMS systems. Reproducibility of the results was checked by comparing the results for the n-alkane standard mixtures analyzed by both GC–MS and GC-C-IRMS. Here, reproducibility refers to the level of precision among different instruments in different labs (the MUMM laboratory and the Department of Earth and

350

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355

Table 5 n Average values for particulate organic carbon (POC), particulate nitrogen (PN), C: N atom ratio, carbon and nitrogen isotopic composition, and n-alkane indexes at site B for leaves, sheaths, roots, and rhizomes (nd: not detected, – means no data). B

Leaves

Sheaths

Roots

Rhizomes

2007 POC (mg g 1 dw) PN (mg g 1 dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

255.6 ± 83.8 9.3 ± 2.4 11.4 ± 2.5 4.3 ± 0.9 31.9 ± 7.9 51.8 ± 15.9 6.4 ± 0.1 0.9 ± 0.0 0.8 ± 0.0 0.3 ± 0.3 1.2 ± 0.1 5.7 ± 5.2 ND nC19 155.8 ± 114.6

484.2 5.5 14.5 19.8 104 – – – – – – – – – –

– – – – – – – – – – – – – – –

256.9 ± 93.4 6.6 ± 1.9 11.5 ± 2.6 4.7 ± 2 45.6 ± 9.4 – – – – – – – – – –

2009 POC (mg g 1 dw) PN (mg g 1 dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

386.6 ± 179.3 10.8 ± 5.1 11.8 ± 1.4 7.6 ± 3 44.2 ± 13.8 – – – – – – – – – –

428.1 6.1 13.2 17.2 81.3 15.4 ± 6.5 5.3 ± 2.6 1.0 ± 0.1 0.8 ± 0.1 1.2 ± 1.3 1.6 ± 0.0 1.7 ± 1.7 ND nC19 106.4 ± 110.9

– – – – – – – – – – – – – – –

389.9 ± 70.2 8.3 ± 1.8 12.2 ± 1.3 5.7 ± 1 57.7 ± 20.2 0.9 1.6 0.8 0.3 0.7 – 1.4 ND – –

2010 POC (mg/g dw) PN(mg/g dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

221.7 ± 19 11.6 ± 6.8 13.9 ± 0.6 6.2 ± 0.5 40.7 ± 43.9 235.7 ± 9.1 0.9 ± 0.1 0.9 ± 0.1 0.8 ± 0.4 5.5 ± 1.2 1.9 ± 0.1 0.7 ± 0.3 ND nC19 35.1 ± 22.1

234.5 5.5 14.2 12.8 49.8 – – – – – – – – – –

368.31.±31.1 10.1 ± 0.4 12.7 ± 0.3 10.8 ± 0.9 42.6 ± 5.6 140.3 1.6 0.9 1.0 1.9 1.2 2.1 ND nC21 12.3

349.2 ± 122.3 15.1 ± 3.3 13.2 ± 0.4 6.8 ± 0.5 26 ± 13.2 13.5 ± 6.0 2.0 ± 0.5 0.9 ± 0.2 1.0 ± 0.1 2.6 ± 1.1 3.1 ± 3.3 1.3 ± 0.4 ND nC19 12.2 ± 7.1

2011 POC (mg/g dw) PN (mg/g dw) d13C (‰) d15N (‰) C/N (atom ratio) Rn_alkanes (lg/g dw) CPI PAQ1 PAQ2 nC17/PR nC18/PH PR + PH/nC17 UCM/Rn_alkanes Cmax Rn_alkanes/nC16

157.7 4.1 13.8 10.5 44.8 8.3 5.8 0.8 0.8 0.1 0.2 11.3 ND nC19 114.5

1157.1 41.6 14.7 11 32.5 50.5 11.2 0.9 0.6 2.9 6.2 0.4 ND nC19 409.6

– – – – – 7.3 ± 3.8 2.2 ± 2.8 0.6 ± 0.2 0.7 ± 0.1 1.3 ± 1.3 0.6 ± 0.0 2.5 ± 2.9 ND nC19 50.5 ± 65.9

1246.4 23.6 13.5 12.7 61.5 19.4 4.8 0.9 0.4 0.9 3.3 1.2 ND nC17 185.2

Environmental Sciences, KUL). The relative standard deviation (RSD) values were below 0.5% for all concentrations (Supplementary Material 5). To define the limit of detection (LOD), blank samples were subjected to the same analytical protocols as field samples. The concentrations of eight compounds that showed discrete peaks in the blank samples were calculated (nC13, nC21, nC27, nC29, nC31,

nC33, and pristane), revealing that n-alkane concentrations smaller than 18.11 ng g 1 cannot be considered valid. Analysis of the results was performed using IBM SPSS (for Windows v. 20) statistic software (IBM, New York, USA). The values of POC, PN, C:N atom ratio, carbon and nitrogen isotopic composition and n-alkane analysis are presented in Table 3

