Chemosphere 48 (2002) 947–954 www.elsevier.com/locate/chemosphere

Burial, exportation and degradation of acyclic petroleum hydrocarbons following a simulated oil spill in bioturbated Mediterranean coastal sediments V. Grossi *, D. Massias, G. Stora, J.-C. Bertrand Laboratoire d’Oc eanographie et de Biog eochimie, UMR CNRS 6535, Facult e des Sciences de Luminy, Centre d’Oc eanographie de Marseille, Campus de Luminy, Case 901-13288, Marseille Cedex 09, France Received 8 October 2001; received in revised form 18 February 2002; accepted 8 March 2002

Abstract A field study was conducted in a French Mediterranean littoral (Gulf of Fos) in order to determine the role of bioturbation processes during the bioremediation of oil-contaminated sediments. Inert particulate tracers (luminophores) and Arabian light crude oil were deposited at the surface of sediment cores incubated in situ for 2, 6 and 12 months. After incubation, luminophores and hydrocarbons presented roughly similar depth distributions in the sediment, showing a continuous burial of material until 55 mm depth. Short-chain (6 n-C25 ) n-alkanes were totally removed from the sedimentary column after 6 months, whereas
20% of heavier n-alkanes (e.g. n-C30 ) and of isoprenoid hydrocarbons (pristane (Pr) and phytane (Ph)) remained at the end of the experiment. The determination of the degradation constant and the turn-over rate of individual hydrocarbon indicated that C17–25 n-alkanes were degraded two to three times faster than longer homologues and than pristane and phytane. Using the 17a,21b-C30 hopane as an internal inert reference, we could demonstrate that, after 12 months of in situ incubation, 55% of the losses of the n-alkanes 6 C25 and 35% of the losses of the heavier n-alkanes and of Pr and Ph were due to biodegradation processes. These results demonstrate that the activity of benthic organisms can have a significant influence on the qualitative and quantitative fate of acyclic hydrocarbons following a petroleum contamination in marine coastal sediments. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Petroleum hydrocarbons; In situ experiments; Bioturbation; Kinetics of degradation; Mediterranean sea; Oil spill; Bioremediation

1. Introduction Extensive laboratory studies have been performed to understand the fate of petroleum in the marine environment (Leahy and Colwell, 1990; Harayama et al., 1999). In contrast, our knowledge on the weathering of oil under natural marine conditions is rather limited, and has stemmed from studies carried out either after

*

Corresponding author. Tel.: +33-491-829-651; fax: +33491-826-548. E-mail address: [email protected] (V. Grossi).

accidental contamination due to the wreck of tankers such as Arrow, Amoco Cadiz, Tanio, Exxon Valdez and Erika (Bragg et al., 1994; Wang et al., 1994; Swannell et al., 1996; Oudot, 2000), or on chronically contaminated sites such as industrial harbours or offshore platforms (Prince, 1993; Fischer et al., 1996; Le Dreau et al., 1997a). The fate of petroleum in recent sediments is strongly dependent on abiotic and biotic processes. In coastal environments where the overlying water is oxygenated, the activity of benthic infauna, particularly those capable of bioturbation, is known to influence the decomposition of organic matter (OM) (Kristensen and

0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 2 ) 0 0 1 2 2 - 4

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Blackburn, 1987). Sediment reworking can, for instance, alter oxic/anoxic boundaries, affect microbial populations directly or indirectly, cause vertical transport of particles, and lead to movements of particles across oxic and anoxic boundaries. The frequency and duration of these processes may significantly influence degradation rates and net preservation of OM (Aller, 1994). Unfortunately, only few studies have been dedicated to the impact of bioturbation on the fate of petroleum in sediments. During a 6 months experiment in an intentionally contaminated marine coastal ecosystem, Gilbert et al. (1996) have observed that bioturbation processes can play an important role in the burial and the degradation of hydrocarbons. Nevertheless, in this study, the use of descriptive ratios (e.g. n-C17 /pristane, n-C18 /phytane) only allowed a qualitative description of hydrocarbon degradation. The present study was thus designed to examine, for over one year period, the overall impact of a natural benthic assemblage on the qualitative and quantitative fate (burial, kinetics and extent of degradation) of individual acyclic petroleum hydrocarbons following a simulated oil spill in coastal sediments. The concentration of different hydrocarbons were determined as a function of time and sediment depth. The use of the 17a,21b-C30 -hopane as an inert internal reference allowed the part of hydrocarbons which was biodegraded to be distinguished from the one released in the water column.

