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Effects of Bioremediation on Toxicity and Chemical Composition of No. 2 Fuel Oil: Growth Responses of the Brown Alga Fucus vesiculosus
PII: S0025-326X(99)00181-2
Marine Pollution Bulletin Vol. 40, No. 2, pp. 135±139, 2000 Ó 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/00 $ - see front matter
Eects of Bioremediation on Toxicity and Chemical Composition of No. 2 Fuel Oil: Growth Responses of the Brown Alga Fucus vesiculosus MICHELE L. WRABEL and PAULETTE PECKOL* Department of Biological Sciences, Smith College, Northampton, MA 01063, USA We conducted a laboratory experiment to assess the effectiveness of nutrient (N and P) application to indigenous, marine microbial populations as a bioremediation technique to respond to oil spills along the temperate coastline of the western North Atlantic. We investigated the eects of various concentrations (10 ppm to 1 ppt) of No. 2 fuel oil, a commonly transported oil, on growth rates of the intertidal macroalga Fucus vesiculosus. We found growth inhibition after a one-day application of the lowest concentration tested and a clear dosage eect. The sensitivity of F. vesiculosus to oiling suggests that this abundant brown alga may serve as an eective indicator species. Nutrient (N + P) application as a bioremediation technique ameliorated the toxic eects of oil on F. vesiculosus without resulting in enrichment of macroalgal growth. Analysis of treatment samples with gas chromatography indicated greater microbial breakdown of the oil under enriched, compared with unenriched conditions. We measured a complete loss of n-alkanes, the preferred substrate by micro-organisms, from `enriched+oiled' samples after 18 days, while oil introduced to autoclaved seawater showed only minimal degradation. Bioremediation may be a viable clean-up alternative for petroleum contamination of temperate ecosystems, particularly in areas of heavy shipping trac, which probably support a relatively large background population of oil-degrading organisms. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: bioremediation; brown algae; Fucus vesiculosus; oil spills; western N Atlantic. Repeated inputs of both crude and re®ned oils threaten marine ecosystems worldwide, particularly coastal areas within oil shipping lanes (Vandermeulen and Ahern, 1976; Mann and Clark, 1978; Jaworski, 1989; Atlas and *Corresponding author. E-mail address: [email protected]
Cerniglia, 1995). A single spill of greater than 200 000 barrels of crude oil from the oil tanker Exxon Valdez into AlaskaÕs Prince William Sound in 1989 focused attention on the potential hazards of transportation of oil (Atlas, 1991). The tiny state of Rhode Island, which possesses over 400 miles of coastline, is located in a busy shipping zone with an estimated 170 ships entering Narragansett Bay annually and has recently experienced several serious oil spills. Notably, in June 1989 the World Prodigy grounded on Brenton Reef, Rhode Island, spilling nearly 300 000 gallons of No. 2 fuel oil (Jaworski, 1989; Peckol et al., 1990), while the barge North Cape grounded in January 1996 releasing nearly 1 000 000 gallons of No. 2 fuel oil into Block Island Sound and nearby wetland communities. After a spill, petroleum is degraded by a variety of biotic and abiotic processes, including dissolution and dispersion into the water column, evaporation, photooxidation and biodegradation by micro- and macroorganisms (Gearing and Gearing, 1982). Most of the long-term degradation of hydrocarbons is attributable to biological processes, with microbial (bacteria, protozoa, fungi) biodegradation as a major contributor (Walker, 1984). The percentage of petroleum-degrading microorganisms may increase from 1 to 10% of the total population in response to one spill; in fact, populations that have adapted to chronic oil inputs can increase within hours of a spill (Atlas, 1991; Atlas and Cerniglia, 1995). Because booms and skimmers, the primary response techniques, are rendered ineective by even moderate wave action or currents, and chemical dispersants are sometimes more toxic to marine organisms than the oil itself (Burridge and Shir, 1995), bioremediation may oer a less ecologically damaging alternative by taking advantage of these oil-degrading organisms (US Congress, Oce of Technology Assessment, 1991). Since availability of inorganic nitrogen (N) and phosphorus (P) often limits the rate of oil biodegradation by micro-organisms (Walker, 1984), research has focused on nutrient application as a `natural' means of 135
Marine Pollution Bulletin
oil spill mitigation; these received some attention as a means of clean-up in response to the Exxon Valdez spill. In laboratory enrichment experiments, Tabak et al. (1991) found enhanced biodegradation of weathered crude oil from the spill by indigenous micro-organisms; ®eld applications of fertilizer along 74 miles of aected shoreline supported these laboratory ®ndings (Pritchard, 1991; Pritchard et al., 1992; Atlas and Cerniglia, 1995). Two possible adverse consequences of nutrient application, eutrophication and ammonium toxicity, were found not to be signi®cant (Pritchard, 1991). Based on such laboratory and ®eld tests, bioremediation was established as an important treatment for oil spills on rocky intertidal shorelines of the type found in Alaska (Bragg et al., 1994). Here, we explored the eectiveness of nutrient (N and P) application as a bioremediation technique for the rocky temperate coastline of the western North Atlantic. We ®rst investigated the eects of various concentrations of No. 2 fuel oil, a commonly transported oil, on growth rates of the brown alga Fucus vesiculosus L. This fucoid alga is particularly suitable as a monitor species because it a dominant, perennial component of the intertidal North Atlantic ¯ora (Luning, 1990). Fucoid algae occupying the upper shore may be more vulnerable to oiling due both to direct contact after a spill and because such species often experience slower growth rates and hence slower recovery times (Thomas, 1978; De Vogelaere and Foster, 1994 and references cited therein). We evaluated any changes in the toxicity and chemical composition of the bioremediated oil. Estimations of sensitivity to oiling and bioremediation can provide data useful for determining community response and appropriate clean-up technology in the event of an oil spill along temperate rocky shores.