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355

Fig. 2. Temporal trends of median nC15–nC35 n-alkane concentrations (ng g 1 dw) in sediments from all 3 coastal sites (A–C). The box and whisker diagram illustrates the spread of a set of the data.

for bulk marine and river sediments and in Tables 4 and 5 for P. oceanica tissues at sites A and B, respectively. For marine sediments at sites A, B, and C, median nC15–nC35 n-alkane concentrations (not including Unresolved Complex Mixture compounds which were observed mainly in 2010 and 2011) were higher in 2007 and 2010 (Fig. 2) compared to 2009 and 2011 with differences between years being significant (p 6 0.04) (see Supplementary Material 6).

351

A similar pattern is observed when n-alkane concentrations are normalized to sediment POC content, i.e., median normalized nC15–nC35 concentrations were generally higher in 2007 and 2010, than in 2009 and 2011 (Fig. 3), indicating that in 2007 and 2010 there was a marked organic input in the area. However, variations between sites A (p = 0.49), B (p = 0.62) and C (p = 0.31) for the different years were not significant (see Supplementary Material 6, Table 12). The median nC15–nC35 n-alkane concentration relative to POC in river sediments for 2010 and 2011 increases downstream from D1 over D2 to D3 (Fig. 3d), but the differences between D1 and D2 and between D2 and D3 are not statistically significant (p > 0.05), while they are between D1 and D3 (p = 0.003; Supplementary Material 6). For marine sediments the Carbon Preference Index (CPI, see Table 1) which is used to determine the degree of biogenic versus petrogenic input (Gogou et al., 2000; Jeng, 2006; Heemken et al., 2000) ranged from 0.9 ± 0.8 to 2 for all sites and years, except for site C in 2007, which had very high CPI values (17.7 ± 12.2, Table 3). CPI values larger than 5 imply that the n-alkane concentrations at site C in 2007 can be attributed to terrigenous material. Sediment CPI values are in agreement with the trends seen for n-alkane Cmax values. For most sediment samples at site C in 2007 Cmax is associated with nC29 alkanes and the n-alkane-based proxy 1 (Paq 1; Table 1) value < 0.3, revealing

Fig. 3. Temporal trends of median nC15–nC35 n-alkane concentrations relative to POC content (ng g 1 POC) in sediments from coastal sites A (a), B (b), and C (c); spatial trends in Evros River sediments at sites D1 to D3 (d) (note that for Evros River there are only data for 2010 and 2011).

352

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355

significant terrestrial influence at this site. The nC18 alkane is dominant for all marine sediments from 2010 (Table 3), indicating abundance of petroleum hydrocarbons (Readman et al., 1987; Venkatesan et al., 1980; Bouloubassi et al., 2001; Gao et al., 2007). Furthermore, the nC18/phytane ratios in marine sediments for 2010 (>5.9) and the nC17/pristane for 2011 (>10.6) are high for all sites (Table 3), indicating an influx of freshly supplied petroleum hydrocarbons (Gao et al., 2007). The CPI values for riverine sediments range from 2.1 ± 0.9 to 4.6 ± 4.9 (Table 3). The TAR ratio (see Table 1) was introduced to better resolve any terrestrial influence in sediments at the three riverine sites (D1, D2, D3). All locations exhibited high TAR values (>14.7) in 2010, which is an indication of strong terrestrial influences. For these samples, the n-alkane-based proxies 1 and 2 (Paq 1 and Paq 2; see Table 1) are <1, confirming a terrestrial influence. The Rn-alkanes/nC16 ratio is generally >15, except for a few samples, suggesting that the river is not a major route for petrogenic pollutants to the coastal area. This is reasonable since there is little boating activity in Evros River. For all samples, nC17/pristane and nC18/phytane ratios are >1, reinforcing this assumption (Table 3). P. oceanica n-alkane contents show a similar temporal trend as for marine sediments, with increased levels for 2007 and 2010 (compare Figs. 2 and 3 for sediments and Fig. 4 for P. oceanica whole plant). Seagrass median nC15–nC35/POC ratios at site B, next to the more polluted site C, tend to be larger than at site A, though differences are not statistically significant (p > 0.5; see Supplementary Material 6). Comparing Figs. 3 and 4 it appears that n-alkane contents expressed relative to POC content are much higher in sediments than in seagrasses. In addition to being subject to anthropogenic inputs, alkanes are more resistant to degradation (especially the shorter-chain alkanes) compared to bulk sedimentary organic matter (Versteegh et al., 2010), possibly resulting in higher alkane/POC ratios in sediments. In addition, small (tens of ppm) additions of petroleum to the sediment cause the ratio to increase dramatically, since n-alkanes (ng/g) increase and POC (mg/g) does not (Boehm et al., 1987). In our study, the Carbon Preference Index (CPI, see Table 1) which is used to determine the degree of biogenic versus petrogenic input (Gogou et al., 2000; Jeng, 2006; Heemken et al., 2000) range widely from 0.7 ± 0.7 to 11.2 (Tables 4 and 5) but they are mostly > 1. CPI values < 1 (rhizomes, site A, 2010, Table 4 and leafs, site B, 2010, Table 5) may be an indication of petrogenic hydrocarbons presence. High levels of nC16 alkanes in seagrasses may indicate oil pollution, as reported by Botello and Mandelli (1979) for the seagrass Thalassia testudinium from a polluted site