2. Experimental 2.1. Field experiment Experiments were carried out in the Carteau Cove (Gulf of Fos, Mediterranean Sea, Fig. 1) at 5 m depth, where bioturbation processes have been extensively studied (e.g. Gerino, 1990; Gilbert et al., 1996, 1998). The sediment is classified as muddy sand sediment and is occupied by a macrofauna assemblage characteristic of muddy sand in sheltered areas (Gilbert et al., 1998). Organisms larger than 250 lm have a density of 6100  2000 individuals/m2 , and more than 90% are located in the first 10 cm of sediment. Polychetes are the most representative class of macrofauna (40–60% of total macrofauna), essentially represented by the Cirratulideae Tharyx heterochaeta and the Spionideae Paradoneis lyra. The experimental site (43°23N–4°53E) was divided into three fields and seven PVC cores (25 cm length  11 cm diameter) were inserted into the sediment of each field by scuba diving (Fig. 1). Three series of cores were defined as followed: Set A: nine contaminated cores (three per field) for the analysis of petroleum hydrocarbons;

Fig. 1. Study site location.

Set B: nine contaminated cores (three per field) for the study of bioturbation processes; Set C: three control cores (one per field) for the analysis of background hydrocarbons. The contamination of cores A and B was realised under the form of petroleum cakes (1 cm of thickness; diameters were the same as the cores) made by homogenising sieved sediment (1 mm) and Arabian light crude oil (BAL) to a final concentration of 41:1  5:8 g hydrocarbons kg1 dry sediment. For the study of bioturbation processes (cores B), the cakes were supplemented with luminophores (4 g), which are fluorescent particles (size 25–250 lm) used as conservative tracers (Gerino et al., 1998). In order to standardise the experimental conditions, the third set of cores (cores C) received only sieved sediment cakes where neither petroleum nor luminophores were added. All the frozen cakes were deposited at the sediment–water interface of the cores by scuba diving. After 2, 6 and 12 months of incubation in the field, three cores from sets A and B (one of each per field) and one core from set C were collected by divers. Oxygen profiles were then determined using polarographic minielectrodes (Revsbech and Jørgensen, 1986) with a vertical step of 200 lm, and the linear calibration was performed according to De Wit et al. (1997). Sediment was extruded from each collected core and sliced as follows: 0–3 cm in 0.5 cm intervals and 3–10 cm in 1 cm intervals (beneath this depth, the hydrocarbon content of the contaminated cores reached the background level). Samples were immediately frozen. 2.2. Luminophores counting Each section of the cores from set B was sieved through a 250 lm mesh to remove the biggest particles

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and the macrofauna. The remaining sediment fraction was dried at 50 °C (48 h) and carefully homogenised. The total number of luminophores was counted under UV light (Gerino, 1990). 2.3. Extraction and fractionation of hydrocarbons Hydrocarbons were extracted (24 h) from a known amount of freeze-dried sediment using a Soxhlet apparatus and a mixture of dichloromethane/methanol (DCM/MeOH; 2:1). Elemental sulphur was removed with activated copper curls during the extraction. Each sample was evaporated to dryness to obtain the total extractable organic matter (EOM). A known aliquot of the EOM was then dissolved in a minimum of heptane and applied to a half silica (8 g) and half alumina (8 g) (both deactivated with 5% water) chromatography column (31  1:8 cm i.d.). Aliphatic hydrocarbons (fraction F1) were eluted with 40 ml of heptane, and extracts were then concentrated by rotary evaporation and evaporated to dryness under a stream of nitrogen. 2.4. Analysis of the aliphatic fraction Aliphatic hydrocarbons were identified by GC/MS using a HP 5890 series II plus gas chromatograph equipped with a splitless injector (280 °C), and coupled with a HP 5972 mass spectrometer operated at 70 eV with a mass range m=z 50–700. Separations were performed with a fused silica capillary column (30  0:25 mm i.d.) coated with HP-5MS (0.25 lm film thickness) with helium as the carrier gas. The oven temperature was programmed from 60 to 130 °C at 20 °C/min, then increased to 300 °C at 4 °C/min and held at this temperature for 15 min. The hydrocarbons were then quantified by GC, using a HP 4890 gas chromatograph equipped with an automated splitless injector (280 °C) and a FID detector (290 °C). The column and the oven temperature programs were the same as the ones used for GC/MS analyses, and nitrogen was used as the carrier gas. Alkanes were quantified by internal calibration using two internal standards: hexamethylbenzene (HMB) for C14–23 alkanes and squalane (Sq) for C24–34 alkanes.

