Danica C. Mueller, James S. Bonner, and Robin L. Autenrieth. Texas A&M University, Civil Engineering Department. College Station, Texas 77843-3136.
THE TOXICITY OF OIL-CONTAMINATED SEDIMENTS DURING BIOREMEDIATION OF A WETLAND Danica C. Mueller, James S. Bonner, and Robin L. Autenrieth Texas A&M University, Civil Engineering Department College Station, Texas 77843-3136 Kenneth Lee Fisheries and Oceans Canada Maurice Lamontagne Institue Mont-Joli, Quebec G5H 3Z4, Canada
Kenneth Doe Environmental Sciences Centre Environment Canada Moncton, New Brunswick El A 3E9, Canada
Methods
ABSTRACT: Inorganic nutrients were applied to oiled wetland sediments during an experiment to assess the effect of enhanced bioremediation on petroleum degradation and toxicity reduction. For a period of 6 months after the controlled application of oil to experimental plots, oiled wetland sediment samples were tested for acute toxicity. The three treatments evaluated were an oiled control, inorganic nutrient addition, and inorganic nutrient addition plus an alternate electron acceptor. Sediment toxicity was assessed using Microtox® and amphipod bioassays. The Microtox® 100% Test detected significant sediment toxicity up to 29 days after oil and treatment application while the Solid-Phase Test detected significant sediment toxicity up to Day 15. The Microtox 100%® Test showed elevated toxicity on Day 8 for nutrient plus alternate electron acceptor plots and reduced toxicity on Day 15 for nutrient plots, relative to oiled controls. The observed decrease in sediment toxicity from the Microtox® assays correlated with petroleum losses. Oiled sediments were initially highly toxic to amphipods with average mortality rates ranging from 86 to 92%. Amphipod mortality decreased significantly 71 days after treatment to between 47 and 28%. However, a significant increase in mortality was observed at Day 140 in plots receiving nutrient amendments, possibly due to elevated ammonia levels.
Oil was applied under controlled conditions to wetland sediments in a designated research site located off the San Jacinto River near Houston, Texas. Twenty-one sampling plots were delineated along the shoreline of the study site. Of these plots, 3 were left unoiled, 6 served as oiled controls, and the remaining 12 were oiled and evenly divided among two bioremediation amendments. One amendment consisted of nitrogen and phosphorus in the form of diammonium phosphate (NP). The other amendment contained nitrogen and phosphorus in the same amount plus nitrate (potassium nitrate) as an alternate electron acceptor (NP+AEA). Samples were collected for background analysis (Day -6) before the day of oil application (Day -4). Amendments were first applied on Day 0, following sample collection. Sediment toxicity was assessed using the Microtox® 100% Test, Microtox® Solid-Phase Test, and an amphipod bioassay. Hydrocarbon concentrations in sediment were estimated with gas chromatography/mass spectrometry (GC/MS) total target saturate (single-bonded chain) and aromatic (doublebonded ring) measurements. Microtox® bioassays. The Microtox® bioassay is a standardized toxicity testing system using luminescent bacteria, Vibrio fisheri, as the toxicity indicator. Toxicity is indicated by a decrease in light production by the bacteria, which is easily measured. For testing, 7 g of wet sediment and 35 ml Microtox® diluent were vigorously mixed, then centrifuged to separate the liquid phase from the solid sediment. The resulting liquid phase (elutriate) was then removed and tested according to the Microtox® 100% Test protocol (Microbics Corporation, 1992). Responses were reported as the effective concentration reducing the reagent light output by 25%, also known as the EC25- Liquid/sediment suspensions were tested with the Microtox® SPT according to the small-sample procedure described by Microbics Corporation (Microbics Corporation, 1994). SPT responses were reported as the effective concentration that reduced the reagent light output by 50% (EC»). Amphipod bioassay. The amphipod bioassay analyzed the lethality of oiled sediments using the sediment-burrowing amphipod, Eohaustorius estuarius, as the test organism. The assay is a 10-day, static assay performed according to standard methods set by Environment Canada (1992). Toxicity is measured by the number of dead or missing animals at the end of the 10-day experiment.
Introduction A multi-phase bioremediation project was initiated following the rupture of several oil and gas pipelines during extensive flooding of the San Jacinto River near Houston, Texas, in October of 1994. A small cove and its associated wetlands were excluded from clean-up efforts and set aside for a petroleum bioremediation study. Phase I research began in December of 1994 and monitored sediment toxicity associated with intrinsic petroleum degradation at the study site. The Microtox® 100% Test was used in this phase to evaluate sediment elutriate toxicity as described by Mueller, et al. (1999). Phase Ð research began in March 1996 with the controlled application of weathered oil and selected nutrient amendments to evaluate treatment effects on petroleum degradation and toxicity reduction. This manuscript summarizes toxicity methods and results from Phase II research.