in the Gulf of Mexico. Since biological samples have high ratios of Rn-alkanes/nC16 exceeding (>50; see e.g., Adedosu et al., 2012), ratios < 15, as observed here for marine sediment, can thus be taken to indicate petroleum contamination. For 2010, all P. oceanica tissues (one leaf sample at site B excepted) have Rn-alkanes/nC16 ratios < 15, strengthening the assumption that the hydrocarbon source in 2010 was probably oil (Tables 3 and 4). The Paq 1 values in most seagrass samples are close to 1, except in root samples where they are <1 (Paq 1 = 0.7 ± 0.1), indicating the occurrence of distinct n-alkane distributions in the different P. oceanica tissues. However, such values are in the range reported for other seagrasses by Mead et al. (2005). The highest marine sediment d15N values (from +3.3‰ to +14.8‰) were observed at site C (Table 3), which might be subject to both terrestrial and sewage input. Published d15N values for marine particulate organic matter from the Mediterranean Sea usually range from +3‰ to +12‰ with a mean value of +6‰ (Lepoint et al., 2004). d15N values larger than +12‰ are often attributed to inputs of sewage or terrestrial sources (Wada and Hattori, 1991; Muller and Voss, 1999; Maksymowska et al., 2000; Hu et al., 2006; Higgins et al., 2010). For seagrasses the observed range of d15N values was rather large, from +1.7 ± 0.2‰ for roots (site A, 2007, Table 4), to +19.8‰ (site B, 2007; Table 5) for sheaths compared to a range of 2‰ to +12‰ reported in the literature (Fourqurean et al., 1997; Anderson and Fourqurean 2003; Lepoint et al., 2004). P. oceanica sheaths shelter primary producers (multicellular photosynthetic organisms and diatoms), and animals (crustaceans, molluscs, polychaetes) with reduced mobility (Gacia et al., 2009; Personnic et al., 2014) and likely due to their trophic position and diet, the nitrogen isotopes are increased. The d13C values for bulk sedimentary organic matter in the coastal area ranged from 23.0 ± 2.8‰ (site C, 2007) to 16.3 ± 1.7‰ (site A, 2010), and from 25.0 ± 0.8‰ (D1, 2010) to 19.4 ± 1.5‰ (D3, 2010) for Evros River (Table 3). Low d13C values in the upstream parts of rivers and higher values in the downstream parts reflect predominance of terrestrial and marine organic matter, respectively (Fry and Sherr, 1984; Hu et al., 2009). The rather low sediment d13C values at site C, where seagrasses are absent, reflect significant input of terrestrial matter, probably advected from Evros River, in agreement with the n-alkane proxy indications discussed above. In contrast to sediments, P. oceanica tissues show a quite homogenous d13C composition, with an average value of 13.8 ± 0.3‰. Similar values have been reported for western Sicily (Vizzini et al., 2003), the Iberian coast and the Balearic

Fig. 4. Temporal trends of median nC15–nC35 n-alkane concentrations relative to POC content (ng g

1

POC) in bulk P. oceanica tissue from sites A (a), and B (b).

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355

353

Fig. 5. d13C signatures of individual n-alkanes for (a) for marine sediments and (b) P. oceanica tissues (whole plant).