Methods Oiling eects We collected healthy, vegetative fronds of F. vesiculosus from intertidal regions at Fort Wetherill, Jamestown, Rhode Island, USA (see Brady-Campbell et al., 1984 for location), during July and October 1996, to evaluate eects of various concentrations of No. 2 fuel oil on growth rates. Algae were held at ambient temperatures during transport; laboratory experiments were initiated soon after return. Apical regions (15 cm) of F. vesiculosus were excised, cleaned of macroscopic epiphytes, and allowed to wound-heal for several hours in seawater prior to oil exposure. We added No. 2 fuel oil to 2800 ml Fernbach ¯asks containing seawater to achieve a range in concentrations, 10 ppm to 1 ppt. These were chosen based on the oil toxicity limit established by the US Environmental Protection Agency Oce of Research and Development (1 ppm) and on oil concentrations (including 100 ppm and 1 ppt) used in laboratory bioremediation tests (Tabak et al., 1991). Flasks, held at ambient tempera136
tures and a 14:10 L:D cycle (photon ¯ux density 150 lE mÿ2 sÿ1 ), were continuously shaken. Oil exposure lasted for a one-day period to simulate a onetime contact. After exposure to oil, the algae were removed from the oiled and control treatments (n 10, each treatment) and rinsed thoroughly in clean seawater, returned to clean ¯asks containing unoiled seawater, and allowed to grow for the remaining days (totaling three days). Growth rates were measured as doublings dÿ1 (Rhee, 1980) from initial and ®nal fresh weights after spinning similarly sized fronds (3 gfw) in a lettuce spinner to remove gravitational seawater. Bioremediation eects We tested nutrient application as a bioremediation technique during October and December 1996 using natural (indigenous) microbial populations present in the Narragansett Bay seawater. During the ®eld tests of fertilizers applied to areas aected by the Exxon Valdez spill, visual dierences in oil coverage occurred 10 to 14 days following nutrient application (Pritchard et al., 1992). To simulate this eect in the laboratory, we allowed natural microbial populations to grow in ®ltered seawater at ambient temperatures for 2 weeks prior to adding algal fronds under the following treatment conditions: (1) oiled to 100 ppm or (2) oiled and enriched. For the latter treatment the seawater was enriched weekly to 10 lM NH4 Cl:1 lM NaH2 PO4 , based on the 10.6 N:1 P ratio of nutrients applied to beaches during the Exxon Valdez bioremediation ®eld tests (US Environmental Protection Agency Oce of Research and Development, 1994). A third treatment (handled as the above two treatments), in which the seawater was autoclaved, then oiled and enriched as above, was used to measure oil toxicity in the absence of microorganisms. An autoclaved, enriched treatment without oiling was used to assess any enrichment eect on macroalgal growth; an unoiled control treatment contained only ®ltered seawater and macroalgae. We measured growth rates (n 10, each treatment condition) of F. vesiculosus after six days under the 5 treatment conditions using methods described above. Oil analysis Seawater samples from treatments described above were analyzed by gas chromatography (GC). Three extractions were performed with hexane to remove the water-accommodated fraction of No. 2 fuel oil from each treatment. A rotary evaporator was used to evaporate each sample to an average volume of 2 ml. The samples (n 2±3, each treatment) were transferred to glass vials, and GC was performed.
Results Eects of No. 2 fuel oil Fucus vesiculosus exposed to No. 2 fuel oil for one day showed decreasing growth rates with increasing oil concentrations during July and October (Fig. 1). All
Volume 40/Number 2/February 2000
Fig. 1 Mean (SE) growth rates (doublings dayÿ1 ) of F. vesiculosus following a one-day exposure to various concentrations of No. 2 fuel oil during July (WT 18°C) and October (WT 16°C) 1996.
treatments varied signi®cantly from one another during July (ANOVA, F 37.3, p < 0.0001; TukeyÕs, p < 0.05) in a clear dosage eect. During October, a cooler time of the year, signi®cant decreases (ANOVA, F 21.1, p < 0.0001) in growth rates occurred in response to oiling, and weight losses were evident at all oil concentrations above 10 ppm. Growth rates during October compared with July were lower across treatment for F. vesiculosus, and oiling had a greater eect during autumn.
greatest toxicity (TukeyÕs, p < 0.05) occurring in the oiled treatment without micro-organisms (`autoclaved, oiled and enriched'). The enriched bioremediation treatment (N and P addition to 100 ppm oil) signi®cantly (TukeyÕs, p < 0.05) ameliorated the negative effect of oiling during both experimental periods. Nutrient enrichment did not result in increased growth rates of F. vesiculosus (TukeyÕs, p > 0.05), thus, any ameliorating eects of bioremediation would be due to microbial breakdown of oil rather than a fertilization eect.