3. Results 3.1. Hydrocarbon and luminophore distribution Fig. 2 shows the depth profiles of luminophores and of the saturated hydrocarbon fraction in contaminated cores after 2, 6 and 12 months of in situ incubation. The distributions show a continuous burial of material throughout the experiment, under the form of accumu-

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lation zones. These zones were localised between 10–20, 20–30 and 20–55 mm depth after 2, 6 and 12 months respectively, while only the uppermost mm of sediment (
2–5 mm) was shown to be permanently oxygenated (Fig. 3). On a quantitative point of view, the amount of the saturated hydrocarbon fraction in the entire sedimentary column decreased during the first 6 months of the experiment, and seemed to stabilise afterwards. The amount deposited was equal to 1901  84 mg F1 (n ¼ 3) whereas the amount recovered in the cores (after correction from background) was 1793  54 (n ¼ 3), 1227  281 (n ¼ 3) and 1385  182 (n ¼ 2; a core could not be satisfactorily sampled) mg F1 after 2, 6 and 12 months respectively. The amount present in the non-contaminated cores (background hydrocarbons) was 88  4 mg F1, and remained constant throughout the experiment. 3.2. Composition of the saturated hydrocarbon fraction Fig. 4 represents the chromatograms of the saturated hydrocarbon fraction in the contaminated and the control cakes which were deposited at the beginning of the experiment. The hydrocarbon distribution of the contaminated cakes is representative of the BAL (Fig. 4A), whereas the hydrocarbon distribution of the noncontaminated cakes shows the preponderance of C-odd long-chain n-alkanes characteristic of biogenic sources (Fig. 4B). This was confirmed by the determination of specific hydrocarbon ratios such as n-C17 /n-C29 (Colombo et al., 1989) which was equal to 0.3 and 8.1 in the control and the contaminated cakes respectively. Examples of chromatogram of the saturated fraction at the depth of maximum petroleum concentration after 2, 6 and 12 months of experiment are shown in Fig. 2. The distribution observed after 2 months resembles one of the original BAL (Figs. 2A and 4A), while the distribution observed after 12 months shows characteristic of a strongly weathered petroleum (Fig. 2C). This, in spite of the remains of
60% of the F1 fraction. The depletion of acyclic hydrocarbons was followed through the study of seven individual components characteristic of the n-alkanes (n-C17 , n-C18 , n-C20 , n-C25 and n-C30 ) and of isoprenoid hydrocarbons (pristane and phytane). For each compound, the comparison of the amount recovered in the entire sediment cores after incubation (corrected from the background) with the amount deposited initially at the surface allowed the determination of its extent of total loss, its total loss rate constant (k) and its corresponding turn-over time (s, defined as the reciprocal of the rate constant) (Table 1). After 6 months, most (>95%) of the linear alkanes ranging from C17 to C25 have disappeared from the sedimentary column while
20% of the longer-chain nalkanes and of the isoprenoid compounds remained at the end of the experiment (Fig. 5). For those latter

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Fig. 2. Depth profiles of luminophore ( ) and of saturated hydrocarbon fraction (r) and chromatograms of the saturated hydrocarbon fraction in the zone of oil accumulation in contaminated cores after (A) 2, (B) 6 and (C) 12 months of in situ incubation. HMB (hexamethylbenzene) and Sq ðsqualaneÞ ¼ internal standards; Pr ¼ pristane; Ph ¼ phytane; Cn ¼ n-alkanes with n carbon atoms. The black rectangles on the low x axes indicate the concentration in the petroleum cakes deposited.

