AQUATIC MICROBIAL ECOLOGY Vol. 19: 285-295,1999 Published October 27 Aquat Microb Ecol Biogeochemical transformations and potential polyaromatic hydrocarbon degradation in macrofaunal burrow sediments W. K. Chung, Gary M. King* Darling Marine Center, University of Maine. Walpole. Maine 04573, USA ABSTRACT- A variety of polyaromatic hydrocarbons (PAH) includmg naphthalene, phenanthrene, acenaphthene, and dibenzothiophene were degraded with little or no lag in oxic slurries of marine sed- iment~from burrow walls constructed by benthic macrofauna. PAH degradation potentials of burrow sediments from the polychaetes Nereis virens and Clymenella torquata, the mollusc Mya arenaria and the enteropneust Saccoglossus bromophenolosus were generally greater than potentials for non-bur- row sedirnents; relative rates of degradation varied among the burrow wall sediments depending on the PAH assayed. Comparisons of the effects of available electron acceptors (oxygen, nitrate, ferric iron, sulfate) indicated that significant degradation of benzene, hexadecane and PAH occurred only in the presence of molecular oxygen. However, the capacity for oxic phenanthrene degradation was sta- ble during incubations with alternating oxic and anoxic conditions, suggesting significant anoxia toler- ance. Although burrow wall sediments were biogeochemically distinct with respect to rates of sulfate reduction, potential denitrification and potential ammonia oxidation, these patterns were not related to those of PAH degradation. KEY WORDS. Polycyclic aromatic hydrocarbon . PAH . Benthic . Biodegradation . Macrofauna INTRODUCTION Results from early n~icrobiological analyses sug- gested that PAH degradation depends on molecular Polycyclic aromatic hydrocarbons (PAH) account for oxygen to initiate ring cleavage (e.g. Delaune et al. a significant fraction of crude oil and petroleum-based 1981, Bauer & Capone 1985, 1988, Mihelic & Luthy products (e.g. creosote; Sharak-Genther et al. 1997). 1988). However, some monoaromatics, e.g. benzene In addition, PAH are derived from numerous natural and toluene, are labile under anaerobic conditions sources, perhaps the most important of which is bio- (e.g. Grbic-Galic & Vogel 1987, Lovley et al. 1989, mass burning (Jenkins et al. 1996). As a consequence Evans et al. 1991, Edwards et al. 1992). In addition, of extensive fossil fuel use and pollution, PAH occur naphthalene and phenanthrene have been degraded ubiquitously in marine systems, especially coastal under sulfate-reducing conditions in chronically conta- systems that are subject to numerous intense point minated sediment (Coates et al. 1997). Nevertheless, sources and less intense but widespread diffuse numerous recent studies indicate that aerobic transfor- sources. Since many PAH are mutagenic or carcino- mations result in more extensive and rapid degrada- genic (Menzie et al. 1992),their fate has been the focus tion of a greater diversity of PAH, including those with of extensive geochemical, biochemical, physiological high molecular weights (Delaune et al. 1981, Boldrin et and ecological analyses. al. 1993, Fritzsche et al. 1994, Kanaly et al. 1997). In coastal marine sediments, high rates of benthic oxygen uptake limit molecular oxygen to the upper 'Addressee for correspondence several mm of sediment (e.g. Blackburn & Blackburn E-mail: [email protected] 1993, Fenchel et al. 1998), thereby substantially con- O Inter-Research 1999 286 Aquat Microb Ecol 19: 285-295, 1999 straining the potential for aerobic PAH degradation. MATERIALS AND METHODS Nonetheless, macrofaunal burrow ventilation with oxygenated water may facilitate sub-surface aerobic Sediment collection. Burrow and non-burrow sedi- degradation, and in some cases inhibit anaerobic ments were collected from the intertidal zone of metabolism (Banta et al. 1999). The importance of Lowes Cove, Maine. This site has been described burrows and burrow ventilation is evident from bur- previously (e.g. Andersen et al. 1981, King et al. row surface area and ventilation rates. Kristensen 1983, Rice 1986, Hansen et al. 1996); it is not signi- (1984) has estimated that Nereis virens burrows at a ficantly contaminated. Clymenella torquata burrows modest density (700 m-') increase the sediment-water (sefi-rigid tubes penetrating about 8 to 10 cm into interface area by 150 %; individual burrow ventilation the sediment surface) were collected intact since they rates up to 400 m1 h-' have been reported consist of sediment cemented into a stable structure. (Scott 1975). Ventilation with oxygenated surface C. torquata tubes were later homogenized for use in water undoubtedly accounts for well-documented assays other than sulfate reduction. Mya arenaria increases in aerobic ammonia oxidation rates in the burrow