Eects of bioremediation During October and December, F. vesiculosus showed similar responses to treatment conditions in the bioremediation experiments (Table 1). Negative growth rates were measured under all oiled (100 ppm) treatment conditions (for October, ANOVA, F 36.3, p < 0.0001; for December, ANOVA, F 74.11, p < 0.0001), with
Appearance and chemistry of No. 2 fuel oil with bioremediation Visual dierences among treatments in the bioremediation experiments were obvious. For example, oil held in contact with microbial populations, namely the `100 ppm oil' and `enriched + 100 ppm oil' treatments, became more dispersed throughout the water column compared with the autoclaved (`autoclaved, oiled and enriched') treatment, eventually forming globules on the water surface. Microbial degradation of No. 2 fuel oil varied according to treatment, as re¯ected in GC results (Table 2). The nC14/UCM ratio re¯ects the loss of n-(normal) alkanes, compared to loss of the unresolved complex mixture (UCM), composed of compounds that cannot be resolved by this technique. Pristane (pr) and phytane (phy), two isoprenoid hydrocarbons that indicate the extent of microbial breakdown, are also measured in relation to n-alkanes. As microorganisms preferentially degrade the n-alkanes, the reported ratios (nC17/pr and
TABLE 1 Mean (SE) growth rates (doublings dayÿ1 ) of F. vesiculosus following a six-day exposure to 100 ppm oil under various treatments (see text for description of treatment preparation) conditions (n 10, each treatment) during October (water temperature, WT 16°C) and December (WT 8°C) 1996. Treatment Control Autoclaved, enriched 100 ppm 100 ppm, enriched 100 ppm, autoclaved, enriched
October
December
0.014 0.001 0.016 0.002 )0.019 0.003 )0.010 0.002 )0.021 0.005
0.007 0.002 0.006 0.002 )0.022 0.002 )0.015 0.004 )0.030 0.004
137
Marine Pollution Bulletin TABLE 2 Gas chromatographic analysisa of treatment samples (n 2±3) of 100 ppm No. 2 fuel oil.b Sample No. 2 fuel oil Autoclaved enriched +100 ppm 100 ppm Enriched +100 ppm
nC14/UCM
nC17/pr
nC18/phy
Range of n-alkanes
Degree of degradation
6.5 4.5 1.0 (No C14)
1.4 1.5 1.3 0.4
2.2 1.8 1.4 0.5
nC9±nC26 nC12±nC26 nC13±nC26 loss of all n-alkanes
None Mild Substantial High
a
Based on analysis and interpretation of E. Van Vleet. The n-alkanes used for diagnostic determination of microbial degradation are labelled as nC14, nC17, and nC18. Presence of UCM (Unresolved Complex Mixture), pristane (pr) and phytane (phy) (two isoprenoid hydrocarbons) are compared with these n-alkanes as shown in the abbreviated ratios. Lower nC14/UCM, nC17/pr, and nC18/phy ratios and smaller ranges of n-alkanes indicate a greater extent of degradation, as summarized under ``Degree of Degradation.''
b
nC18/phy) decrease. We used these ratios for comparison among treatments on the assumption that most nonbiological processes (e.g. physical weathering, volatilization) will not produce dierential losses of normal and branched hydrocarbons (Kennicutt, 1988; Pritchard et al., 1992). The number of n-alkanes remaining in each sample also indicates degradation. Analysis of the enriched (+N and P) bioremediation treatment samples revealed almost complete loss of alkanes and low nC17/ pristane and nC18/phytane ratios, indicating signi®cant microbial degradation of the oil. Oil subjected to microbial breakdown without N and P addition also was altered in relation to reference No. 2 fuel oil. Van Vleet (pers. comm.) attributed these changes to evaporation and some microbial degradation. Oil exposed to N and P in autoclaved seawater (without microorganisms) showed the least degradation of the three oiled treatments, with 25±30% of n-alkanes lost primarily to evaporation (Van Vleet, pers. comm.).