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1999 INTERNATIONAL OIL SPILL CONFERENCE which was reflected by a decrease in toxicity with both Microtox® bioassays. Total target saturates and aromatics showed a strong correlation with Microtox® 100% toxicity test results and followed a similar pattern of reduction (Figure 3). The SPT provided a stronger correlation with target saturates and aromatics with R2 equal to 0.77 and 0.75, respectively. Sediments to which oil was applied were initially highly toxic to amphipods with average mortality rates ranging from 86 to 92%. Amphipod mortality decreased significantly 71 days after treatment to between 47 and 28%. After day 71 a significant increase in amphipod mortality was observed in the nutrient plots (Figure 4). Although not confirmed, this observed latent mortality may be due to ammonia build-up in plots due to repeated nitrogen addition. Amphipods are recognized as being highly sensitive to ammonia, which is a potential byproduct of microbial nitrogen metabolism (EPA, 1994).
Results The Microtox® Bioassay on both elutriates and solid phases indicated a rapid decline in acute sediment toxicity. In the elutriate analysis, 18 of the oiled plots demonstrated nondetectable responses by Day 29 (Figure 1). The Microtox® 100% Test showed toxicity that was significantly elevated in NP+AEA plots on Day 8. However, by Day 15 these plots were not statistically different from the oiled controls. On Day 15 the 100% Test showed significant toxicity reduction for NP plots equivalent to that of the unoiled controls. The Microtox® SolidPhase Test (SPT) detected elevated toxicity in oiled plots compared with unoiled controls until Day 113 (Figure 2). However, the SPT test showed no statistical difference in toxicity between the bioremediation treatments and oiled controls. GC/MS analysis showed significant removal of hydrocarbons,
more toxic ■ Unoiled -NP ■NP+AEA -Oiled less toxic 19
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Day of experiment Figure 1. Acute toxicity trends over time determined using the Microtox® 100% Test on sediment elutriates. The plot shows the results of the two treatments: nitrogen/phosphorus, and nitrogen/phosphorus plus alternate electron acceptor, in comparison with unoiled and oiled plots. Day 0 marks the first day of treatment application.
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Day of experiment Figure 2. Acute toxicity trends over time in sediment from the research site, determined using the Microtox® Solid Phase Test. The plot shows the results of the two treatments: nitrogen/phosphorus, and nitrogen/phosphorus plus alternate electron acceptor, in comparison with unoiled and oiled plots. Day 0 marks the first day of treatment application.
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Figure 3. Acute toxicity of elutriates, determined using the Microtox® 100% Test, is plotted alongside GC/MS total target saturates and total target aromatics.
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Day of experiment Figure 4. Acute toxicity trends over time in sediment from Parker's Cove, determined using an amphipod bioassay. The plot shows the results of the two treatments: nitrogen/phosphorus, and nitrogen/phosphorus plus alternate electron acceptor, in comparison with unoiled and oiled plots. Conclusions
Acknowledgements
The Microtox® and amphipod bioassays demonstrated their usefulness as indicators of petroleum contamination and treatment effectiveness. The Microtox® bioassays showed a rapid decrease in toxicity with significant reduction observed for NP amended plots and increased toxicity for NP+AEA plots, relative to oiled controls. The amphipods were more sensitive than the Microtox® bacteria to toxicity changes in the sediments. Toxicity decreased rapidly to day 70, at which time amphipod mortality significantly increased unexpectedly. This effect was possibly due to ammonia build-up in the test plots due to repeated nutrient addition.
Special thanks to the Texas General Land Office for funding this research.
Biography Danica Mueller is a graduate student at Texas A&M University. Her primary interests include the areas of environmental science and toxicology.
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References 1.
2.
Environment Canada (EC), 1992. Biological test method: Acute test for sediment toxicity using marine or estuarine amphipods. EPS l/RM/26. Environment Canada, Environmental Protection, Canada. EPA (U.S. Environmental Protection Agency). 1994. Methods for assessing the toxicity of sediment-associated contaminants with estuarine and marine amphipods. EPA/600/R-94/025. Office of Research and Development, Washington, DC.
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Microbics Corporation, 1992. Microtox Manual: A Toxicity Testing Handbook. Microbics Co. Carlsbad, CA, USA. Microbics Corporation, 1994. Microtox M500 Manual. Microbics Corporation. Carlsbad, CA, USA. Mueller, Danica C , James S. Bonner, Susanne J. McDonald, and Robin L. Autenrieth, 1999. Acute toxicity of estuarine wetland sediments contaminated by petroleum. Environmental Technology. In press.