sediments were collected from upper Lowes burrows of diverse macrofauna (e.g. Sayama & Kuri- Cove after exposing the animals with a clam fork. hara 1983, Kristensen et al. 1985, 1991, Kristensen M. arenana was typically found at a depth of 15 to 1988, Pelegn & Blackburn 1996), and may contribute 20 cm; burrows were formed by extension of the to enhanced transformations of iron, manganese, sul- animal's siphons to the sediment surface. Only the fur, and organic matter. innermost (1 to 2 mm thick) oxidized, light brown Anaerobic transformations are also enhanced in at layer of sediment from 5 to 10 cm depth surrounding least some burrows. This is due in part to the fact that the siphons was scrapped off with a spatula. Bur- ventilation and oxic conditions occur only intermit- row sediments of Saccoglossus bromophenolosus and tently (e.g. Scott 1975, AUer 1982, Kristensen 1988, Nereis virens were collected similarly. In all cases, Bussman & Reichardt 1991). In the case of denitrifica- burrow identification was based on the animal occu- tion, enhanced activity is due to increased nitrate pants and burrow morphologies. availability from nitrification during oxic periods and Sulfate reduction. Sulfate reduction was measured close coupling between these processes (Blackburn et by direct injection of carrier-free 35S042-(approxi- al. 1994). Patterns of sulfate reduction vary more than mately 37 kBq [l pCi] cm-3) into 3 cm3 disposable those of nitrification and denitrification, with enhanced syringes modified by removing the luer tips. For bur- activity near burrows in some instances and apparent rows of Mya arenaria, Nereis virens, and Saccoglossus inhibition in others (Hansen et al. 1996, Giray & King bromophenolosus, sediments (inner I mm) were col- 1997). In this case, parameters other than ventilation lected using a spatula and then loaded into the and oxygen, e.g. organic matter supply, may be impor- syringes. For Clymenella torquata, bulk sediments tant. Regardless, burrows may be sites of enhanced were removed from intact burrow tubes; the tubes, anaerobic aromatic degradation by sulfate-reducing with walls about 1 mm thick, were subsequently bacteria (Edwards et al. 1992, Rabus et al. 1993, Beller loaded into syringes. Modified 3 cm3 syringes were et al. 1996, Bedessem et al. 1997, Coates et al. 1997) also used to obtain cores perpendicular to the burrow or denitrifiers (Bossert & Young 1986, Archangeli & walls of N. virens, M. arenaria, and C. torquata. These Arvin 1995). cores were collected by carefully inserting syringe In the present study, we examine PAH degradation barrels into the sediment while holding the plunger potentials for burrow sediments from the polychaetes steady at the inner surface of the burrow wall. Sub- Clymenella torqua ta and Nereis virens, the mollusc sequent to incubation with a radiotracer, the cores Mya arenana and the enteropneust Saccoglossus were extruded and sectioned at 2 mm intervals or bromophenolosus. Radiotracers were used to com- greater. Vertical profiles of non-burrow sedirnents pare degradation for benzene and hexadecane under were obtained using acrylic cores (7.5 cm inner dia- oxic and anoxic conditions; stable substrates were meter) carefully positioned between obvious burrow used to assay oxic PAH degradation and to deter- openings at the sediment surface. These cores were mine responses to periodic changes in oxygen avail- subsampled at 1 cm intervals over a depth of 15 cm. ability. A suite of biogeochemical transformations, Details of radiotracer injection, sediment processing by including sulfate reduction and denitrification, and chromous chloride distillation for radiosulfide forma- an aerobic process, ammonia oxidation, were also tion after 3.5 to 4 h incubation and estimation of pore- measured to determine the extent to which different water sulfate concentrations follocv Hansen et al. burrow and ventilation characteristics affect pro- (1996) and Giray & King (1997).All radi.oassays were cesses that may contribute to hydrocarbon biode- conducted with a Wallac LKB model 1217 scintillation gradation. counter using internal standardization. Chung & King- Polyaromatic hydrocarbon degradation potentials of burrow sedlments 2 87 Potential denitrification rate. Slurries of burrow tles were incubated with shaking (-150 rpm) at room sediments collected as above were prepared by mixing temperature in the dark. Center wells were removed 10 cm3 of sediment with 20 cm3 of seawater in 160 cm3 from triplicates in each set at intervals; the well con- serum bottles. The bottle headspaces were flushed tents were added to scintillation vials containing 10 m1 with nitrogen and then the bottles were sealed with of Scintiverse (Fisher Scientific, Inc.).Radioactivity was butyl stoppers.