compounds, it may be that a longer incubation time would have resulted in further losses. The total losses of hydrocarbons were fitted using least square regression of amount vs. time to yield rate constants (k) using the simple first-order equation QðtÞ ¼ Q0 ekt , where QðtÞ is the amount of a component in the entire core at time t (corrected from the background), and Q0 the amount deposited on top of the cores (Table 1). Fitted experimental curves generally correspond well with the data as shown by the correla-

tion coefficients (r2 ; Bravais–Pearson, n ¼ 8, a ¼ 0:05). The apparent first-order total loss constants of the studied hydrocarbons ranged from 1.4 year1 for n-C30 to 4.7 year1 for n-C25 (Table 1). The losses of hydrocarbons during our in situ incubations can be attributed to (i) the exportation of compounds in the water column, and (ii) diagenetic processes such as microbial mineralization and/or transformation, digestion by benthic fauna and to a lesser extent abiotic reactions. In order to distinguish these

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Fig. 3. Example of oxygen profile measured in a contaminated core after in situ incubation.

two parts (exportation vs. (bio)degradation), we used the 17a,21b-C30 -hopane (C30 H) as an inert internal reference (Bragg et al., 1994; Venosa et al., 1996, 1997; Le Dreau et al., 1997b; Oudot et al., 1998; Wang et al., 1999). The comparison of the amount of C30 H recovered in the entire sediment cores after incubation with the amount deposited initially at the surface gave the part of hydrocarbons that was exported. A similar approach has been applied by Venosa et al. (1996). Following normalisation to the C30 H, we could estimate the extent of (bio)degradation after each incubation time and the degradation rate constants for the entire experiment period of the seven individual hydrocarbons considered (Fig. 5 and Table 1). After 12 months,
55% of the losses of C17–25 n-alkanes and
35% of the losses of longer-chain n-alkanes and of the isoprenoid hydrocarbons could be attributed to (bio)degradation processes. The apparent first-order decay constants ranged from 0.38 year1 for n-C30 to 0.96 year1 for n-C25 .

4. Discussion In spite of a massive petroleum contamination at the water–sediment interface, the activity of benthic organ-

Fig. 4. Chromatograms of the saturated hydrocarbon (F1) fraction in (A) the contaminated and (B) the control sediment cakes deposited at the water–sediment interface. HMB (hexamethylbenzene) and Sq ðsqualaneÞ ¼ internal standards; Pr ¼ pristane; Ph ¼ phytane; Cn ¼ n-alkanes with n carbon atoms.

isms remained significant in the polluted cores as demonstrated by the consequent burial of hydrocarbons and luminophores (Fig. 2). It was previously shown that in the absence of macrofauna, the petroleum was not buried (Gilbert et al., 1996). The amount of C30 H recovered after each incubation time compared with the amount deposited (80%, 59% and 55% after 2, 6 and

Table 1 Apparent first-order constants (k, year1 ), determination coefficients (r2 ; n ¼ 8) and turn-over times (s, days) for total losses and (bio)degradation of linear and isoprenoid hydrocarbons during the in situ incubation of petroleum contaminated sediment cores Hydrocarbon n-C17 n-C18 n-C20 n-C25 n-C30 Pristane Phytane

Total losses (exportation þ degradation) 2

k

r

3.3 3.2 2.8 4.7 1.4 1.5 1.7

0.85 0.83 0.64 0.99 0.83 0.96 0.96

Degradation

s

k

r2

s

110 114 130 78 261 243 214

0.84 0.74 0.69 0.96 0.38 0.45 0.55

0.86 0.86 0.66 0.86 0.56 0.96 0.98

435 493 529 380 960 811 664

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Fig. 5. Percentages of total losses (exportation þ degradation) and proportion attributed to (bio)degradation processes of linear and isoprenoid hydrocarbons during the in situ incubation of petroleum contaminated sediment cores (average of triplicate cores for 2 and 6 months, and of duplicate cores for 12 months).