Discussion Results of this study indicate a clear, dosage-related inhibition of growth of F. vesiculosus by No. 2 fuel oil at concentrations as low as 10 ppm. The high sensitivity of F. vesiculosus to oiling suggests that this alga may serve as an eective indicator species (Levine, 1984). Bioremediation, nutrient enrichment of indigenous microbial populations, ameliorated the toxic eects of the oil without resulting in enrichment eects on macroalgal growth. GC analysis indicated greater microbial breakdown of the fuel oil under enriched conditions, suggesting that bioremediation with natural populations could be more widely used to respond to oil spills, particularly in areas of heavy shipping trac, which probably support a relatively large background population of oil-degrading organisms. The bioremediation experiments revealed that treatments enriched with N and P resulted in less deleterious eects of oiling on F. vesiculosus; GC analysis indicated substantial microbial breakdown of oil in the bioremediated treatments. The extent to which oil is degraded, or weathered, will in¯uence its toxic eects on macroalgae because weathered oil contains proportionately 138
less light normal alkanes than crude oil (Sjùtun and Lein, 1993). Analysis of the `enriched + 100 ppm oil' sample indicated nearly complete loss of all n-alkanes. This biodegradation occurred rapidly, within18 days, at both 8°C (December) and 16°C (October). As suggested by a ®eld study along the western North Atlantic (Venosa et al., 1996), bioremediation in a temperate climate can take place rapidly during most seasons, unlike the more limited treatment window for boreal locations like Alaska (Pritchard et al., 1992). Rapid oil disappearance brought about by application of fertilizers should thus make oiled coastlines safer for local populations. Our results also suggest that the indigenous petroleum-degrading micro-organisms of Narragansett Bay, Rhode Island, may be nutrient-limited. In fact, Howarth (1988) cites Narragansett Bay as an ecosystem that is largely N-limited, reporting a 6 N:1 P ratio. Some works (Bragg et al., 1994; Venosa et al., 1996) suggest that the decision to apply nutrients to enhance biodegradation of petroleum hydrocarbons should in part depend on the ambient nutrient levels available at the contaminated site, with conditions of optimal (>10°C) temperatures and low background nutrients arguing for bioremediation. Because limited, regional application of nutrients following a spill may not adversely aect the macroalgal populations, bioremediation may therefore be a viable clean-up alternative for petroleum contamination of temperate ecosystems such as Narragansett Bay. We thank E. Van Vleet for the gas chromatographic analysis and interpretation; P.H. Pritchard provided valuable suggestions for the bioremediation design. Financial support was provided by Smith College. Atlas, R. M. (1991) Microbial hydrocarbon degradation ± bioremediation of oil spills. Journal of Chemical Technology and Biotechnology 52, 149±156. Atlas, R. M. and Cerniglia, C. E. (1995) Bioremediation of petroleum pollutants. Biological Science 45, 332±338. Brady-Campbell, M. M., Campbell, D. B. and Harlin, M. M. (1984) Productivity of kelp (Laminaria spp.) near the southern limit in the northwestern Atlantic Ocean. Marine Ecology and Progressive Series 18, 78±88. Bragg, J. R., Prince, R. C., Harner, E. J. and Atlas, R. M. (1994) Eectiveness of bioremediation of the Exxon Valdez oil spill. Nature 368, 413±418. Burridge, T. R. and Shir, M. (1995) The comparative eects of oil dispersants and oil/dispersant conjugates on germination of the
Volume 40/Number 2/February 2000 marine macroalga Phyllospora comosa (Fucales: Phaeophyta). Marine Pollution Bulletin 31, 446±452. De Vogelaere, A. P. and Foster, M. S. (1994) Damage and recovery in intertidal Fucus gardneri assemblages following the Exxon Valdez oil spill. Marine Ecology and Progressive Series 106, 263±271. Gearing, P. J. and Gearing, J. N. (1982) Behaviour of No. 2 fuel oil in the water column of controlled ecosystems. Marine Environmental Research 6, 115±132. Howarth, R. W. (1988) Nutrient limitation of net primary productivity in marine ecosystems. Australian Review of Ecological System 19, 89±110. Jaworski, C. (1989) A close call: the Narragansett Bay oil spill. Maritimes 33, 12±13. Kennicutt, M. C. (1988) The eect of biodegradation on crude oil bulk and molecular composition. Oil Chemistry Pollution 4, 89±112. Levine, H. G. (1984) The use of seaweeds for monitoring coastal waters. In Algae as Ecological Indicators, ed. E. L. Shubert, pp. 189± 210. Academic Press, London. Luning, K. (1990) Seaweeds: Their Environment, Biogeography, and Ecophysiology. Wiley, New York, 527 p. Mann, K. H. and Clark, R. B. (1978) Long-term eects of oil spills on marine intertidal communities. Journal of the Fisheries Research Board of Canada 35, 791±795. Peckol, P., Levings, S.C. and Garrity, S. D. (1990) Kelp response following the World Prodigy oil spill. Marine Pollution Bulletin 21, 473±476. Pritchard, P. H. (1991) Bioremediation as a technology: experiences with the Exxon Valdez oil spill. Journal of Hazardous Material 28, 115±130. Pritchard, P. H., Mueller, J. G., Rogers, J.C., Kremer, F. V. and Glaser, J. A. (1992) Oil spill bioremediation: experiences, lessons, and results from the Exxon Valdez oil spill in Alaska. Biodegradation 3, 315±335.
