Molecular Markers in Environmental Geochemistry - American

Chapter 18

Downloaded by NORTH CAROLINA STATE UNIV on October 4, 2012 | http://pubs.acs.org Publication Date: July 1, 1997 | doi: 10.1021/bk-1997-0671.ch018

Aliphatic Hydrocarbons, Linear Alkylbenzenes, and Highly Branched Isoprenoids in Molluscs of the Adriatic Sea, Italy G. P. Serrazanetti, R. Artusi, and C. Pagnucco Section of Veterinary Biochemistry, Department of Biochemistry, University of Bologna, Via Tolara di Sopra 50,40064 Ozzano Emilia, Bologna, Italy

Aliphatic hydrocarbons and linear alkylbenzenes (LABs) were determined in four species of molluscs of the Central Adriatic Sea: two bivalves, Mytilus galloprovincialis, Solen vagina and two gastropods, Patella vulgata, Cassidaria echinophora. The concentrations of total aliphatic hydrocarbons determined in the mollusc samples were between 35.0 and 68.1 μg g dry wt, and they may be considered within normal limits for areas reported to be mildly polluted. In P. vulgata and in C. echinophora over 70% and 50%, respectively, of the total aliphatic hydrocarbons are represented by highly branched isoprenoids (HBIs). In M. galloprovincialis 30% is made up of squalene. These hydrocarbons are usually considered of recent biogenic origin, and in particular HBIs found in P. vulgata support previous suggestions that these alkenes might be considered molecular markers of the presence of some diatom species. LAB concentrations are between 3.2 and 15.9 μg g dry wt and show that all four mollusc species are contaminated by these molecular tracers of domestic wastes. Based on their composition, the LABs appear to have been recently discharged into the marine environment. The high concentration of external isomers indicates that LABs have not been biodegraded. This is probably due to the fact that the wastes were not treated or were only partially treated prior to reaching the sampling area. -1

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Oil pollution in some coastal areas of the Mediterranean Sea is considered to be serious requiring specific studies on the sources, effects, and fate of fossil fuel compounds discharged in this environment (7). Research has been carried out to understand the origins of hydrocarbons. In particular, it is of interest to know whether the fossil hydrocarbons indicative of petroleum contamination are present along with recent biogenic hydrocarbons in order to understand what role these compounds 276

© 1997 American Chemical Society

In Molecular Markers in Environmental Geochemistry; Eganhouse, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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Hydrocarbons and Alkylbenzenes in Molluscs

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might play as biochemical markers in the marine environment. Moreover, it is known that the determination of linear alkylbenzenes (LABs) allows one to verify the presence of municipal waste contamination because these compounds are manufactured for production of linear alkylbenzenesulfonate (LAS) surfactants used in commercial detergent formulations (2, 3). Previous research has shown that bivalves concentrate many pollutants to a marked degree above sea water concentrations. This ability provides an alternative to collecting large volumes of sea water and transporting them to the laboratory for extraction and analysis (4). The determination of hydrocarbons in molluscs is consequently of great interest both because organisms belonging to this phylum play an important role as monitors of contamination in the coastal zone (4, 5) and because several species are consumed by humans. Therefore, we decided to analyze aliphatic hydrocarbons with gas chromatographic (GC) retention times from w-C to »-C and LABs in two species of filter-feeding bivalves: Mytilus galloprovincialis, widely distributed and frequently utilized in environmental studies, and Solen vagina, a species living in the sandy sea floor. Both species are widespread along the Italian coasts, and they are also interesting to analyze as they constitute human seafood (6). Also, we deemed it useful to determine the hydrocarbons in two species of gastropod molluscs that occupy different positions in the marine trophic chain (7). Specifically, we analyzed specimens of the herbivorous gastropod Patella vulgata, which lives on intertidal rocks, and the carnivorous gastropod, Cassidaria echinophora, which lives on the sea floor. All specimens of the four species were collected in the Adriatic Sea. This is a semienclosed basin with poor exchange, heavy traffic from oil tankers and a lot of human activity associated with urban centers and harbors located along its coasts. In this work we determined saturated and unsaturated aliphatic hydrocarbons with branched or straight chains and LABs in different species of molluscs because information on concentrations of these compounds in organismsfromthe Adriatic Sea is limited. This research aims to establish the presence in these organisms of biogenic hydrocarbons, in order to obtain information on the transfer of the organic matter to the marine trophic chain, of fossil hydrocarbons to detect the presence of oilcontamination, andfinallyof LABs because they are to be considered very important as molecular tracers of domestic wastes. 15

