Use of PAHs and n-Alkanes Biomarkers for Source Identification of Organic Matters of Qeshm Mangrove Forests

Introduction
Persian Gulf is a warm, hypersaline, shallow and semi-enclosed sea with low hydrodynamic energy and ideal conditions for deposition of particle materials.
Qeshm Island is the largest island of Persian Gulf, in the mouth of strategic Hormoz Strait. Every day, many tankers and ships that transport oil production of Persian Gulf throughout the world, cross near this island. Thus, Qeshm and Mangrove forests located on northwest of it, are continually subjected to oil contamination.
Mangrove ecosystems because of their natural characters have the highest sensitivity to oil contamination relative to other costal environments. Mangroves are highly productive intertidal ecosystems. These forests are well known for its suitable sedimentation environment and therefore these forests behave like a reservoir for non-polar suspense load. Oil contamination has been established as one of the most serious health hazards to coastal environments, essentially mangrove ecosystems due to having anoxic and saturated sediments. As a result, Mangrove ecosystems have become interesting local ecosystems for checking the type and origin of pollutants and organic matters.
Tracing organic matter in sediment samples is important to understand the input sources in organic matter cycling in aquatic environments. Polycyclic aromatic hydrocarbons (PAHs) are one group of persistent organic pollutants (POPs). PAHs are listed by the United States Environmental Protection Agency (USEPA) as priority pollutants. Due to toxic, carcinogenic and mutagenic properties of some of these compounds, studying their levels, sources and impacts on environment is interesting. Petroleum and petroleum products are main origins for entering these compounds to marine environments. PAHs discharged to the environment are present in complex mixtures and include both low and high molecular weight PAHs. In this research, the profile and the characteristic ratios of different individual PAHs in sediments such as phenanthrene/anthracene and fluoranthene/pyrene, have been used to determine origins of PAHs.
In the recent years, Straight chain alkanes (n-alkanes) have been applied to assess the petroleum pollution in the marine environments. Significant difference in chain lengths n-alkanes of petroleum products, marine and terrestrial plants could be utilized for recognizing sources of organic matter in environmental samples. Distribution of hydrocarbons could be varied in different environments. In order to determine source of organic matters some of hydrocarbonic indices have been proposed. We used them to assess organic inputs of Qeshm mangrove forests and geochemical conditions of this valuable ecosystem. 
Materials and methods
Our study area is within the mangrove forests in the northwest of Qeshm Island which form a vast area of the Hara Protected Area. This forest is the largest marine protected area in Iran and designated as a Protected Area in 1972, the entire area was designated as a Ramsar Site in 1975 as well as a Biosphere Reserve in 1976.
The surface sediment samples were collected from 18 stations of the mangrove forest nearby the Qeshm Island and were analyzed for PAHs and normal alkanes. Sampling was carried out randomly at low tides with four replicate samples, from nearest sediments of mangrove aerial roots by boat, using solvent-cleaned stainless steel spoons. The sediments were transferred in aluminum foils to reduce any contamination and after being labeled, were placed in icebox and transported to the laboratory for further analysis. Upon reaching the laboratory, the samples were stored in the Cold Room (-20°C) until further analysis.
Firstly, 10 g freeze-dried sediments were used from each sample. Sediment samples were extracted with 320 ml distilled dichloromethane for 10 h with a Soxhlet apparatus. After soxhlet extraction, immediately 100µl of deuterated PAH surrogate internal standard mixture (naphthalene-d₈, anthracene-d₁₀, chrysene-d₁₂ and perylene-d₁₂) was added to the sample extracts for quality control of PAH analyses, and  activated copper pieces were also added to DCM that were contained extracted organic fraction of each sediment sample, after extraction, for removal of sulphur from samples. Thereafter, two stages of silica gel column chromatography were applied. In first stage a range of hydrocarbons containing aliphatic and aromatic ones were eluted by means of 20 ml hexane/dichloromethane (3:1). In the next stage, the aliphatic hydrocarbon fraction was isolated from the aromatic lipids by fully activated silica gel column chromatography using 4ml hexanes. Also, hexane/dichloromethane (3:1) was used to get PAHs fraction.
Gas chromatographic-mass spectrometry (GC-MS) analysis was carried out with Agilent Technologies instrument model 7890 A gas chromatograph, coupled to a 5975 C quadrupole mass spectrometer. A 30 meter fused silica capillary column with 0.25 mm i.d. and 0.25 µm film thickness with helium was used as gas carrier in the analyses. GC-MS operating conditions were 70eV ionization potential with the source at 200°C and electron multiplier voltage at ~2000 eV.A selected ion monitoring method was employed after a delay of 3 min for analyzing PAHs and scan method for n-alkanes. Twenty- three of PAH compounds that including EPA PAH compounds were analyzed.