Rhee, G-Yull. (1980) Continuous culture in phytoplankton ecology. In Advances in Aquatic Microbiology, eds. M. R. Droop and H. W. Jannash, pp. 151±203, Academic Press, New York. Sjùtun, K. and Lein, T. E. (1993) Experimental oil exposure of Ascophyllumnodosum (L.) Le Jolis. Journal of Experimental Marine Biology and Ecology 170, 197±212. Tabak, H. H., Haines, J. R., Venosa, A. D., Glaser, J. A., Desai, S. and Nisamaneepong, W. (1991) Enhancement of biodegradation of Alaskan weathered crude oil components by indigenous microbiota with the use of fertilizers and nutrients. In Proceedings of the 12th International Oil Spill Conference, pp. 583±590. San Diego, California. Thomas, M. L. H. (1978) Comparison of oiled and unoiled intertidal communities in Chedabucto Bay, Nova Scotia. Journal of Fisheries Research Board of Canada 35, 707±716. US Environmental Protection Agency Oce of Research and Development (1994) Cooperative research and development agreement with Exxon Company, USA.: bioremediation of oil in Prince William Sound, Alaska. EPA Publication EPA/600/F±94/036. US Congress, Oce of Technology Assessment (1991) Bioremediation of Marine Oil Spills-Background Paper, OTA-BP-O-70, Government Printing Oce, Washington, DC, 31 p. Vandermeulen, J. H. and Ahern, T. P. (1976) Eect of petroleum hydrocarbons on algal physiology: review and progress report. In Eects of Pollutants on Aquatic Organisms, ed. A. P. M. Lockwood, pp. 107±125. Cambridge University Press, London. Venosa, A. D., Suidan, M. T., Wrenn, B. A., Strohmeier, K. L., Haines, J. R., Eberhart, B. L., King, D. and Holder, E. (1996) Bioremediation of an experimental oil spill on the shoreline of Delaware Bay. Environmental Science and Technology 30, 1764± 1775. Walker, J. D. (1984) Chemical fate of toxic substances: biodegradation of petroleum. Marine Technology Society Journal 18, 73±86.
139
Marine Pollution Bulletin Vol. 40, No. 2, pp. 135±139, 2000 Ó 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/00 $ - see front matter
Eects of Bioremediation on Toxicity and Chemical Composition of No. 2 Fuel Oil: Growth Responses of the Brown Alga Fucus vesiculosus MICHELE L. WRABEL and PAULETTE PECKOL* Department of Biological Sciences, Smith College, Northampton, MA 01063, USA We conducted a laboratory experiment to assess the effectiveness of nutrient (N and P) application to indigenous, marine microbial populations as a bioremediation technique to respond to oil spills along the temperate coastline of the western North Atlantic. We investigated the eects of various concentrations (10 ppm to 1 ppt) of No. 2 fuel oil, a commonly transported oil, on growth rates of the intertidal macroalga Fucus vesiculosus. We found growth inhibition after a one-day application of the lowest concentration tested and a clear dosage eect. The sensitivity of F. vesiculosus to oiling suggests that this abundant brown alga may serve as an eective indicator species. Nutrient (N + P) application as a bioremediation technique ameliorated the toxic eects of oil on F. vesiculosus without resulting in enrichment of macroalgal growth. Analysis of treatment samples with gas chromatography indicated greater microbial breakdown of the oil under enriched, compared with unenriched conditions. We measured a complete loss of n-alkanes, the preferred substrate by micro-organisms, from `enriched+oiled' samples after 18 days, while oil introduced to autoclaved seawater showed only minimal degradation. Bioremediation may be a viable clean-up alternative for petroleum contamination of temperate ecosystems, particularly in areas of heavy shipping trac, which probably support a relatively large background population of oil-degrading organisms. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: bioremediation; brown algae; Fucus vesiculosus; oil spills; western N Atlantic. Repeated inputs of both crude and re®ned oils threaten marine ecosystems worldwide, particularly coastal areas within oil shipping lanes (Vandermeulen and Ahern, 1976; Mann and Clark, 1978; Jaworski, 1989; Atlas and *Corresponding author. E-mail address: [email protected]
Cerniglia, 1995). A single spill of greater than 200 000 barrels of crude oil from the oil tanker Exxon Valdez into AlaskaÕs Prince William Sound in 1989 focused attention on the potential hazards of transportation of oil (Atlas, 1991). The tiny state of Rhode Island, which possesses over 400 miles of coastline, is located in a busy shipping zone with an estimated 170 ships entering Narragansett Bay annually and has recently experienced several serious oil spills. Notably, in June 1989 the World Prodigy grounded on Brenton Reef, Rhode Island, spilling nearly 300 000 gallons of No. 2 fuel oil (Jaworski, 1989; Peckol et al., 1990), while the barge North Cape grounded in January 1996 releasing nearly 1 000 000 gallons of No. 2 fuel oil into Block Island Sound and nearby wetland communities. After a spill, petroleum is degraded by a variety of biotic and abiotic processes, including dissolution and dispersion into the water column, evaporation, photooxidation and biodegradation by micro- and macroorganisms (Gearing and Gearing, 1982). Most of the long-term degradation of hydrocarbons is attributable to biological processes, with microbial (bacteria, protozoa, fungi) biodegradation as a major contributor (Walker, 1984). The percentage of petroleum-degrading microorganisms may increase from 1 to 10% of the total population in response to one spill; in fact, populations that have adapted to chronic oil inputs can increase within hours of a spill (Atlas, 1991; Atlas and Cerniglia, 1995). Because booms and skimmers, the primary response techniques, are rendered ineective by even moderate wave action or currents, and chemical dispersants are sometimes more toxic to marine organisms than the oil itself (Burridge and Shir, 1995), bioremediation may oer a less ecologically damaging alternative by taking advantage of these oil-degrading organisms (US Congress, Oce of Technology Assessment, 1991). Since availability of inorganic nitrogen (N) and phosphorus (P) often limits the rate of oil biodegradation by micro-organisms (Walker, 1984), research has focused on nutrient application as a `natural' means of 135