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Materials and Methods Study Site. All four mollusc species were collected in summer by divers, well away from motor boats, in the central Adriatic Sea about 100 m off the coast opposite the town of Pescara. In this area the water depth is about 5 m, a north/south current is present and in this season the water temperature is always over 20°C (Figure 1). The specimens of P. vulgata were sampled on the artificial protective reef made up of natural rocks, those of M. galloprovincialis on the same reef at a water depth of about 3 m (subtidal rocks) and those of S. vagina and C. echinophora on the sandy sea floor at a water depth of about 5 m. Sample Treatment. All samples were manually collected, washed with distilled water, enclosed in aluminium foil and kept at about 4°C. At the end of the sampling,



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MOLECULAR MARKERS IN ENVIRONMENTAL GEOCHEMISTRY

Figure 1 - The sampling area in the central Adriatic Sea coast.


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SERRAZANETTI E T A L .



the specimens were frozen and kept at -20°C until dissections were performed. After thawing, the soft tissues were excised, and surplus fluid from the molluscs was discarded. The total soft tissues were homogenized with a Waring blender so that for each of the four species we could prepare 3 aliquots of whole soft parts from which approximately 5g were used for dry weight determination and lOOg were used for the lipid extraction. The dry weight of the tissues was determined gravimetrically after freeze drying. Lipid Extraction. All glassware and steel tools were carefully washed, rinsed with distilled water, acetone, ethyl ether/w-hexane then dried at 100°C for an hour at least, (all solvents were obtainedfromCarlo Erba, Milano, Italy). The lipids were extracted from wet tissues with chloroform-methanol (2:1) (8) with propyl gallate as antioxidant, measured gravimetrically and saponified (40°C) by refluxing for 2 h in 150 ml round-bottom flask with methanolic (GHsOHiI^O = 95:5) KOH (2N). The flask was cooled at room temperature, and then 50 ml of w-hexaneethyl ether (1:1) were poured into the flask. The contents were transferred to a separatory funnel, and the flask wasrinsedwith 50 ml of w-hexane-ethyl ether (1:1) which was then combined with the digestates in the funnel. Distilled water (100 ml saturated with NaCl) was subsequently added to the funnel. The digestates were shaken for 2 min and allowed to settle. The clearly separated digestates were extracted again twice with 60 ml of w-hexane-ethyl ether (1:1). The combined extracts were backwashed three times with 100 ml distilled water. After dehydrating with anydrous sodium sulphate, the combined extracts were transferred to a round-bottom flask and concentrated in a rotary evaporator (40°C) under vacuum. Hydrocarbon Separation. The hydrocarbons were separated from the other lipid classes of unsaponifiable matter (about 40 mg per plate) by thin layer chromatography (?) using w-hexane-ethyl ether (1:1) as the mobile phase, and «-C , squalene, 1phenyldodecane and chrysene as external standards to check the positions of the hydrocarbons on the plates. After elution, the plates were sprayed with 2,7 dichlorofluoroscein (0.02% in EtOH) and examined under an ultra-violet lamp. The band with Rf higher than 0.90 containing aliphatic hydrocarbons, squalene and LABs was scraped off and extracted with w-hexane-ethyl ether (1:1). The aromatic hydrocarbons with two or three rings, which could have been collected along with the unsaturated aliphaticfractionand the polycyclic aromatic hydrocarbons that show Rf values lower than 0.90, were present in amounts not detectable by GC-MS analysis. Quantitative and qualitative analyses were carried out by means of capillary gas chromatography/mass spectrometry (GC/MS). 19

Gas Chromatography. The hydrocarbons were analyzed by gas chomatography using a Carlo Erba HRGC 5160 MEGA, equipped with aflameionization detector (FID) and a fused silica capillary column coated with OV1 (25m x 0.32mm; film thickness = 0.4u.m). The oven temperature was programmed from 120°C to 295°C at the rate of 3.5°C min- and final isothermal hold of 4 min. The injector (splitter) and detector temperature was 310°C, the carrier gas flow was 1.8 ml min- and the split ratio was 1:80. 1