Islands (Papadimitriou et al., 2005; Fourqurean et al., 2007). Bulk sheath material appears to have the lowest d13C values, with about a 1.5‰ offset relative to other seagrass tissues. d13C signatures of individual nC12–nC36 n-alkanes were obtained for 28 sediment samples as well as for 8 leaf, 7 rhizome, 6 sheath and 3 root samples. Fig. 5 shows the average d13C signatures of individual n-alkanes for sediments and for bulk P. oceanica tissue. The average n-alkane values for bulk seagrass material are less depleted in 13C (d13C is less negative) compared to sediments. n-Alkane d13C values varied between 32.2 and 29.1 ± 1.6‰ for marine sediments and between 33.7 ± 2.5‰ and 28.6 ± 0.1‰ for river sediments. The d13C offset between n-alkanes and bulk organic carbon varied between 4‰ and 12‰ and between 8‰ and 10‰ for marine and riverine sediments, respectively (Table 6). Such differences are in accordance with values reported in the literature (Degens, 1969; Naraoka and Ishiwatari, 1999). The

Table 6 Average d13C composition of individual n-alkanes in marine and riverine sediments (for 2007, 2009, 2010 and 2011) and in P. oceanica tissues (for all years together); d13C for sediments and bulk tissue; last column gives difference between n-alkanes and bulk d13C. Year

Site

2007

A B C A B A B C D1 D2 D3 A B D1 D3

2009 2010

2011

Tissues (for all years) Leaves A B Rhizomes A B Sheaths A B Roots A B

n-alkanes d13C

bulk OC d13C

D n-alkane-bulk (‰)

29.3 ± 0.1 29.8 ± 0.4 30.0 ± 0.4 29.9 ± 1.0 29.6 ± 1.3 30.3 ± 0.2 29.3 ± 0.3 29.1 ± 1.6 33.7 ± 2.5 30.3 ± 0.7 30.6 ± 1.8 29.5 32.2 29.3 ± 0.8 28.6 ± 0.1

17.5 ± 0.5 22.3 ± 3.7 22.5 ± 2.3 18.9 ± 1.9 18.3 ± 1.6 21.5 ± 0.8 17.3 ± 0.2 20.0 ± 2.8 24.0 ± 1.5 20.4 ± 2.0 20.6 ± 2.1 20.3 20.3 21.1 ± 0.8 20.0 ± 0.8

12.3 ± 0.0 7.5 ± 3.5 7.6 ± 2.0 11.0 ± 1.4 11.3 ± 1.2 8.8 ± 1.0 12.1 ± 0.5 9.2 ± 1.2 9.8 ± 1.1 9.9 ± 2.1 10.0 ± 0.4 9.2 11.9 8.3 ± 0.1 8.8 ± 0.6

24.2 ± 0.9 23.5 ± 0.9 29.0 ± 0.8 28.7 ± 0.6 20.9 ± 1.0 20.6 ± 0.4 28.4 28.4 ± 0.7

13.9 ± 0.6 13.3 ± 1.3 13.1 ± 0.6 13.4 ± 0.9 14.1 ± 0.5 14.7 ± 0.5 13.8 13.7 ± 0.3

10.4 ± 0.6 10.2 ± 1.4 14.7 ± 0.4 14.5 ± 1.0 7.5 ± 0.5 7.7 ± 0.1 14.6 14.7 ± 0.4

lower d13C values of n-alkanes compared to bulk organic matter are attributed to isotope fractionation during n-alkane biosynthesis (De Niro and Epstein, 1977; Naraoka and Ishiwatari, 1999). The n-alkane in P. oceanica roots and rhizomes are more depleted in 13C (more negative d13C values) than sheaths (Table 6; Fig. 6). MANOVA analysis reveals that differences are significant (at a confidence level of 95%) between leaves and rhizomes (p = 0.001), leaves and sheaths (p = 0.007), roots and rhizomes (p = 0.003), and roots and sheaths (p = 0.000) (Supplementary Material 7). From Fig. 6 it also appears that n-alkane d13C values from the different P. oceanica tissues and sediments are more depleted in 13C (lower d13C values) than corresponding bulk organic matter. The d13C offset between n-alkanes and seagrass bulk organic carbon varies between 7‰ and 14‰ (the former d13C values are more negative; Table 6). For estuarine plants, Hernandez et al. (2001) and Canuel et al. (1997) report that the difference between isotopic signatures of bulk organic carbon and n-alkanes is approximately 3–5‰, with n-alkanes always being more depleted in 13C. Though the average n-alkane d13C values for seagrass leaves and sheaths are quite different from those for sediments, with the latter having more negative d13C values, they are quite similar between rhizomes, roots and sediments (Fig. 6). The differences between sediments and leaves, sheaths may result either from the presence of terrestrial plant material (with their n-alkanes depleted in 13C) in the sediments or from n-alkane

Fig. 6. Comparison between d13C composition of bulk organic matter and individual n-alkanes for sediments and P. oceanica tissues (the line shows the regression between sediments in sites A and B and seagrass leaves and sheaths).