Results and discussion
Mean value of PAHs concentration in Qeshm sediment samples were 1344±306 ng g^-1 dry weight which was moderate relative to other marine environments. The analyzed sediment samples had higher percentages of lower molecular weight PAHs. Naphthalene were predominant compound in all of the PAH profiles which constitutes a significant fraction of crude oils and petroleum products with lighter fractions. Therefore, it can be used as an authentic indicator of petrogenic source of PAH contamination. In addition, lower molecular weight PAHs were dominant ones. This is another proof of petrogenic source for PAHs in surface sediment samples.
The sources of PAHs, whether from pyrolytic or from petrogenic contamination, may be identified by ratios of individual PAH compounds based on peculiarities in PAH composition and distribution pattern. Ratios value of specific PAH compounds such as phenanthrene/anthracene (Phe/Ant), fluoranthene/pyrene (Flu/Pyr), benzo(a)anthracene/chrysene (BaA/Chr) , fluoranthene to fluoranthene plus pyrene (Fl/Fl+Py), benzo(a)anthracene to the sum of benzo(a)anthracene and chrysene, (BaA/BaA+Chr), methylphenanthrene/phenanthrene (MP/P), were calculated to evaluate the possible source of PAH contamination in sediment samples. These ratios obviously suggest that the surface sediments of Qeshm mangrove have a petrogenic input of PAHs.
The mean of total n-alkanes concentrations (ΣHC) were 2766.40±1782.20 µg/g in surface sediment samples of the study area. GC-MS chromatograms of analyzed Qeshm samples showed two different distribution patterns of n-alkanes. Three predominant components (n-C₂₃, n-C₂₅, n-C₂₇) were obviously identified. In the sediments of these stations, long chain n-alkanes strongly outweighed short chain ones, 
with strong odd-to-even preference, suggesting a predominant contribution from recent higher plant sources.
Stations that are located just near the Qeshm Island have received terrigenous contribution from the mangrove covers. Therefore, mangrove leaves are the major sources of organic matters in surface sediments of Qeshm mangrove forests. High CPI, providing good evidence for a major contribution of epicuticular waxes from higher plants and also indicating negligible petroleum inputs. TAR index for sediments samples of Qeshm stations, similar to CPI is valuable index for source identification of organic matters. High CPI and TAR for some of sediment samples, clearly show dominance of higher plant-derived organic material.
Presence of UCM in chromatograms for Qeshm samples which indicate that stations far from Qeshm Island have high amounts of petrogenic compounds beside biogenic hydrocarbon inputs of higher plant. However, UCM was minor in the sediments of costal Qeshm Island.
Also, Pristane and phytane present in high concentrations confirm petroleum contamination in Qeshm mangrove sediments. In addition, low values for n-C₁₇/Pr and n-C₁₈/Ph could suggest relative petroleum contamination in surface sediments of Qeshm mangrove forests.
Distribution pattern of n-alkanes and the diagnostic hydrocarbonic ratios in analyzed sediment samples such as CPI, TAR and U/R, all revealed that n-alkanes have dominant biogenic source in Qeshm mangrove mudflat that origin of mangrove covers. However, some samples which have more distance from the island have shown a combined condition of petrogenic and biogenic origins. On the other hand, observed values for U/R, Pr/Ph, n-C₁₇/Pr, n-C₁₈/Ph  and UCM in all of the analyzed samples demonstrates that Qeshm mangrove forests was contaminated by a background oil contamination as a result of continuous oil spills and past wars in Persian Gulf.
In summary, PAHs and n-alkanes have not shown the same origin for sedimentary organic matters of Qeshm surface sediments. The reason of this consequence could attribute to this fact that PAHs compound, unlike n-alkanes, cannot display natural source of organic matter in environmental samples. In this study case, deduction is only based on PAHs due to navigation of these compounds, so that we distinguish dominant petrogenic source for organic matters of Qeshm samples, wrongly. However, n-alkanes biomarkers showed biogenic source as the dominant source of organic matters in studied mangrove forest truly. Therefore, combined application of various molecular biomarkers covered such insufficiency of special biomarkers to revealing real source of organic matters in environmental samples and getting a complete and reliable viewpoint of geochemical conditions. This could be important for having a successful management on valuable ecosystems.