Marine Pollution Bulletin
oil spill mitigation; these received some attention as a means of clean-up in response to the Exxon Valdez spill. In laboratory enrichment experiments, Tabak et al. (1991) found enhanced biodegradation of weathered crude oil from the spill by indigenous micro-organisms; ®eld applications of fertilizer along 74 miles of aected shoreline supported these laboratory ®ndings (Pritchard, 1991; Pritchard et al., 1992; Atlas and Cerniglia, 1995). Two possible adverse consequences of nutrient application, eutrophication and ammonium toxicity, were found not to be signi®cant (Pritchard, 1991). Based on such laboratory and ®eld tests, bioremediation was established as an important treatment for oil spills on rocky intertidal shorelines of the type found in Alaska (Bragg et al., 1994). Here, we explored the eectiveness of nutrient (N and P) application as a bioremediation technique for the rocky temperate coastline of the western North Atlantic. We ®rst investigated the eects of various concentrations of No. 2 fuel oil, a commonly transported oil, on growth rates of the brown alga Fucus vesiculosus L. This fucoid alga is particularly suitable as a monitor species because it a dominant, perennial component of the intertidal North Atlantic ¯ora (Luning, 1990). Fucoid algae occupying the upper shore may be more vulnerable to oiling due both to direct contact after a spill and because such species often experience slower growth rates and hence slower recovery times (Thomas, 1978; De Vogelaere and Foster, 1994 and references cited therein). We evaluated any changes in the toxicity and chemical composition of the bioremediated oil. Estimations of sensitivity to oiling and bioremediation can provide data useful for determining community response and appropriate clean-up technology in the event of an oil spill along temperate rocky shores.
Methods Oiling eects We collected healthy, vegetative fronds of F. vesiculosus from intertidal regions at Fort Wetherill, Jamestown, Rhode Island, USA (see Brady-Campbell et al., 1984 for location), during July and October 1996, to evaluate eects of various concentrations of No. 2 fuel oil on growth rates. Algae were held at ambient temperatures during transport; laboratory experiments were initiated soon after return. Apical regions (15 cm) of F. vesiculosus were excised, cleaned of macroscopic epiphytes, and allowed to wound-heal for several hours in seawater prior to oil exposure. We added No. 2 fuel oil to 2800 ml Fernbach ¯asks containing seawater to achieve a range in concentrations, 10 ppm to 1 ppt. These were chosen based on the oil toxicity limit established by the US Environmental Protection Agency Oce of Research and Development (1 ppm) and on oil concentrations (including 100 ppm and 1 ppt) used in laboratory bioremediation tests (Tabak et al., 1991). Flasks, held at ambient tempera136
tures and a 14:10 L:D cycle (photon ¯ux density 150 lE mÿ2 sÿ1 ), were continuously shaken. Oil exposure lasted for a one-day period to simulate a onetime contact. After exposure to oil, the algae were removed from the oiled and control treatments (n 10, each treatment) and rinsed thoroughly in clean seawater, returned to clean ¯asks containing unoiled seawater, and allowed to grow for the remaining days (totaling three days). Growth rates were measured as doublings dÿ1 (Rhee, 1980) from initial and ®nal fresh weights after spinning similarly sized fronds (3 gfw) in a lettuce spinner to remove gravitational seawater. Bioremediation eects We tested nutrient application as a bioremediation technique during October and December 1996 using natural (indigenous) microbial populations present in the Narragansett Bay seawater. During the ®eld tests of fertilizers applied to areas aected by the Exxon Valdez spill, visual dierences in oil coverage occurred 10 to 14 days following nutrient application (Pritchard et al., 1992). To simulate this eect in the laboratory, we allowed natural microbial populations to grow in ®ltered seawater at ambient temperatures for 2 weeks prior to adding algal fronds under the following treatment conditions: (1) oiled to 100 ppm or (2) oiled and enriched. For the latter treatment the seawater was enriched weekly to 10 lM NH4 Cl:1 lM NaH2 PO4 , based on the 10.6 N:1 P ratio of nutrients applied to beaches during the Exxon Valdez bioremediation ®eld tests (US Environmental Protection Agency Oce of Research and Development, 1994). A third treatment (handled as the above two treatments), in which the seawater was autoclaved, then oiled and enriched as above, was used to measure oil toxicity in the absence of microorganisms. An autoclaved, enriched treatment without oiling was used to assess any enrichment eect on macroalgal growth; an unoiled control treatment contained only ®ltered seawater and macroalgae. We measured growth rates (n 10, each treatment condition) of F. vesiculosus after six days under the 5 treatment conditions using methods described above. Oil analysis Seawater samples from treatments described above were analyzed by gas chromatography (GC). Three extractions were performed with hexane to remove the water-accommodated fraction of No. 2 fuel oil from each treatment. A rotary evaporator was used to evaporate each sample to an average volume of 2 ml. The samples (n 2±3, each treatment) were transferred to glass vials, and GC was performed.