12 months respectively) indicated that most of the exportation of petroleum in the water column appeared during the first 6 months of experiment. This suggests that, following its deposition at the sediment interface, the burial of oil by bioturbation reduces its chance of being released in the water column and thus, enhances its possibility of being preserved within the sediment. This is supported by the recovery of most of the remaining saturated fraction of the BAL between 20 and 55 mm depth after 12 months. However, since the individual aliphatic hydrocarbons constitutive of the BAL were completely degraded during the experiment (Figs. 2 and 5), the part of the saturated fraction that was preserved (appearing on the chromatogram as an unresolved complex mixture) exhibits a strong recalcitrance towards diagenetic processes. Although most of the acyclic hydrocarbons considered in this study disappeared after 12 months of experiment, significant differences in rates of loss were observed between the components (Table 1). Linear alkanes with a chain-length shorter than C25 exhibited comparable degradation rates, but disappeared two to three times faster than the longer-chain n-alkanes and the isoprenoid Pr and Ph. Branched alkanes are known to be more resistant toward biodegradation than linear alkanes (Swannell et al., 1996; Venosa et al., 1996; Le Dreau et al., 1997b). In the present study, the chainlength of the linear alkanes also appeared to influence their degradation rate. This is in good agreement with previous observations made during other field studies, which showed a chain-length dependence of the reactivity of linear alkanes (De Jonge et al., 1997; Del’Arco and de Francßa, 2001). The degradation rate constants of individual alkanes determined in the present work are up to 20 times lower than the one calculated by Venosa et al. (1996), during a

bioremediation experiment conducted in an oil-polluted sandy beach. These differences are likely due to the environmental conditions prevailing in both studies. The experiment of Venosa et al. (1996) was performed on a dry and permanently oxygenated sandy matrix. In our muddy sediment cores, despite a very thin oxygenated layer (Fig. 3), the activity of macrobenthic organisms in the reworked zone (0–10 cm) likely induced intermittent oxygen penetration into the anoxic deeper sediment. For this reason, it is possible that the reworked alkanes were degraded in the presence of oxygen. Nevertheless, the examination of the depth profiles of both oxygen and alkanes (Figs. 2 and 3) suggests that these latter compounds were subjected to oscillating redox conditions; the implication of anaerobic degradation processes cannot thus be ruled out. Besides displacing hydrocarbons from oxic to anoxic layers, macrobenthic organisms can also act on hydrocarbon evolution by producing digestive solubilizers which enhance hydrocarbon’s bioavailability, and may increase their exportation in the water column. Biosurfactants are present in high amounts in the digestive tract of benthic macrofauna (Mayer et al., 1997), which ingests the surface sediment several times a year (Myers, 1977). Consequently, sediment-bound hydrocarbons are solubilized during the gut transit of the organisms. Ahrens et al. (2001) have demonstrated that digestive surfactants can be responsible for the solubilization of sediment-bound organic contaminants. Similarly, Gilbert et al. (2001) have observed the solubilization of aliphatic hydrocarbons during Nereis virens feeding. The solubilization by benthic-surfactants has a positive effect on biodegradation, which depends on the dispersion state of hydrocarbons. This is optimum when hydrocarbons are dissolved or (pseudo)-solubilized (Bertrand et al., 1993; Bonin and Bertrand, 2000). This work shows that bioturbation processes due a natural benthic community can have a significant influence on the qualitative and quantitative fate of acyclic hydrocarbons following a petroleum contamination. These results are supported by the high density of macrobenthic organisms observed in the Carteau Cove sediments (Gerino et al., 1994). Complementary investigations will investigate the role of different functional groups (e.g. biodiffusors, upward and downward convoyers; Francßois et al., 2002) in the bioremediation of contaminated marine sediments.

Acknowledgements We would like to express our appreciation to the divers Christian Marschal, Roland Graille and Jacques Millet, and the crew of the N/O Antedon and of the Asbesto for help with fieldwork. Dr. F. Gilbert and Ms. K. Miyagawa are gratefully acknowledged for helpful

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