Occurrence of aliphatic and polyaromatic hydrocarbons (PAHs) in Mytillus galloprovincialis from the traditional market in Marseille, France, by Gas Chromatography triplequadropole tandem Mass Spectrometry (GC-QQQ/MS)

Mediterranean mussel, Mytillusgalloprovincialis collected from the traditional market in Marseille, France,have been analysed using GC-QQQ/MS for their hydrocarbons (n-alkanes and polyaromatic hydrocarbons (PAHs)) extentwith two different solvent extraction, such as heptane:dichloromethane (HEP:DCM;1:1) and heptane:acetone (HEP:ACE; 1:1). The results showed hydrocarbons yielded from heptane:acetone extractionwere 28335 μg.kg- 1mussels dw (Ʃ n-alkanes C15-34) and 202 μg.kg-1mussels dw(ƩPAHs) while the yield from heptane:DCM extract was lower ca. 27026 μg.kg-1musselsdw and 133 μg.kg-1 mussels dw respectively from the Ʃn-alkanesC15-34and ƩPAHs. High hydrocarbon levels can be affected by the presence of lipids or other metabolites in mussels that have the same polarity with hydrocarbon compounds which has interferred the measurement. Several ratio parameter of n- alcanes and PAHs source in the mussels were evaluated to asses the origins of their hydrocarbons in mussels from which we suggested origins of hydrocarbons were pyrolytic and biogenic rather than petrogenic.


Introduction
Petroleum hydrocarbons compounds i.e. aliphatic hydrocarbons and polyaromatic PAHs that known as toxic pollutants to the environment e.g.rivers, lagoons, and marine, and some of them have mutagenic and carcinogenic properties [1,2,6,7]. Those compounds may be derived from petroleum or oil spills rafinery product (petrogenic sources), incomplete combustion of organic matter, and biomass fossile fuels (pyrolytic source) [1,3,6,7,10]. For that reason, petroleum hydrocarbons analysis within the organims are important to monitoring activity of pollutant in environment as one integrative approach [14].This study used Mediteranean mussel (M.galloprovincialis) to monitor marine ecosystems quality. Hydrocarbons can absorb into sediment via particulate or colloid and potentially ingested by marine benthic organisms in marine environment [2,9] [2,4,5,8,9]. Bivalves such as Mussels had used as sentinel organisms of marine pollution and have the capability to reflect the environment quality which related to their habitat, food chains, and pollutant hydrophobicity properties that have the tendency to be absorbed into their living tissues [2,9]. Mytillus families widely used since 1990's and become one of the most successful model organisms for time-integrated responses to complex mixture of pollutants [14], also as bioindicator reference of hydrocarbons level [8,13]. This study focus on the hydrocarbons content in mussel from traditional market that have direct impact to human, because as we know that the traditional market sell unknown source of mussels which can be from pollutant site or propre site for human consumption.
The significance of this study related to the extraction, fractination, and analysis method using GC-QQQ/MS [1,2,6,7,10]. Quadropole can be used in scanning or filtering mode. Triple quadropole system contains of three quadropoles. Quadropole 1 and 3 as mass filter while quadropole 2 as collision cell [15].Several studies used heptane: dichloromethane (1:1 v/v) [1,6,7,9,10] and heptane:acetone (1:1, v/v) to extract the hydrocarbons [4,13]. Thematrice of mussels tissues are more complex than sediment and can influence the interference effect to separate the hydrocarbons content from their matrices.This study aimed to analyze the content of hydrocarbons in M.galloprovincialis mussel species that derived from traditional markets in Marseille, France, by using protocol analysis of hydrocarbon sediments and to compare the different extraction solution to extract hydrocarbons that can be optimized using GC-QQQ/MS.

Sampling
Approximately 3 kg wet mussels, M. galloprovincialis have taken from Noailles traditional market, Marseille, France. Fresh seafood market is located about 300 meters from Mediteranean port. After purchased, the wet mussel stored in freezer -20 0 C.

Sampel Preparation
The tissues taken and placed in buchner. Water content in wet tissues removed by gravitation for 30 minutes [3], then wetmass weighed with microbalance (Perkin Elmer AD2Z, Marseille France, weighed approximately ± 250 gr). The tissues were freeze-dried under pressure 0.080 bar and freeze at -50 0 C for 48 h. Then it cutted and crashed by mortar to small size and the dry mass weighed and stored under temperature ambient before the extraction [8,13].