354

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355

biosynthesis in the seagrass leaves and sheaths (Chikaraishi and Naraoka, 2003). Since sediment n-alkane d13C values are similar (close to 30‰) between the coastal sites, independent from seagrass presence or not, it is likely that rhizomes and roots have acquired their n-alkane d13C signatures from the sediments. This study examined the n-alkane and stable C & N isotope compositions as identifiers of organic matter sources in P. oceanica meadows. Overall, n-alkane indexes and d13C composition of sedimentary organic material in Alexandroupolis Gulf indicate an influx of petroleum-derived alkanes in 2010 and 2011, while in 2007 and 2009 terrestrial material constituted the dominant contribution. n-Alkane indexes and concentrations in bulk P. oceanica tissues and sediments show a similar temporal trend, highlighting the fact that P. oceanica tissues are useful indicators of n-alkane sources in the coastal environment. Accumulation of terrestrial plant detritus and potential oil contaminants in coastal sediments or n-alkane biosynthesis in P. oceanica results into less negative average n-alkane values in d13C compared to marine sediments. The results presented here provide baseline information about P. oceanica n-alkane contents and source-related n-alkane indexes for the North East Aegean Sea.

Acknowledgments We express our gratitude to Gijs Coulier, Eveline Demeulemeester, Marijke Neyts, Daniël Saudemont, Marc Knockaert and Edwige Devreker at MUMM laboratory in Ostend, Koen Parmentier at ECOCHEM group (Ecosystems PhysicoChemistry) of the Royal Belgian Institute of Natural Sciences, as well as to Steven Bouillon and Zita Kelemen at the Department of Earth & Environmental Sciences, Katholieke Universiteit Leuven, for guidance and assistance during n-alkane isotopic analyses. We are grateful also to Natacha Brion, David Verstraeten, Michael Korntheuer, Leen Rymenans, Veronique Woule Ebongue, Virginie Riou, and Perrine Mangion, at AMGC laboratory, for assistance during laboratory work at VUB. The authors acknowledge also the Management Body of Evros Estuary for assistance during samples collection in from Evros River and the Greek Ministry of Education and Religious Affairs for offering study leave and financial support to Maria-Venetia Apostolopoulou.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.marpolbul.2015. 07.033.

References Adedosu, T.A., Sonibare, O.O., Jincai Tuo, J., Ekundayo, O., 2012. Biomarkers, carbon isotopic composition and source rock potentials of Awgu coals, middle Benue trough, Nigeria. J. Afr. Earth Sci. 66–67, 13–21. Anderson, W.T., Fourqurean, J.W., 2003. Intra- and interannual variability on seagrass carbon and nitrogen stable isotopes from south Florida, a preliminary study. Org. Geochem. 34, 185–194. Apostolopoulou, M.V., Monteyne, E., Krikonis, K., Pavlopoulos, K., Roose, P., Dehairs, F., 2014. Monitoring polycyclic aromatic hydrocarbons in the Northeast Aegean Sea using Posidonia oceanica seagrass and synthetic passive samplers. Mar. Pollut. Bull 87, 338–344. Blumer, M., Mullin, M.M., Thomas, D.W., 1963. Pristane in zooplankton. Science 140, 974. Boehm, P.D., Steinhauer, M.S., Crecelius, E.A., Neff, J., Tuckfield, C., 1987. Analysis of trace metals and hydrocarbons from OSC activities. Final Report on the Beaufort Sea Monitoring Program. Outer Continental Shelf Study MMS 87-0072. U.S. Department of Interior, Minerals Management Service, Anchorage, Alaska. Botello, A.V., Mandelli, E.F., 1979. Distribution of normal paraffins in the leaves of Thalassia testudinium from the Gulf of Mexico. Bull. Mar. Sci. 29, 436–440.