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MOLECULAR MARKERS IN ENVIRONMENTAL GEOCHEMISTRY

Mass Spectrometry (MS). Mass spectra were obtained by GC-MS using either a Carlo Erba Mega QMD 1000, 70 eV coupled to a Carlo Erba MEGA 5300 gas chromatograph with a fused silica capillary column coated with SE52 (25m x 0.25mm; film thickness = 0.20|im) or a Finnigan MAT ITS40 coupled to a Varian 3300/3400 GC with a fused silica capillary column coated with SPB5 (30m x 0.25mm; film thickness = 0.25 nm). The aliphatic hydrocarbon concentrations were determined by GC using W-C24 as an internal standard, and the LAB concentrations were determined using two daily injected solutions of 1-phenyldodecane, at known concentration, close to those of the analytes, as an external standard. All samples were injected at least three times and linearity of detector response was periodically checked. Procedural blanks were determined at the start of the work and for every six samples. Blanks and samples with W-C24 and 1-phenyldodecane were processed as real samples. No contaminations were evident as well as no LABs were determined in several samples of macroalgae analyzed with the same method in our laboratory. Recoveries for H-C24 were 87 ± 9% and for 1-phenyldodecane were 85 ± 11% (n-3). As we calculated the recoveries only for H-C24 among the aliphatic hydrocarbons, and for 1-phenyldodecane among the LABs, none of the data presented here were adjusted for recovery. The detection limits are 200 ng of total aliphatic hydrocarbons and 100 ng of total LABs. Aliphatic hydrocarbon identification was performed by the use of retention indices and coinjection with a standard mixture containing all A2-alkanesfromH-C12 to H-C30 (minus 25, 27, 29 w-alkanes), pristane, phytane and «-C20:i. By GC-MS, previous identifications were confirmed, and the identification of the different olefins and of LABs was carried out. The use of columns with slightly different polarity has permitted us to avoid interferences with coeluting peaks. Hereafter, LAB isomers will be symbolized as n-C , where n = position of phenyl substitution along the linear alkyl chain and m = number of carbon atoms in the alkyl chain. m

Total Aliphatic Hydrocarbons The total aliphatic hydrocarbon concentrations determined in the four species of molluscs are reported in Table I. The concentrations of these compounds appear rather uniform and range between 35.0 and 68.1 u,g g - dry wt. Our results are difficult to compare with those obtained by other researchers because the only available data concern Mytilus spp. and not the species analyzed in this study. However, it is known that the hydrocarbon concentrations reported in the literature for molluscs may vary by as much as a factor of 10 (70) because the data on these compounds include different kinds of hydrocarbons (saturated, unsaturated, only aliphatic, aliphatic plus aromatic, etc.). Comparing the concentrations of total aliphatic hydrocarbons in the whole soft tissues of molluscs from the central Adriatic Sea with those in specimens of M galloprovincialisfromother areas affected to various degrees by oil pollution, we consider those given in Table I (ranging from 8 to 20 u,g g" wet wt) to be within normal limits for areas regarded as mildly polluted (1-46 u.g g' wet wt) (77). However, Ameijeiras et al. (11) only examined saturated aliphatic hydrocarbons, whereas the present work also includes unsaturated hydrocarbons of recent biological 1

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SERRAZANETTI ET AL.


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origin (for instance squalene and highly branched isoprenoids). For this reason, the apparent level of contamination by petroleum of some species examined here is considered to be low. The concentration of w-alkanes found in M. galloprovincialis from the Adriatic Sea (17.2 jig g" dry wt) is similar to that of M. californianus (14.8 ug g" dry wt) exposed to a moderate level of pollution (12), and is equivalent to that of M. edulis (17.3 jug g' dry wt) exposed to low levels of chronic oil contamination (12) and lower than those found in certain polluted areas of Australia: Cockburn Sound (50 jig g' ) (13) and Westernport Bay (over 100 ug g" ) (14). 1

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Table I. Concentrations of total lipids, aliphatic hydrocarbons, and linear alkylbenzenes in Molluscs of Adriatic Sea Solen Mytilus vagina galloprovincialis

Patella vulgata

Cassidaria echinophora

wet wt / dry wt

5.05

5.35

3.55

3.35

Total lipids (mg g - dry wt)

44 ± 6 *

68 ± 10*

129 ± 5*

165 ± 16*

38.7 ± 2.6

35.0 ± 15.8

68.1 ± 19.5

49.3 ± 18.3

(ng mg - lipid)

879 ± 59

515 ±232

528 ± 151

299 ± 111

E w-alkanes (ug g dry wt)

29.8 + 2.1

17.2 ± 5.9

9.3 ± 2.6

15.2 ±3.4

ZLABs (ug g dry wt)

15.9 ± 2.9°

6.3 ± 2.6

3.2 ± 1,4

4.7 ±2.7

(ng mg " lipid)

361 ± 85

93 ±38

25 ± 11

29 ± 17

I/E ratio

1.12

1

Aliphatic hydrocarbons (ug g dry wt) _ 1

1

_ 1

_ 1

J

0.98

0.91

1.18

Values are means ± SD (n = 3) * All the values are significatively different (P < 0.05) ° Value significatively higher than others (P < 0.05) I/E = [6(|)-C + 5(|>-C ] / [4-C + 3

Molecular Markers in Environmental Geochemistry - American

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