Result and discussion
Extractable Organic Matter (EOM) concentrations, Total Hydrocarbons Content (THC), quantities of F1 and F2, comparing between THC and MOE, F1 and F2 from two different solvant extraction are given gravimetrically in Table 1 Generally, n-alkanes are nonpolar compounds and semipolar for PAHs, that can extracted and separated using fractionation method with comparing different polarity of eluent. GC/MS analysis with Single Ion Monitoring (SIM)mode has found 20 peaks n-alkanes (n-C 15-34 ) and 8 peaks identic of PAHs (Figure 1 and Figure 2). According from chromatograms, both of chromatograms have similar profile. Besides n-alkanes derivatives, isomer found from n-alkanes such Pristane, Phytane which indicate the presence of crude oils that might be from natural (biogenic or diagenetic) and isomer from n-alkenes, squalene which derived from microbial degradation [1]. Figure 1 showed that there are two high peaks (n-C 29 and n-C 31 ) in the last chromatograms and n-C 17 , n-C 18 , n-C 19 and n-C 20 have high peaks in the beginning.  Total of n-alkanes (n-C 15-34 ) and PAHs respectively were 27026 and 133μg.kg -1 tissues.dw for those extracted with HEP:DCM (1:1, v/v) and for HEP:ACE (1:1, v/v), ∑n-C 15-34 and ∑PAHs respectively have value 28335 and 202μg.kg -1 tissues.dw (see Table 2). The high level of n-alkanes content have the possibilities that originated from disrupting of lipid and metabolites, or these mussels have high contamination in mussels tissues from Mediteranean sea.  The mixed solvents HEP:ACE (1:1, v/v) known to be more polar than HEP:DCM (1:1, v/v).From this result, hydrocarbons content from M.galloprovincialis were determined. The properties of mussels tissues are presumably more complex because of their macromolecules content such as lipid, protein, and other metabolite [4]. Such compounds are capable to interference while separating process and these metabolites macromolecules can affect the extraction yield since they have the same polarity with hydrocarbons compounds. For that reason, hydrocarbons can associated with this macromolecul and have the same ion fragmentation with ion target [4]. The method of fractionation need another method to separate these macromolecules before the hydrocarbons fractination process. One of method for macromolecules separation is permeable gel chromatography, which is the macromolecule can be removed from mussel tissues before the fractionation process. Our finding showed aliphatic and PAHs extraction yield within the same greater magnitude that compared to previous studies using sediment as environmental matrices. Partially concluded thatthe mussels from traditional market were potentially polluted by hydrocarbons [1,6,7 and 10]. Obviously, further research is needed to revalidate and to find out the effective method to separate another molecules prior for the environmental analysis. In addition, high concentration of the hydrocarbons in M. Galloprovincialis has been observed, compare to another previous studies concerning PAHsin Mytillus edulis (27,6-442 μg.kg -1 mussels dw ) [12]; M.galloprovincialis from coastal of Saronikos, Gulf, Greece, (17PAHs: 219-1487 μg.kg -1 mussels tissues dw) [13] and for n-alkanes in M.galloprovincialis from Galicia coast (n-C 8-35 : 89.46-5098.01 μg.kg -1 mussels tissues dw) [16]. 7 ratios for n-alkanes used to assess the potential sources of hydrocarbons contamination: CPI (Carbon Preference Index), NAR (Natural alkane ratio, nC18/phyt, nC17/phyt, prist/phyt,TAR (Terrigenous Aquatic Ratio), and LMW/HMW (Low Molecular Weight/High Molecular Weight), while PAHsindices are derived from comparison identical molecular weight such as (anthracene/∑m/z 178; Fluorene/∑m/z 202; Bz(a) anthracene/∑m/z 228; indeno (123 cd)pyrene/∑m/z 176). Generally, there are two main sources of hydrocarbons, origin from anthropogenic activities and from biogenic process (natural process) or biological activites. Concern tobiogenic source, it may be derived from plant or animal residualor both of them. Parameters index and their values of hydrocarbons are given at Table 3 for n-alkanes and Table 4 for PAHs.  CPI: Carbon Preference Index = 2*(n-C 25 +n-C 27 +n-C 29 +n-C 31 )/(n-C 24 +2*(n-C 26 +n-C 28 +n-C 30 ) +n-C 32 ); NAR: Natural n-alkane ratio = ((∑n-C 19-32 )-2∑even n-C 20-32 )/(∑n-C 19-32 ); TAR: Terrigenous/Aquatic Ratios = (n-C 27 +n-C 29 +n-C 31 )/(n-C 15 +n-C 17 +n-C 19 ); Pris: Pristane; Phy: Phytane; LMW/HMW (Low Molecular Weight/High Molecular Weight) = (n-C 15-21 )/(n-C 22-34 ) [1,6,7,10,16] The results showed an apportionment of the n-alkanes level from biogenic sources, probable from aquatic organism or terrestrial environment with index (CPI, n-C17/Pr, n-C18/Phy, and TAR ) >1, for NAR is close to zero < 1, indicates for petroleum hydrocarbons, LMW/HMW< 1 usually represent nalkanes that are produced by higher plants, marine animals and sedimentary bacteria.there are found parameters ratio from oil which origin from n-alkanes isomer (Pristane/Phytane) < 2. The source nalkanes from this experiment are shown in Table 5. Source of PAHs Fluoranthene/ (Fluoranthene+pyrene) (Fl/∑202) is often used to discriminate PAH sources (1, 10). Ratio<0.5 is generally characteristic as petrogenic sources and ratio > 0.5 founded in kerosene, grass, coal, and wood combustion samples. The ratio of anthraceene/(Anthracene+Phenanthrene) ratio (An/∑178) < 0.1 indicates a petrogenic source while a ratio > 0.1 reflects a combustion source [1,2,6,10]. Benz (a) anthracene/chrysene (Benzo(a)Anthracene/Benzo(a) anthracene+Chrysene) ratio (BzA/∑228) < 0.2 indicates petroleum sources whileratio > 0.35 indicates combustion sources and values between 0.2-0.35 cannot used to discriminate petroleum and/or combustions sources [1,6].
The index indicates the source of PAHs in mussels from traditional market derived as natural or biogenic sources which origin from the burning of fossil or living creatures biosynthesis results with given indices < 0.5 (anthracene / Σm/z 178, benzo (a) anthracene / Σm/z 228; indeno (123cdi) pyrene / Σ m/z 176 and it also has the probability that those indices came from oil with indices > 0.5 (Fluorene / Σ m/z 202) parameters source of PAH resulting from the two solutions of different extracts that shown in Table 6. The profile of capillary column gas chromatograms of n-alkanes fraction in mussels extract between HEP:ACE (1:1, v/v) and HEP: DCM (1:1, v/v) as solvent extraction have same profile. n-C 15-34 were found with different yield.

Conclusion
The