Bouloubassi, I., Fillaux, J., Saliot, A., 2001. Hydrocarbons in surface sediments from the Changjiang (Yangtze River) Estuary, East China Sea. Mar. Pollut. Bull. 42, 1335–1346. Bourbonniere, R.A., Telford, S.L., Ziolkowski, L.A., Lee, J., Evans, M.S., Meyers, P.A., 1997. Biogeochemical marker profiles in cores of dated sediments from large North American lakes. In: Eganhouse, R.P. (Ed.), Molecular Markers in Environmental Geochemistry. ACS Symposium Series, Washington D.C., pp. 133–150. Canuel, E.A., Freeman, K., Wakeham, H., Stuart, G., 1997. Isotopic compositions of lipid biomarker compounds in estuarine plants and surface sediments. Limnol. Oceanogr. 42, 1570–1583. Chikaraishi, Y., Naraoka, H., 2003. Compound-specific dD–d13C analyses of n-alkanes extracted from terrestrial and aquatic plants. Phytochemistry 63, 361–371. Clarck Jr., R.C., Blumer, M., 1967. Distribution of n-paraffins in marine organisms and sediment. Limnol. Oceanogr. 12, 79–87. Cranwell, P.A., 1973. Chain length distribution of n-alkanes from lake sediments in relation to post glacial environment change. Freshwater Biol. 3, 259–265. Dauby, P., 1989. The stable carbon isotope ratios in benthic food webs of the Gulf of Calvi, Corsica. Cont. Shelf Res. 9, 181–195. Degens, E.T., 1969. Biochemistry of stable carbon isotope. In: Eglinton, G., Murphy, M.J. (Eds.), Organic Geochemistry—Methods and Results. Springer-Verlag, pp. 304–356. DeNiro, M.J., Epstein, S., 1977. Mechanism of carbon isotope fractionation associated with lipid synthesis. Science 197, 261–263. Duan, Y., 2000. Organic geochemistry of recent marine sediments from the Nansha Sea, China. Org. Geochem. 31, 159–167. Fahl, K., Stein, R., 1999. Biomarkers as organic-carbon-source and environmental indicators in the late quaternary Arctic ocean: problems and perspectives. Mar. Chem. 63, 293–309. Fourqurean, J.W., Marba, N., Duarte, C.M., Diaz-Almela, E., Ruiz-Halpern, S., 2007. Spatial and temporal variation in the elemental and stable isotopic content of the seagrasses Posidonia oceanica and Cymodocea nodosa from the Illes Balears, Spain. Mar. Biol. 151, 219–232. Fourqurean, J.W., Moore, T.O., Fry, B., Hollibaugh, J.T., 1997. Spatial and temporal variation in C:N: P ratios, d15N, and d13C of eelgrass Zostera marina as indicators of ecosystem processes, Tomales Bay, California, USA. Mar. Ecol. Prog. Ser. 157, 147–157. Friligos, N., Krasakopoulou, E., 2001. Chemical observations in the seawater and surface sediments of the Aegean Sea. In: Proceedings of 7th International Conference on Environmental Science and Technology, Ermoupolis, Syros island, Greece – September 2001. Friligos, N., Moriki, A., Hatzianestis, J., Barbetseas, S., Sklivagou, E., Krasakopoulou, E., 1998. Chemical characteristics of the surface sediments of the Aegean Sea, 3rd MTP-MATER Workshop, Rhodos, 15–17 October 1998, pp. 208–209. Fry, B., Sherr, E.B., 1984. d13C measurements as indicators of carbon flow in marine and freshwater ecosystems. Mar. Sci. 27, 13–47. Gacia, E., Costalago, D., Prado, P., Piorno, D., Tomas, F., 2009. Mesograzers in Posidonia oceanica meadows: an update of data on gastropod-epiphyte-seagrass interactions. Bot. Mar. 52, 439–447. Gao, X., Chen, S., Xie, X., Long, A., Ma, F., 2007. Non-aromatic hydrocarbons in surface sediments near the Pearl River estuary in the South China Sea. Environ. Pollut. 148, 40–47. Garg, A., Bhosle, N.B., 2004. Abundance of macroalgal OM in biofilms: evidence from n-alkane biomarkers. Biofouling 20, 155–165. Gogou, A., Bouloubassi, I., Stephanou, E., 2000. Marine organic geochemistry of the Eastern Mediterranean: 1. Aliphatic and polyaromatic hydrocarbons in Cretan Sea surficial sediments. Mar. Chem. 68, 265–282. Grimalt, J., Albaiges, J., 1987. Sources and Occurrence of C12–C22 n-alkane distributions with even carbon number preference in sedimentary environments. Geochim. Cosmochim. Acta 51, 1379–1384. Guo, W., He, M., Yang, Z., Lin, C., Quan, X., 2011. Characteristics of petroleum hydrocarbons in surficial sediments from the Songhuajiang River (China): spatial and temporal trends. Environ. Monit. Assess. 179, 81–92. http:// dx.doi.org/10.1007/s10661-010-1720-0. Guo, Z.G., Li, J.Y., Feng, J.L., Fang, M., Yang, Z.S., 2006. Compound-specific carbon isotope compositions of individual long-chain n-alkanes in severe Asian dust episodes in the North China coast in 2002. Chin. Sci. Bull. 51, 2133–2140. Han, J., McCarthy, E., Hoeven, V., Calvin, M., Bradley, W., 1967. A preliminary report on the distribution of aliphatic hydrocarbons in algae, in bacteria, and in a recent lake sediments. Org. Geochem. Stud., II, 59, 1, 59. Heemken, O.P., Stachel, B., Theobald, N., Wenclawiak, B.M., 2000. Temporal variability of organic micropollutants in suspended particulate matter of the river Elbe at Hamburg and the River Mulde at Dessau, Germany. Arch. of Environ. Contam. Toxicol. 38, 11–31. Hemminga, M.A., Mateo, M.A., 1996. Stable carbon isotopes in seagrasses: variability in ratios and use in ecological studies. Mar. Ecol. Prog. Ser. 140, 285–298. Hernandez, M.E., Mead, R., Peralba, M.C., Jaffé, R., 2001. Origin and transport of nalkane-2-ones in a subtropical estuary: potential biomarkers for seagrassderived organic matter. Org. Geochem. 32 (1), 21–33. Higgins, M.B., Robinson, R.S., Carter, S.J., Pearson, A., 2010. Evidence from chlorine isotopes for alternating nutrient regimes in the Eastern Mediterranean Sea. Earth Planet. Sci. Lett. 290, 102–107.