Results Eects of No. 2 fuel oil Fucus vesiculosus exposed to No. 2 fuel oil for one day showed decreasing growth rates with increasing oil concentrations during July and October (Fig. 1). All
Volume 40/Number 2/February 2000
Fig. 1 Mean (SE) growth rates (doublings dayÿ1 ) of F. vesiculosus following a one-day exposure to various concentrations of No. 2 fuel oil during July (WT 18°C) and October (WT 16°C) 1996.
treatments varied signi®cantly from one another during July (ANOVA, F 37.3, p < 0.0001; TukeyÕs, p < 0.05) in a clear dosage eect. During October, a cooler time of the year, signi®cant decreases (ANOVA, F 21.1, p < 0.0001) in growth rates occurred in response to oiling, and weight losses were evident at all oil concentrations above 10 ppm. Growth rates during October compared with July were lower across treatment for F. vesiculosus, and oiling had a greater eect during autumn.
greatest toxicity (TukeyÕs, p < 0.05) occurring in the oiled treatment without micro-organisms (`autoclaved, oiled and enriched'). The enriched bioremediation treatment (N and P addition to 100 ppm oil) signi®cantly (TukeyÕs, p < 0.05) ameliorated the negative effect of oiling during both experimental periods. Nutrient enrichment did not result in increased growth rates of F. vesiculosus (TukeyÕs, p > 0.05), thus, any ameliorating eects of bioremediation would be due to microbial breakdown of oil rather than a fertilization eect.
Eects of bioremediation During October and December, F. vesiculosus showed similar responses to treatment conditions in the bioremediation experiments (Table 1). Negative growth rates were measured under all oiled (100 ppm) treatment conditions (for October, ANOVA, F 36.3, p < 0.0001; for December, ANOVA, F 74.11, p < 0.0001), with
Appearance and chemistry of No. 2 fuel oil with bioremediation Visual dierences among treatments in the bioremediation experiments were obvious. For example, oil held in contact with microbial populations, namely the `100 ppm oil' and `enriched + 100 ppm oil' treatments, became more dispersed throughout the water column compared with the autoclaved (`autoclaved, oiled and enriched') treatment, eventually forming globules on the water surface. Microbial degradation of No. 2 fuel oil varied according to treatment, as re¯ected in GC results (Table 2). The nC14/UCM ratio re¯ects the loss of n-(normal) alkanes, compared to loss of the unresolved complex mixture (UCM), composed of compounds that cannot be resolved by this technique. Pristane (pr) and phytane (phy), two isoprenoid hydrocarbons that indicate the extent of microbial breakdown, are also measured in relation to n-alkanes. As microorganisms preferentially degrade the n-alkanes, the reported ratios (nC17/pr and
TABLE 1 Mean (SE) growth rates (doublings dayÿ1 ) of F. vesiculosus following a six-day exposure to 100 ppm oil under various treatments (see text for description of treatment preparation) conditions (n 10, each treatment) during October (water temperature, WT 16°C) and December (WT 8°C) 1996. Treatment Control Autoclaved, enriched 100 ppm 100 ppm, enriched 100 ppm, autoclaved, enriched
October
December
0.014 0.001 0.016 0.002 )0.019 0.003 )0.010 0.002 )0.021 0.005
0.007 0.002 0.006 0.002 )0.022 0.002 )0.015 0.004 )0.030 0.004
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Marine Pollution Bulletin TABLE 2 Gas chromatographic analysisa of treatment samples (n 2±3) of 100 ppm No. 2 fuel oil.b Sample No. 2 fuel oil Autoclaved enriched +100 ppm 100 ppm Enriched +100 ppm
nC14/UCM
nC17/pr
nC18/phy
Range of n-alkanes
Degree of degradation
6.5 4.5 1.0 (No C14)
1.4 1.5 1.3 0.4
2.2 1.8 1.4 0.5
nC9±nC26 nC12±nC26 nC13±nC26 loss of all n-alkanes
None Mild Substantial High
a
Based on analysis and interpretation of E. Van Vleet. The n-alkanes used for diagnostic determination of microbial degradation are labelled as nC14, nC17, and nC18. Presence of UCM (Unresolved Complex Mixture), pristane (pr) and phytane (phy) (two isoprenoid hydrocarbons) are compared with these n-alkanes as shown in the abbreviated ratios. Lower nC14/UCM, nC17/pr, and nC18/phy ratios and smaller ranges of n-alkanes indicate a greater extent of degradation, as summarized under ``Degree of Degradation.''