M.-V. Apostolopoulou et al. / Marine Pollution Bulletin 99 (2015) 346–355 Hostettler, F.D., Pereira, W.E., Kvenvolden, Keith A., van Geen, A., Luoma, S.N., Fuller, Ch.C., Anima, R., 1999. A record of hydrocarbon input to San Francisco Bay as traced by biomarker profiles in surface sediment and sediment cores. Mar. Chem. 64, 115–127. Hu, J., Peng, P., Jia, G., Mai, B., Zhang, G., 2006. Distribution and sources of organic carbon, nitrogen and their isotopes in sediments of the subtropical Pearl River estuary and adjacent shelf, Southern China. Mar. Chem. 98 (2–4), 274–285. Hu, L., Guo, Z., Feng, J., Yang, Z., Fang, M., 2009. Distributions and sources of bulk organic matter and aliphatic hydrocarbons in surface sediments of the Bohai Sea, China. Mar. Chem. 113, 197–211. Jansen, B.E., van Loon, E., Hooghiemstra, H., Verstraten, J.M., 2010. Improved reconstruction of palaeo-environments through unravelling of preserved vegetation biomarker patterns. Palaeogeo. Palaeoclim. Palaeoeco. 285, 119–130. Jeng, W., 2006. Higher plant n-alkane average chain length as an indicator of petrogenic hydrocarbon contamination in marine sediments. Mar. Chem. 102, 242–251. Keeley, J.E., Sandquist, D.R., 1992. Carbon: freshwater plants. Plant Cell. Environ. 15, 1021–1103. Lepoint, G., Dauby, P., Gobert, S., 2004. Applications of C and N stable isotopes to ecological and environmental studies in seagrass ecosystems. Mar. Pollut. 49 (11–12), 887–891. Maberly, S.C., Raven, J.A., Johnston, A.M., 1992. Discrimination between 12C and 13C by marine plants. Oecologia 91, 481–492. Maksymowska, D., Richard, P., Piekarek-Jankowska, H., Riera, P., 2000. Chemical and isotopic composition of the organic matter sources in the Gulf of Gdansk (Southern Baltic Sea). Estuar., Coastal Shelf Sci. 51, 585–598. Marguillier, S., Van der Velde, G., Dehairs, F., Hemminga, M.A., Rajagopal, S., 1998. Trophic relationships in an interlinked mangrove-seagrass ecosystem as traced by d13C and d15N. Mar. Ecol. Prog. Ser. 151, 115–121. Mead, R., Xu, Y., Chong, J., Jaffe, R., 2005. Sediment and soil organic matter source assessment as revealed by the molecular distribution and carbon isotopic composition of n-alkanes. Org. Geochem. 36, 363–370. Meyers, P.A., 1994. Preservation of elemental and isotopic source identification of sedimentary organic matter. Chem. Geol. 144, 289–302. Meyers, P.A., Ishiwatari, R., 1993. Lacustrine organic geochemistry – an overview of indicators of organic matter sources and diagenesis in lake sediments. Org. Geochem. 20, 867–900. Muller, A., Voss, M., 1999. The palaeoenvironments of coastal lagoons in the southern Baltic Sea: II. d13C and d15N ratios of organic matter-sources and sediments. Palaeogeo., Palaeoclimatol., Palaeoecol. 145, 17–32. Naraoka, H., Ishiwatari, R., 1999. Carbon isotopic compositions of individual longchain n-fatty acids and n-alkanes in sediments from river to open ocean: Multiple origins for their occurrence. Geochem. J. 33, 215–235. Nishimura, M., Baker, E.W., 1986. Possible origin of n-alkanes with remarkable even to-odd predominance in recent marine sediments. Geochim. Cosmochim. Acta 50, 299–305. Osmond, C.B., Valaane, N., Haslam, S.M., Votila, P., Roksandie, Z., 1981. Comparison of d13C values in leaves of aquatic macrophytes from different habitats in Britain and Finland some implications for photosynthetic processes in aquatic plants. Oecologia (Berl.) 50, 117–124. Pancost, R.D., Boot, C.S., 2004. The palaeoclimatic utility of terrestrial biomarkers in marine sediments. Mar. Chem. 92, 239–261. Papadimitriou, S., Kennedy, H., Kennedy, D.P., Duarte, C.M., Marba, N., 2005. Sources of organic matter in seagrass-colonized sediments: a stable isotope study of the silt and clay fraction from Posidonia oceanica meadows in the western Mediterranean. Org. Geochem. 36, 949–961.

355

Personnic, S., Boudouresque, C.F., Astruch, P., Ballesteros, E., Bellan-Santini, D., Bonhomme, P., Feunteun, E., Harmelin-Vivien, M., Pergent, G., Pergent Martini, C., Renaud, F., Thibaut, T., Ruitton, S., 2014. An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow. PLoS One 9 (6), e98994. http://dx.doi.org/10.1371/ journal.pone.0098994. Readman, J., Mantoura, R., Rhead, M., 1987. A record of polycyclic aromatic hydrocarbons (PAH) pollution obtained from accreting sediments of the Tamar estuary, UK: evidence for non-equilibrium behaviour of PAH. Sci. Total Environ. 66, 73–94. Redfield, A.C., Ketchum, B.H., Richards, F.A., 1963. The influence of organisms on the composition of sea water. In: Hill, M.N. (Ed.), The Sea. Wiley, New York, pp. 26– 77. Salas, N., Ortiz, L., Gilcoto, M., Varela, M., Bayona, J.M., Groom, S., Alvarez-Salgado, X.A., Albaiges, J., 2006. Fingerprinting petroleum hydrocarbons in plankton and surface sediments during the spring and early summer blooms in the Galician coast (NW Spain) after the Prestige oil spill. Mar. Environ. Res. 62, 388–414. Scholz-Böttcher, B.M., Ahlf, S., Vázquez-Gutiérrez, F., Rullkötter, J., 2008. Sources of hydrocarbon pollution in surface sediments of the Campeche Sound, Gulf of Mexico, revealed by biomarker analysis. Org. Geochem. 39, 1104–1108. Schubert, C.J., Calvert, S.E., 2001. Nitrogen and carbon isotopic composition of marine and terrestrial organic matter in Arctic Ocean sediments: implications for nutrient utilization and organic matter composition. Deep-Sea Res. 48, 789– 810. Sonibari, O.O., Sojinu, O.S., 2009. Chemical composition of leaf lipids of angiosperms: origin of land plant-derived hydrocarbons in sediments and fossil fuel. Eur. J. Sci. Res. 25, 192–199. Sulzman, E.W., 2007. In: Michener, Lajtha, Kate (Eds.), Stable Isotopes in Ecology and Environmental Science, second ed. Blackwell Publishing (Chapter 1 pp1). Venkatesan, M.I., Brenner, S., Ruth, E., Bonilla, J., Kaplan, I.R., 1980. Hydrocarbons in age-dated sediment cores from two basins in the Southern California Bight. Geochim. Cosmochim. Acta 44, 789–802. Versteegh, G.J.M., Zonneveld, K.A.F., de Lange, G.J., 2010. Selective aerobic and anaerobic degradation of lipids and palynomorphs in the Eastern Mediterranean since the onset of sapropel S1 deposition. Mar. Geol. 278, 177–192. Viet, K.P., 2006. Distribution and carbon isotope composition of n-alkanes as source indicators of organic matter in mangrove and seagrass sediments. MSc thesis, Vrije Universiteit Brussel, 86pp. Vizzini, S., Sarà, G., Mateo, M.A., Mazzola, A., 2003. d13C and d15N variability in Posidonia oceanica associated with seasonality and plant fraction. Aquat. Bot. 76, 195–202. Viso, A.C., Pesando, D., Bernard, P., Marty, J.C., 1993. Lipid components in the Mediterranean seagrass Posidonia oceanica. Phytochemistry 34, 381–387. Volkman, J.K., Holdsworth, D., Neill, G., Bavor, H., 1992. Identification of natural, anthropogenic and petroleum hydrocarbons in aquatic sediments. Sci. Total Environ. 112, 203–219. Wada, E., Hattori, A., 1991. Nitrogen in the Sea: Forms, Abundances, and Rate Processes. CRC Press, Boca Raton, FL, p. 208. Wang, Z., Fingas, M., Page, D.S., 1999. Oil spill identification. J. Chromatogr. A 843, 369–411. Zaghden, H., Kallel, M., Elleuch, B., Oudot, J., Saliot, A., 2007. Sources and distribution of aliphatic and polyaromatic hydrocarbons in sediments of Sfax, Tunisia, Mediterranean Sea. Mar. Chem. 105, 70–89.