b
nC18/phy) decrease. We used these ratios for comparison among treatments on the assumption that most nonbiological processes (e.g. physical weathering, volatilization) will not produce dierential losses of normal and branched hydrocarbons (Kennicutt, 1988; Pritchard et al., 1992). The number of n-alkanes remaining in each sample also indicates degradation. Analysis of the enriched (+N and P) bioremediation treatment samples revealed almost complete loss of alkanes and low nC17/ pristane and nC18/phytane ratios, indicating signi®cant microbial degradation of the oil. Oil subjected to microbial breakdown without N and P addition also was altered in relation to reference No. 2 fuel oil. Van Vleet (pers. comm.) attributed these changes to evaporation and some microbial degradation. Oil exposed to N and P in autoclaved seawater (without microorganisms) showed the least degradation of the three oiled treatments, with 25±30% of n-alkanes lost primarily to evaporation (Van Vleet, pers. comm.).
Discussion Results of this study indicate a clear, dosage-related inhibition of growth of F. vesiculosus by No. 2 fuel oil at concentrations as low as 10 ppm. The high sensitivity of F. vesiculosus to oiling suggests that this alga may serve as an eective indicator species (Levine, 1984). Bioremediation, nutrient enrichment of indigenous microbial populations, ameliorated the toxic eects of the oil without resulting in enrichment eects on macroalgal growth. GC analysis indicated greater microbial breakdown of the fuel oil under enriched conditions, suggesting that bioremediation with natural populations could be more widely used to respond to oil spills, particularly in areas of heavy shipping trac, which probably support a relatively large background population of oil-degrading organisms. The bioremediation experiments revealed that treatments enriched with N and P resulted in less deleterious eects of oiling on F. vesiculosus; GC analysis indicated substantial microbial breakdown of oil in the bioremediated treatments. The extent to which oil is degraded, or weathered, will in¯uence its toxic eects on macroalgae because weathered oil contains proportionately 138
less light normal alkanes than crude oil (Sjùtun and Lein, 1993). Analysis of the `enriched + 100 ppm oil' sample indicated nearly complete loss of all n-alkanes. This biodegradation occurred rapidly, within18 days, at both 8°C (December) and 16°C (October). As suggested by a ®eld study along the western North Atlantic (Venosa et al., 1996), bioremediation in a temperate climate can take place rapidly during most seasons, unlike the more limited treatment window for boreal locations like Alaska (Pritchard et al., 1992). Rapid oil disappearance brought about by application of fertilizers should thus make oiled coastlines safer for local populations. Our results also suggest that the indigenous petroleum-degrading micro-organisms of Narragansett Bay, Rhode Island, may be nutrient-limited. In fact, Howarth (1988) cites Narragansett Bay as an ecosystem that is largely N-limited, reporting a 6 N:1 P ratio. Some works (Bragg et al., 1994; Venosa et al., 1996) suggest that the decision to apply nutrients to enhance biodegradation of petroleum hydrocarbons should in part depend on the ambient nutrient levels available at the contaminated site, with conditions of optimal (>10°C) temperatures and low background nutrients arguing for bioremediation. Because limited, regional application of nutrients following a spill may not adversely aect the macroalgal populations, bioremediation may therefore be a viable clean-up alternative for petroleum contamination of temperate ecosystems such as Narragansett Bay. We thank E. Van Vleet for the gas chromatographic analysis and interpretation; P.H. Pritchard provided valuable suggestions for the bioremediation design. Financial support was provided by Smith College. Atlas, R. M. (1991) Microbial hydrocarbon degradation ± bioremediation of oil spills. Journal of Chemical Technology and Biotechnology 52, 149±156. Atlas, R. M. and Cerniglia, C. E. (1995) Bioremediation of petroleum pollutants. Biological Science 45, 332±338. Brady-Campbell, M. M., Campbell, D. B. and Harlin, M. M. (1984) Productivity of kelp (Laminaria spp.) near the southern limit in the northwestern Atlantic Ocean. Marine Ecology and Progressive Series 18, 78±88. Bragg, J. R., Prince, R. C., Harner, E. J. and Atlas, R. M. (1994) Eectiveness of bioremediation of the Exxon Valdez oil spill. Nature 368, 413±418. Burridge, T. R. and Shir, M. (1995) The comparative eects of oil dispersants and oil/dispersant conjugates on germination of the
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